RNA Viruses: Host Gene Responses to Infections

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Decheng Yang University of British Columbia, Canada

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Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

Library of Congress Cataloging-in-Publication Data RNA viruses : host gene responses to infections / editor, Decheng Yang. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-981-283-379-2 (hardcover) ISBN-10: 981-283-379-X (hardcover) 1. RNA viruses. 2. Viral genetics. I.Yang, Decheng. [DNLM: 1. Host-Pathogen Interactions. 2. RNA Viruses--physiology. 3. Gene Expression Regulation, Viral. 4. RNA Interference. 5. RNA Viruses--genetics. QW 168 R6268 2009] QR395.R584 2009 579.2'5--dc22 2008035064

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

Copyright © 2009 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

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Contributors

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Preface Section I

xxiii Retrovirus

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1 Efforts to Characterize Host Response to HIV-1 Infection Monty Montano & Paola Sebastiani

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2 Signaling Pathways Activated by HIV and Their Impact on Immune Responses Katrina Gee, Sasmita Mishra, Wei Ma, Marko Kryworuchko & Ashok Kumar

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3 Host Immune Responses in HIV Infection Robert D. Allison & Shyam Kottilil

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4 HIV-1 and RNA Interference — Examining a Complex System Zachary A. Klase, Kuan-Teh Jeang & Fatah Kashanchi

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Section II

Negative Single-Stranded RNA Virus

5 Signaling Pathways Induced by Influenza Viruses Stephan Pleschka, Stephan Ludwig, Oliver Planz & Thorsten Wolff

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6 Host Immune Response to Influenza Virus Thomas M. Moran & Carolina B. López

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7 Signaling Pathways in the Host Cell Response to RSV Infection Allan R. Brasier, Ping Liu, Bing Tian & Sanjeev Choudhary

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8 Protective and Pathologic Host Responses to Pulmonary Respiratory Syncytial Virus Infection Dennis M. Lindell & Nicholas W. Lukacs

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9 Innate Recognition of Viral Infection and the Involvement of Autophagy Balaji Ramanathan & Akiko Iwasaki

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10 VSV Infection Elicits Distinct Host Responses in the Periphery and the Brain Carol Shoshkes Reiss

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11 Hantavirus Infection and Innate Immunity Nandini Sen, Adrish Sen, Peter Alff, Irina Gavrilovskaya & Erich R. Mackow

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12 Measles Virus Captures Specific Host Machineries to Cause Immunosuppression and Disease Bumsuk Hahm & Michael B. A. Oldstone

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Section III

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Positive Single-Stranded RNA Virus

13 Severe Acute Respiratory Syndrome Coronavirus Induces Differential Host Gene Expression Responses Associated with Pathogenesis Vincent T. K. Chow & W. F. Leong

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14 Characterization of Signaling Pathways in Cells Infected with SARS-CoV Tetsuya Mizutani

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15 Impact of Filovirus Infection upon Cellular Signaling Pathways Christopher F. Basler

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16 Responses to Ebola and Marburg Virus Infections Thomas W. Geisbert, Friederike Feldmann, Joan Geisbert & Heinz Feldmann

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17 Host Gene Expression Triggered by West Nile Virus Infection Janusz T. Pawe ska

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18 West Nile Virus-host Interaction: An Immunological Prospective Raghavan Bhuvanakantham & Mah-Lee Ng

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19 Hepatitis C Virus (Flaviviridae): Host Immune Responses to Hepatitis C Virus Christoph Neumann-Haefelin & Robert Thimme

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20 Host responses to Hepatitis C Virus Infection: Gene Expression Profiles and Viral Pathogenesis Melanie Lambotin, Françoise Stoll-Keller & Thomas F. Baumert

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21 Differential Gene Expression in Coxsackievirus Infection and Its Effect on Viral Pathogenesis Huifang M. Zhang, Travis Lim, Honglin Luo, Bruce McManus & Decheng Yang

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22 Host Signaling Responses to Coxsackievirus Infection Guang Gao, Decheng Yang, Bruce M. McManus & Honglin Luo

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23 Endothelial Damage Response by Dengue Virus Infection Irene Bosch, Katherine J. Martin, Aniuska Becerra Artiles & Rajas V. Warke

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24 Molecular Pathogenesis of Human Norovirus Ming Tan, Tibor Farkas & Xi Jiang

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25 Host Responses During Sindbis Virus Encephalomyelitis in Mice: New Implications for Understanding the Pathogenesis of Alphavirus Infections of the Central Nervous System David N. Irani & Natalie A. Prow

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Section IV

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Double-Stranded RNA Virus

26 Host Signaling Responses to Reovirus Infection Da Pan, Paola Marcato, Maya Shmulevitz & Patrick W. Lee

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27 Rotavirus Antagonism of the Host Innate Immune Response John T. Patton & Mario Barro

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Index

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Peter Alff Molecular and Cellular Biology Graduate Program Department of Medicine, Stony Brook University, Stony Brook N.Y. 11794 USA Robert D. Allison Infectious Diseases and Immunogenetics Section Department of Transfusion Medicine Clinical Center, NIH Building 10, Room 1C711, 900 Rockville Pike Bethesda, MD 20892 USA Aniuska Becerra Artiles University of Massachusetts Medical School Center for Infectious Disease and Vaccine Research 55 Lake Ave. North, Worcester MA 01655 USA Mario Barro Global Vaccines, Inc. 7020 Kit Creek Road, Research Triangle Park, NC, 27709-4827 USA Christopher F. Basler Department of Microbiology Box 1124, Mount Sinai School of Medicine 1 Gustave L. Levy Place New York, NY 10029 USA ix

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Thomas. F. Baumert Inserm, U748, Strasbourg, France Université Louis Pasteur Strasbourg France Service d’Hépatogastroentérologie Hôpitaux Universitaires de Strasbourg Strasbourg France Raghavan Bhuvanakantham Flavivirology Laboratory Department of Microbiology 5 Science Drive 2 National University of Singapore 117597 Singapore Irene Bosch University of Massachusetts Medical School Center for Infectious Disease and Vaccine Research 55 Lake Ave. North Worcester, MA 01655 USA Allan R. Brasier Departments of Medicine Biochemistry and Molecular Biology, and the Sealy Center for Molecular Medicine The University of Texas Medical Branch Galveston, TX 77555-1060 USA Sanjeev Choudhary Departments of Medicine, Biochemistry, and Molecular Biology, and the Sealy Center for Molecular Medicine The University of Texas Medical Branch Galveston, TX 77555-1060 USA

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Vincent T. K. Chow Infectious Diseases Program Department of Microbiology Yong Loo Lin School of Medicine National University of Singapore 5 Science Drive 2 Kent Ridge 117597, Singapore Tibor Farkas Division of Infectious Diseases Cincinnati Children’s Hospital Medical Center University of Cincinnati, College of Medicine 3333 Burnet Avenue Cincinnati, OH45229, USA Friederike Feldmann Special Pathogens Program National Microbiology Laboratory Public Health Agency of Canada Winnipeg, Manitoba, Canada Heinz Feldmann Special Pathogens Program National Microbiology Laboratory Public Health Agency of Canada Winnipeg, Manitoba Canada Department of Medical Microbiology University of Manitoba Winnipeg, Manitoba, Canada Current address: Laboratory of Virology Rocky Mountain Laboratories National Institute of Allergy and Infectious Diseases National Institute of Health 903 South 4th Street, Hamilton Montana 59840 USA

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Guang Gao The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research Department of Pathology and Laboratory Medicine University of British Columbia Providence Heart + Lung Institute St. Paul’s Hospital Vancouver, BC V6Z 1Y6, Canada Irina Gavrilovskaya Molecular and Cellular Biology Graduate Program Department of Medicine, Department of Molecular Genetics, and Microbiology Stony Brook University Stony Brook, N.Y. 11794 Northport VA Med. Center Northport N.Y. 11768 USA Katrina Gee Department of Microbiology & Immunology Queen’s University, Kingston Ontario, Canada Joan Geisbert National Emerging Infectious Diseases Laboratories Institutes Boston University School of Medicine, Boston, MA, USA Thomas W. Geisbert National Emerging Infectious Diseases Laboratories Institutes Boston University School of Medicine Boston, USA

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Bumsuk Hahm Viral-Immunobiology Laboratory Department of Immunology and Microbial Science The Scripps Research Institute La Jolla, CA 92037, USA Current Address: Departments of Surgery and Molecular Microbiology and Immunology Center for Cellular and Molecular Immunology University of Missouri-Columbia School of Medicine One Hospital Dr. Columbia MO 65212, USA David N. Irani Department of Neurology University of Michigan Medical School Biomedical Sciences Research Building Room 4007, 109 Zina Pitcher Place Ann Arbor Michigan 48109-2200, USA Akiko Iwasaki Department of Immunobiology Yale University School of Medicine 300 Cedar Street TAC S655B New Haven CT 06520 USA Kuan-Teh Jeang National Institute of Allergy and Infectious Diseases National Institutes of Health Molecular Virology Section, LMM Bethesda MD, 20892 USA

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Xi Jiang Division of Infectious Diseases Cincinnati Children’s Hospital Medical Center University of Cincinnati College of Medicine 3333 Burnet Avenue Cincinnati, OH45229, USA Fatah Kashanchi The George Washington University Medical Center Department of Immunology Microbiology and Tropical Medicine 2300 I Street NW Washington, DC 20037 USA Zachary A. Klase The George Washington University Medical Center Department of Immunology Microbiology and Tropical Medicine 2300 I Street NW Washington, DC 20037 USA Shyam Kottilil Laboratory of Immunology National Institute of Allergy & Infectious Diseases NIH, Building 10, Room 11N204, 900 Rockville Pike Bethesda, MD 20892 USA Marko Kryworuchko Infectious Disease and Vaccine Research Centre Research Institute, Division of Virology Children’s Hospital of Eastern Ontario Departments of Pathology and Laboratory Medicine, and Biochemistry, Microbiology and Immunology 401 Smyth Road, University of Ottawa, Ottawa Ontario, Canada

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Ashok Kumar Infectious Disease and Vaccine Research Centre Research Institute, Division of Virology Children’s Hospital of Eastern Ontario Departments of Pathology and Laboratory Medicine, and Biochemistry, Microbiology and Immunology 401 Smyth Road, University of Ottawa, Ottawa Ontario, K1H 8L1 Canada Melanie Lambotin Inserm, U748, Strasbourg, France Université Louis Pasteur Strasbourg, France Patrick W. K. Lee Department of Microbiology & Immunology Faculty of Medicine Sir Charles Tupper Medical Building 7th floor, Room 7-P 5850 College Street Halifax, NS B3H 1X5, Canada W. F. Leong Infectious Diseases Program Department of Microbiology Yong Loo Lin School of Medicine National University of Singapore 5 Science Drive 2 Kent Ridge 117597, Singapore Travis Lim Department of Pathology and Laboratory Medicine University of British Columbia The iCAPTURE Center Providence Heart + Lung Institute St. Paul’s Hospital Vancouver, BC V6Z 1Y6, Canada

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Dennis M. Lindell Department of Pathology University of Michigan Medical School Ann Arbor, MI 48109 USA Ping Liu Departments of Medicine, Biochemistry and Molecular Biology and the Sealy Center for Molecular Medicine The University of Texas Medical Branch Galveston, TX 77555-1060 USA Carolina B. López Department of Microbiology Mount Sinai School of Medicine New York, 10029 NY USA Stephan Ludwig Institute of Molecular Virology (IMV) ZMBE, Westfälische-Wilhelms-University Von-Esmarch Str. 56, D-48149 Münster, Germany Nicholas W. Lukacs Department of Pathology University of Michigan Medical School Ann Arbor MI 48109 USA Honglin Luo The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research Department of Pathology and Laboratory Medicine, University of British Columbia Providence Heart + Lung Institute, St. Paul’s Hospital Vancouver, BC V6Z 1Y6, Canada

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Wei Ma Biochemistry, Microbiology and Immunology 401 Smyth Road, University of Ottawa, Ottawa Ontario, Canada Erich R. Mackow Molecular and Cellular Biology Graduate Program Department of Medicine, Department of Molecular Genetics and Microbiology Stony Brook University Stony Brook, N.Y. 11794 Northport VA Med. Center Northport N.Y. 11768 USA Paola Marcato Department of Microbiology & Immunology Faculty of Medicine, Sir Charles Tupper Medical Building 7th floor, Room 7-P 5850 College Street Halifax NS B3H 1X5, Canada Katherine J. Martin University of Massachusetts Medical School Center for Infectious Disease and Vaccine Research 55 Lake Ave. North Worcester, MA 01655 USA Bruce McManus The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research Department of Pathology and Laboratory Medicine University of British Columbia Providence Heart + Lung Institute, St. Paul’s Hospital Vancouver, BC V6Z 1Y6, Canada

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Sasmita Mishra Biochemistry, Microbiology and Immunology 401 Smyth Road, University of Ottawa, Ottawa Ontario, Canada Tetsuya Mizutani Department of Virology I National Institute of Infectious Diseases Gakuen 4-7-1, Musashimurayama Tokyo 208-0011, Japan Monty Montano Center for HIV-1/AIDS Care and Research 650 Albany Street, Boston University School of Medicine & Harvard School of Public Health Boston, MA 02118 USA Thomas M. Moran Department of Microbiology, Mount Sinai School of Medicine New York, 10029 NY USA Christoph Neumann-Haefelin Department of Medicine II University Hospital Freiburg Hugstetter Strasse 55 D-79106 Freiburg Germany Mah-Lee Ng Flavivirology Laboratory, Department of Microbiology 5 Science Drive 2 National University of Singapore 117597 Singapore

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Michael B.A. Oldstone Viral-Immunobiology Laboratory Department of Immunology and Microbial Science and Department of Infectology The Scripps Research Institute La Jolla, CA 92037 USA Da Pan Department of Microbiology & Immunology Faculty of Medicine, Sir Charles Tupper Medical Building 7th floor, Room 7-P 5850 College Street Halifax, NS B3H 1X5, Canada John T. Patton Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD 20892 USA Janusz T. Pawe ska Special Pathogens Unit, National Institute for Communicable Diseases of the National Health Laboratory Service Modderfontein Road 1, Sandringham-Johannesburg 2131 South Africa Oliver Planz Friedrich-Loeffler-Institute (FLI) Paul-Ehrlich Str. 28, D-72076 Tübingen Germany Stephan Pleschka Institute for Medical Virology Justus-Liebig-University Frankfurter Str. 107, D-35392 Giessen Germany

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Natalie A. Prow Department of Molecular Microbiology and Immunology The Johns Hopkins University Bloomberg School of Public Health Baltimore Maryland, 21205 USA Balaji Ramanathan Department of Biomedical Sciences Atlantic Veterinary College University of Prince Edward Island 550 University Ave. Charlottetown, PE C1A 4P3, Canada Carol Shoshkes Reiss Biology Department New York University 100 Washington Square East New York, NY 10003, USA Paola Sebastiani Boston University School of Medicine Department of Biostatistics Boston, MA 02118 USA Adrish Sen Molecular and Cellular Biology Graduate Program Department of Medicine Department of Molecular Genetics and Microbiology Stony Brook University Stony Brook, N.Y. 11794 USA

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Nandini Sen Molecular and Cellular Biology Graduate Program Department of Medicine Department of Molecular Genetics and Microbiology Stony Brook University Stony Brook, N.Y. 11794 USA Maya Shmulevitz Department of Microbiology & Immunology Faculty of Medicine Sir Charles Tupper Medical Building 7th floor, Room 7-P 5850 College Street Halifax NS B3H 1X5, Canada Françoise Stoll-Keller Inserm, U748, Strasbourg, France Université Louis Pasteur Strasbourg, France Ming Tan Division of Infectious Diseases Cincinnati Children’s Hospital Medical Center University of Cincinnati, College of Medicine 3333 Burnet Avenue Cincinnati, OH45229, USA Robert Thimme Department of Medicine II University Hospital Freiburg Hugstetter Strasse 55 D-79106 Freiburg, Germany

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Bing Tian Departments of Medicine, Biochemistry and Molecular Biology, and the Sealy Center for Molecular Medicine The University of Texas Medical Branch Galveston, TX 77555-1060 USA Rajas V. Warke University of Massachusetts Medical School Center for Infectious Disease and Vaccine Research 55 Lake Ave. North, Worcester MA 01655 USA Thorsten Wolff Robert-Koch-Institute (RKI) Nordufer 20, D-13353 Berlin Germany Decheng Yang Department of Pathology and Laboratory Medicine University of British Columbia The iCAPTURE Center, Providence Heart + Lung Institute St. Paul’s Hospital Vancouver, BC V6Z 1Y6, Canada Mary H. Zhang Department of Pathology and Laboratory Medicine University of British Columbia The iCAPTURE Center, Providence Heart + Lung Institute St. Paul’s Hospital Vancouver, BC 86Z 1Y6, Canada

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Preface

With the recognition of the first HIV/AIDS case about 25 years ago, a number of emerging and re-emerging infectious diseases of humans and animals have been reported, such as SARS, bird flu, West Nile virus infection, Ebola virus outbreaks, etc. These diseases have caused the death of millions of lives and uncounted economic loss. Interestingly, these infectious diseases are all caused by viral infections and in particular, by RNA viruses. This is probably due to, at least in part, the high mutation rate of RNA viruses, which is attributed to the lack of proofreading activity of viral RNA-dependent RNA polymerase. Thus, in the recent years, research focusing on understanding the molecular pathogenesis of RNA viruses have been very active and made tremendous advances. In this book, we have organized 27 chapters written by highly respective virologists in the field to review the molecular mechanisms of host-virus interactions, particularly on the host gene responses to RNA viral infections. This book is the first volume ever to focus on such a topic in human and animal RNA viruses. It includes chapters on analyses of host differential gene expressional profiles using cutting edge technologies, host innate and adapted immune responses to viral infections, virus-induced signal transduction pathways related to disease occurrence, and recently discovered novel mechanism on RNA interference in regulation of host-virus interactions. These topics cover a number of representatives of almost all major human and animal RNA virus groups, which are divided into four sections, including retrovirus, positive single strand (ss)RNA virus, negative ssRNA virus and double strand (ds)RNA virus. HIV is the most studied virus. As a retrovirus representative, four chapters are prepared in Section I to cover the recent progress on this xxiii

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important virus. The first chapter reviews the analyses of global gene expression profiles induced by HIV-1 infection in different in vitro and in vivo models using gene microarray technology and bioinformatic approach. It illustrates the host signatures that can be identified during HIV infection and discusses the utility and significance of these signatures, including an analysis of the types of these genes and biological categories associated with infection. The second chapter discusses the host immune responses to HIV infection. The author highlights the pathogenesis of HIV infection, the nature of innate and adaptive immune responses to HIV in the control of HIV replication and strategies to develop protective immunity in susceptible individuals. The molecular mechanism by which HIV causes cellular immune dysfunction is further discussed in view of molecular signaling in chapter three. As interactions of viral proteins with the signaling molecules in different pathways can result in the modulation of gene expression in a variety of biological process leading to immune suppression and pathogenesis, this third chapter primarily addresses the role of Tat, Nef and Vpr, the best studied HIV proteins, in viral replication, apoptosis, and cytokine expression. In addition, as HIV studies have led the research in the field of virology and made most significant progress comparing with that of other viruses, a fourth chapter on the siRNA/microRNA-mediated host-virus interactions has been added. This chapter discusses the newly emerging mechanism of gene expressional regulation through RNA interference (RNAi), a so called “new arm of immune response”, in the host responses to HIV infection. The representatives of the negative ssRNA viruses in Section II include influenza virus, respiratory syncitial virus (RSV), vesicular stomatitis virus, Hantavirus and measles virus. This group of viruses, particularly the influenza virus, is one of the most dangerous global health threat to man as well as to many other mammals and birds. This virus once caused millions of death of humans during several pandemic outbreaks in the last century; and in recent years, the bird flu caused by avian influenza virus H5N1 strain re-emerges frequently and has a high potential to reassort (antigenic shift) with human influenza virus genome to generate a new mutant that is lethal to the human population. Thus, our urgent task is to better understand the viral genetics and pathogenesis to prevent the

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outbreak of this deadly virus. Two chapters are arranged to review the recent advances in this field of studies. One chapter provides an overview of the current knowledge of the virus-induced signal pathways, particularly on functional kinase signaling and apoptotic events in influenza virus-infected cells and how these viruses have learned to misuse these cellular responses for efficient replication. The other chapter discusses the multiple elements of immunity that participate in the response to influenza virus infection based on current data obtained from in vitro and in vivo systems. The other viruses in this group are also common human and animal pathogens. For example, RSV is a member of the family Paramyxoviridae and a leading cause of severe lower respiratory tract infection clinically manifesting as pneumonia and bronchiolitis, particularly in children. The first chapter for this virus reviews the impact of RSV disease, the processes that the virus uses to replicate in airway epithelial cells, the signaling pathways responsive to this virus infection, and how non-structural proteins modulate this process. An additional chapter highlights the literature regarding the host immune responses to RSV infection, including viral recognition by host innate immune cells via pattern recognition receptors, resulting in chemokine and cytokine production and the further development of host adaptive immunity. Hantavirus is another pathogen mainly infecting respiratory systems. This virus was first discovered during the Korean War and re-emerged in many states of US in 1990s. It is transmitted via wild rodents and causes two vascular permeability-based diseases: Hemorrhagic Fever with Renal Syndrome and Hantavirus Pulmonary Syndrome. The viral infection and host innate immunity is reviewed in one chapter with the emphasis on the role of pathogenic Hantavirus G1 or Gn proteins in regulating the early innate cellular responses. Vesicular stomatitis virus, a natural epizootic among farm animals which is spread by sand-flies, is often used for experimental acute infections of mice. Two chapters are included to cover its molecular pathogenesis. One summarizes current understanding of host innate and adaptive immune responses and viral evasion of innate responses. In addition, the potential power of this virus for vaccine platforms and for oncolysis is also addressed. The other chapter makes a detailed expansion on the host immune responses in view of virus recognition and autophagy

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signaling pathway. Autophagy is now being appreciated as a pathway used by the innate immune system to recognize and destroy viruses or, in certain viruses, to promote viral replication. This chapter provides a deep discussion on latest studies characterizing novel mechanisms of innate viral recognition and immunity. The last chapter in the negative ssRNA virus section provides updated knowledge on measles virus (MV) pathogenesis with emphasis on virus-induced immunosupression and central nervous system (CNS) diseases. By incorporating outcomes of research on transgenic animal models and clinical studies on host innate immunity as well as data on the MV-dendritic cell interaction and receptor usage, this chapter provides better understanding of the molecular immunology of MV infection. The host gene responses to positive ssRNA virus are discussed in Section III. This group of viruses contains a number of important human and animal pathogens, such as SARS-CoV, Ebola virus, West Nile virus, hepatitis C virus, Coxsackievirus, dengue virus, norovirus, and Sindbis virus. This group of viruses is one of the major sources of emerging and re-emerging pathogens of human and animal diseases. The sudden emerging of Severe Acute Respiratory Syndrome (SARS) in Asia countries in 2002 caused a severe tension on public health worldwide. As the SARSCoV, a new emerging human coronavirus strain, which is predominantly spread by the respiratory route through aerosol particles and has a high fatality rate, its pandemic outbreak will cause an unimagined severe threat to our society. For this reason, a great effort has been made in a number of laboratories all over the world to study this virus during the past six years. Based on the latest advances available, two chapters have been organized. One focuses on discussion of signal transduction pathways, particularly the virus-induced apoptosis signaling pathway and cell survival signaling pathway, as well as their cross-talk to determine the death and survival of virus-infected cells. The other chapter summarizes the global analyses of host gene expression profiles by genomic and proteomic technologies. These studies have revealed alterations in the transcription and translation of genes belonging to various functional groups, which provide new insights into the host-pathogen interactions and pathophysiology of SARS-CoV infection. Marburg and Ebola viruses, in family Filoviridae, are prototype viral hemorrhagic fever pathogens and cause

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a fulminant hemorrhagic disease in humans and nonhuman primates. Since the initial discoveries of these agents over 30 years ago, several large outbreaks have occurred in Africa countries. Today, MARV and EBOV are feared worldwide as highly pathogenic agents that pose a biohazard threat to public health. Two chapters have been included in this volume to focus on either viral infection-activated signal transduction pathways or host immune responses to infection. In addition, the authors have also added brief discussion of their first-hand experience on medical responses to control the infection during their trips to the outbreak sites of Africa countries. West Nile virus, a representative of the family Flaviviridae, originally emerged in Africa and was then transmitted to many other countries including recently to United States and Canada. As this virus is maintained in an enzootic cycle between mosquitoes and birds and enters the CNS from blood, it can cause diseases from febrile illness to fatal encephalitis in humans and other vertebrates. Two chapters are devoted either to the genome-wide analysis of gene expression profile related to pathology or to the host innate and adapted immune responses to viral infection. HCV and Dengue virus are another two species of family Flaviviridae. HCV infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. With an increasing large number of infected individuals in the world, HCV has a major impact on public health. Two chapters draw information together on this virus. One provides a review on DNA microarray analyses of gene expression patterns in host gene responses to HCV infection in state-of-the-art model systems and in HCV-infected patients. Specifically, contribution of gene expression profiling for the understanding of HCV-host interactions during the viral life cycle, outcome of viral infection and host responses to antiviral treatment in vivo are discussed. The other chapter reviews the recent progress on the understanding of innate, humoral, and CD4+ and CD8+ T cells response to HCV infection and discusses the multiple mechanisms by which HCV evades from these responses. Dengue virus has reemerged as a major health problem in the tropics, particularly among children. This mosquito-borne flavivirus infection often results in a febrile illness. Less frequently, infections cause dengue hemorrhagic fever, a potentially fatal vascular leakage syndrome. The chapter on this virus

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reviews the events of dengue vascular leakage and hypotheses that have been put forth. The authors use their data and many others to discuss the genome-wide identification of host gene expression in response to dengue virus-induced damage as well as the central role of certain selected genes in gene expression network in vitro and regulation of disease in vivo. Coxsackie B group viruses are representatives of family Picornaviridae. Coxsackievirus B3 (CVB3) and CVB4 are the two most studied serotypes in this group. CVB3 is the primary pathogens of myocarditis and dilated cardiomyopathy, particularly in children and young adults. CVB4 is the major cause of pancreatitis or type I diabetes mellitus. The first chapter for this virus describes the differential gene expression profiles identified using animal models by genomic and proteomic technologies. These genes or gene groups include cytokine/chemokine profiles, antiviral genes, or genes involved in cell survival, apoptosis, fibrosis, immune responses and others. In addition, the roles of certain viral non-structural genes in regulating host-cell macromolecule synthesis and trafficking are also discussed. The second chapter for CVBs covers the virus-activated signal transduction pathways during viral infection, which include the pathways mediated by tyrosine kinases, phosphatases and MAPKs, particularly the pathway associated with the activation of the ubiquitin-proteasome. The authors also added a discussion of the consequences of these coxsackievirus-activated pathways in viral pathogenesis. The host response to norovirus is thoroughly discussed in a chapter by focusing on molecular pathogenesis. Norovirus is recognized as the major cause of epidemics of gastroenteritis worldwide. The high frequency of norovirus disease can be explained by the low infectious dose, the wide genetic and antigenic variations, the high titre, prolonged shedding of viruses by ill and asymptomatic patients, and the high environmental stability of virions. The recent progress on the understanding of viral replication and virus-host interaction, identification of norovirus receptors, and studies of viral pathogenesis by the development of cell culture and reverse genetics systems are discussed. This section concludes with a chapter on the latest available data on sindbis virus. Sindbis virus, a member of Alphavirus in family Togaviridae, exhibits a broad host range of animals and humans. This viral infection spans a full range of diseases, including asymptomatic infection, self-limited febrile illness, acute

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arthropathy, and rarely, invasion of the central CNS resulting in acute encephalomyelitis. This chapter provides up-to-date knowledge on host responses during sindbis encephalomyelitis in a number of experimental models, with particular emphasis on how they present effective therapeutic targets in these diseases. This volume also includes two chapters in Section IV on host responses to infections of reovirus or rotavirus, the representatives of dsRNA virus group, in the reoviridae family. Although isolated from respiratory and enteric tracts, mammalian reovirus is rarely linked to human disease and thus regarded as benign. Recently, reovirus was found to preferentially kill many types of cancer cells without harming normal cells, raising the prospect of using reovirus as a cancer therapy reagent. Basic studies in reovirus biology reveal that Ras signaling in host cells provides advantages for reovirus replication in transformed cells over normal cells. Thus, the first chapter in this group focuses mainly on reovirus infection in the context of cancer, or transformed cells and highlights the signaling pathways of cancer cells that make them highly infectible by reovirus. Another chapter for this group covers the molecular mechanism of interactions between rotavirus protein and host immune system. Rotavirus is the primary cause of life-threatening diarrhea in young children. Recent studies have revealed that rotaviruses subvert the antiviral effects of interferons through the actions of its NSP1, a viral non-structural protein with a putative N-terminal RING-finger motif and a hypervariable C-terminal region. Its unique structure combined with its manipulation of the proteasomal pathway to degrade the interferon regulatory factors, suggests that rotavirus NSP1 is an E3 ubiquitin ligase and serves as a highly effective broad-spectrum antagonist of the innate immune response. It should be noted that this book is devoted only to human and animal RNA viruses. However, certain aspects of the viral biology and pathogenesis may be useful information for study of other groups of viruses. Since this book is designed primarily as a reference, the authors have attempted to make each chapter comprehensible when read by itself, even though this involves occasional repetition of information in other chapters. In addition, due to the space limitation, I have asked the authors to cite the most recent references, thus I apologise to the authors whose published articles have not been cited in the chapters. I hope that this book will be

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of value, not only to biomedical researchers, undergraduate/graduate students and medical students in virology and viral pathogenesis but also for clinicians in infectious diseases. I thank all of my colleagues who have generously contributed to this book. Particularly, I am grateful to Dr. Bruce McManus for his counsel and encouragement during this work. I would also like to thank the people in my laboratory, particularly Travis Lim and Mary Zhang, for their assistance in editing this book. Decheng Yang

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SECTION I

Retrovirus

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CHAPTER 1

Efforts to Characterize Host Response to HIV-1 Infection Monty Montano & Paola Sebastiani

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) continues to expand among vulnerable and resource-limited populations worldwide, with the global prevalence approaching 40 million. Over the past twenty years, substantial progress has been made in the identification and characterization of the role for specific host factors on infection and disease progression. Furthermore, there is wide recognition that both viral and host genetic variation, as well as environmental and behavioral factors underlie the complexity of host transcriptional response and disease outcome. Despite this complexity, a compelling question is whether common features of host genomic response to infection, i.e., an “HIV-1 induced host signature” can be defined, in a form that would be robust under differing experimental models and clinical conditions. If achieved, this might help to synthesize a growing body of data based on genome-wide expression profiles and would be a useful reference frame for the identification of unique gene expression patterns that are associated with favorable response to infection, or alternatively associated with undesired outcomes. In this review, we will attempt, in the context of a growing body of literature that describes human and viral variation, to describe host response to HIV infection in vitro and in vivo. We will apply methodologies operational in our laboratory to existing HIV infection datasets available in the Gene Expression Omnibus (GEO) database, 3

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derived from expression data obtained in vivo. Our goal will be to illustrate the type of host signatures that can be identified and to discuss the utility and significance of these signatures, including an analysis of the types of genes and biological categories associated with infection and their utility. To achieve this we will employ methodologies based on Bayesian modeling of gene expression data for discovering molecular profiles that characterize host signatures. We will provide examples of HIV induced expression profiles that can be derived from these datasets and present host signatures for infection. We will discuss the strengths and weaknesses of this approach and the intrinsic limitations when challenged with the huge diversity of host-pathogen conditions in biological data derived from in vivo sampling.

1. INTRODUCTION The human immunodeficiency virus type-1 (HIV-1) epidemic remains a significant challenge to global health, despite nearly 25 years of research that has led to countless insights into the molecular, pathobiologic and epidemiologic details associated with its infection. Nevertheless, HIV research has provided fundamental insights that have shed light on multiple viral life strategies and host immune mechanisms engaged in response to infection. As new research tools continue to be developed, they have been invariably applied towards a better understanding of this pandemic. Currently, as a research community, we find ourselves firmly within an era of applied genomic biology, i.e., understanding the variation in human genes and the impact of that polymorphism on host response profiles to many disease states and infections, including HIV infection. In the past ten years, genomic approaches have shed much light onto host variation in response to infection by HIV; however, much remains unknown. A potentially powerful new approach to following pathogenic trajectory in both natural history and drug intervention studies is through the use of genotypic and soluble biomarkers. Biomarkers have the potential for monitoring infection, identifying host correlates for protection, disease progression and profiles for desired treatment outcomes. Identifying and monitoring biomarkers would streamline the testing process for vaccine candidates and therapeutic regimes in current and future trials.

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Furthermore, biomarkers linked to desired outcomes would help to dissect effective adaptive immune response to infection — responses that would likely include the production of neutralizing antibodies, effective virus specific CD4+ and CD8+ T-cells, and a permissive immuno-modulatory environment. The relative contribution of these components of the immune system could be potentially monitored through the use of biomarkers, particularly during disease progression and in intervention trials. Perhaps the earliest biomarker for HIV associated disease progression was the level of CD4 T-cells in the peripheral blood and the association of these levels with opportunistic infections and disease progression. As CD4 numbers decline, there is an associated increase in the risk for tuberculosis, candidiasis, pneumocystis pneumonia and toxoplasmosis.1 Presumably, declining CD4 T-cells represent the level of immune suppression required to succumb to these infectious agents. However, it remains unclear whether these opportunistic infections represent reactivation of latent infections, de novo infection, or commensal pathogens that have become pathogenic. Research tools for dissecting these possibilities represent an area of obvious clinical concern. An important consideration during the onset of this analysis of biomarkers for HIV infection is the changing nature of the HIV epidemic. There are growing populations of longer living adults that represent chronically infected individuals. How chronic infection under treatment changes the potential profile of biomarkers, particularly under conditions of secondary infections and organ drug toxicities, remains a significant challenge to biomedical research. This chapter will summarize the basic features of HIV molecular pathogenesis, including transcriptional control by cellular and viral factors; followed by brief discussions of the role of host genetic variation and viral genetic variation, with the goal of discussing how each of these factors ultimately contribute to overall host response to HIV infection. Our goal in this chapter is to familiarize the reader with the approaches used to evaluate a broad array of genes, to identify genes that were differentially expressed among HIV infected subjects (in our case Africa and the USA) and to determine whether the expression differences in HIV positive compared with HIV negative subjects have shared features that may help to define biomarkers.

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2. MOLECULAR BIOLOGY AND DISEASES ASSOCIATED WITH HIV INFECTION 2.1 General Regulatory Features of HIV-1 Lentiviruses, such as HIV-1 and HIV-2, have been detected in a broad range of vertebrates including cows (bovine immunodeficiency virus), horses (equine infectious anemia virus), cats (feline immunodeficiency virus), sheep (Maedi-visna virus), goats (caprine arthritis-encephalitis virus), and primates. Human primate lentiviruses include HIV-1 and HIV2 (which is less pathogenic than HIV-1). All infectious retroviruses contain genes coding for gag, pro, pol and env transcripts. Gag encodes the internal structural polyprotein of the virus and is proteolytically processed into mature proteins MA (matrix), CA (capsid), NC (nucleocapsid), and various other less well characterized proteins. The viral core contains two copies of single-stranded positive sense RNA bound to NC. Pol encodes the enzyme reverse transcriptase (RT), which contains both DNA polymerase and associated RNaseH activities, and integrase (IN), which mediates insertion of the viral genome into the host genome (see Figures 1 and 2). Pro encodes the viral protease (PR), which acts late in assembly of the viral particle to enzymatically process the proteins encoded by gag, pro, and pol, and in some cases also env. Env codes for the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that selectively interacts with cellular receptor and coreceptor proteins on target cells, primarily including T-cells, monocyte/macrophages and dendritic/langerhan cells. This interaction often leads to a “spring load” fusion of the viral membrane with the cell membrane and subsequent internalization of the virus.

2.1.1 Transcriptional regulation by the cellular NF-κ B heterodimer Once a cell is infected, transcription can be influenced by an inducible class of cellular transcription factors encoded by the NF-κ B/Rel gene family.2,3 Proteins in this family form a variety of homodimers and heterodimers. The NF-κ B heterodimer p50:p65 can be found in the cytoplasm

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Figure 1. Life cycle of HIV-1. Beginning with an HIV-1 infected cell, the stages of viral gene expression, viral release and new infection of target cells are shown.

of unstimulated lymphocytes bound to the negative regulator Iκ B. Binding of NF-κ B to Iκ B sequesters NF-κ B in the cytoplasm. Multiple stimuli can lead to the release of NF-κ B from Iκ B with the subsequent entry of NF-κ B into the nucleus to activate target genes that contain DNA binding sequences related to the κ B enhancer site, GGGACTTTCC. Several lines of evidence have implicated NF-κ B proteins as critically important for HIV transcription. HIV enhancer sites are present in all HIV-1 isolates and are also found in HIV-2 isolates and other viruses (e.g., CMV). Provocatively, HIV-1 isolates contain two to three, and sometimes four copies of this critical enhancer.4–8 Strong support for a role of NF-κ B in HIV-1 transcription

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Figure 2. Organization of the HIV-1 long terminal repeat (LTR) and coding region of the genome in three open reading frames. All 15 proteins are indicated in stippled boxes. Polyprotein precursors are shown. NF-κ B indicates nuclear factor-kappaB; TAR, transactivation response element; LTR, long terminal repeat; MA, matrix; CA, capsid; PR, protease; RT, reverse transcriptase; IN, integrase; SU, surface; TM, transmembrane.

comes from mutational analysis of the NF-κ B binding sites.2 Deletion of the NF-κ B sites results in a loss of part or all of the LTR activation by these stimuli. Activation of latent HIV proviruses in both T-cell and monocyte cell lines by cytokines such as tumor necrosis factor-alpha (TNF-α and interleukin (IL)-1 is correlated with induction of nuclear NF-κ B.9 TNF-α induction of HIV-1 gene expression has also been correlated with the copy number of NF-κ B enhancer sites.5 T-cell lines that contain an HIV provirus containing deletions or mutations in the NF-κ B binding sites fail to undergo activation following cytokine treatment.10

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2.1.2 The retrovirally encoded Tat protein Tat protein is a small (86–102 amino acid) protein derived from the multiple splicing of two exons located within the central region of the HIV-1 genome (exon1) and within the env gene (exon 2).11 Tat is a potent activator of HIV transcriptional elongation. The tat gene appears to be essential for HIV replication because mutations of the tat gene introduced into infectious molecular clones of HIV-1 eliminated HIV-1 production. An interesting feature of Tat is its ability to be released from infected cells and to enter non-infected cells. Exogenous Tat can activate cellular gene expression through its effect on cellular signal transduction pathways and also influences the activity of transcription factors, e.g., AP-112 and NF-κ B.13 Tat has also been shown to upregulate the expression of HIV-1 co-receptors, CCR5 and CXCR4, making cells potentially more susceptible to infection. The mechanism for Tat activation of cellular genes is unknown and requires further study. Efforts to map the Tat-responsive region of the HIV LTR led to the unexpected discovery that Tat binds to an RNA element which forms a stable RNA stemloop structure, referred to as TAR, present at the 5′ end of all HIV-1 RNAs (+1 to +59). The RNA secondary structure is highly conserved and required for Tat-mediated activation of HIV-1. A subdomain of Tat is sufficient for binding to TAR; arginine residues in the basic domain interact with a uridine residue (U23) at the base of the predicted bulge of TAR. The biochemical mechanism by which Tat activates HIV-1 gene expression is not entirely resolved. There is strong evidence that Tat may enhance the efficiency of transcriptional elongation. HIV-1 RNA transcripts that are initiated in the absence of Tat tend to be truncated, suggesting inefficient elongation. Transcripts that are synthesized in the presence of Tat tend to be of full length.

2.1.3 Processing of retroviral RNA Following transcription of the full 9.6 kb HIV-1 provirus, retroviral RNA is subjected to variable processing steps (unspliced, minimally spliced and fully spliced transcripts) to yield 15 currently identified proteins (see Figure 2). The transcript processing involves many of the same processing events that occur for cellular RNAs, including cap addition at the 5′-end of the RNA, cleavage and poly-adenylation at the 3′-end of the

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RNA, and splicing to form sub-genomic, variably spliced RNA molecules. Full-length retroviral RNAs serve two functions: they encode the gag and pol gene products, and they are packaged into progeny virion particles as genomic RNA. Variably spliced, subgenomic sized RNA molecules provide mRNAs for the remainder of the viral gene products. Simple retroviruses splice a subset of the genomic RNA into a transcript that encodes the env gene product.

2.1.4 MicroRNA regulation MicroRNAs (miRNAs) are ∼21–24 nucleotide non-translated RNAs expressed by metazoans and viruses that bind to complementary mRNA binding sites, generally within the 3′ untranslated region (UTR). To date, there are approximately 500 human miRNA sequences with over 4000 entries (miRBase release 10.0, http://microrna.sanger.ac.uk), and each miRNA potentially recognizes and regulates 200 mRNA targets. Binding of miRNA to target mRNA sites is associated with recruitment of the microprocessor silencing machinery, resulting in either the repression of translation and/or degradation of mRNA targets.14,15 Recent data suggest that HIV replication can be promoted by repressing expression of the silencing machinery, notably Dicer and Drosher.16 This suggests that selected host miRNAs may be involved in mediating viral latency, possibly explaining latency in quiescent cells. In support of this possibility, Huang and colleagues have recently identified host miRNAs that appear to target the 3′ UTR of HIV-1 RNAs in resting T-cells and are upregulated in comparison with activated T-cells.17 Interestingly, HIV infection has also been associated with the upregulation of RNA editing enzymes, such as ADAR in vitro and in vivo,18,19 and could potentially alter miRNA target sequences within the viral genome to bypass miRNA-mediated repression. Whether this is relevant to the maintanence of viral latency should be an active area of future research.

2.1.5 Viral entry through cellular receptors HIV-1 enters cells by first attaching to the target cell, followed by fusion between the virus and cell membranes. Both steps are mediated by specific

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receptors with the major host cell receptor being the CD4 molecule, an immunoglobulin (Ig)-like protein expressed on the surface of a subset of T lymphocytes, monocytes, dendritic cells and brain microglial cells. The Env glycoprotein binds CD4 with a high affinity. However, the Env-CD4 interaction alone is not sufficient for viral entry and HIV-1 requires a second receptor belonging to the chemokine coreceptor family (members of the transmembrane G-coupled receptor protein superfamily). CXCR4 (formerly known as fusin) was the first co-receptor identified, and is associated with entry of T-cell line tropic or syncytium-inducing isolates (SI).20 Subsequently, CCR5 was shown to be a major co-receptor for macrophage tropic, non-syncitium-inducing (NSI) viruses.20,21 A nomenclature was proposed based on viral phenotype whereby viruses that utilize the CCR5 coreceptor are referred to as R5 isolates. Alternatively, isolates that utilize CXCR4 are designated X4, and dual tropic viruses that use both CCR5 and CXCR4 are termed R5X4.22 Viruses that are transmitted almost invariably use the CCR5 receptor and the R5 phenotype dominates early in infection.

2.2 Disease Progression A hallmark of AIDS is the progressive loss of CD4+ T-cells. The loss of CD4 T-cells is associated with a loss in adaptive immunity that eventually results in the increased susceptibility to opportunistic infections. Despite the 25 years or so of AIDS research, questions still remain concerning the mechanisms of HIV-induced CD4+ T-cell depletion. The apparent latent period associated with initial HIV infection may actually represent a subclinical viral replication period that eventually results in a symptomatic illness. Over the past few years, a growing body of evidence supports the view that massive viral replication during acute infection in the short course (initial weeks) selectively depletes memory T-cell subsets in mucosal compartments (gut, lung, genital tract) that sets the stage for global CD4+ T-cell decline in the long course (years).23–25 Intriguing new data may also help to explain the inability of HIV-specific cellular immune responses to control viremia. Notably HIV-specific CD8+ T-cell function appears to be impaired through upregulation of the inhibitory receptor programmed death 1 (PD1; also known as PDCD1).26

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Disease progression has often been described as rapid, intermediate or delayed. In the absence of antiretroviral treatment, the majority of individuals experience an intermediate disease progression in which they experience a rise in HIV RNA, a decline in CD4+ T-cells and development of AIDS related illnesses over the course of six to ten years after initial infection. A smaller number of individuals become rapid progressors that have a faster decline in CD4+ T cell count and accelerated AIDS-related events within a few years of infection. A very small percentage of individuals, late progressors, remain healthy without significant changes in CD4 count or HIV RNA for more than ten years. Many of the rate limiting determinants for disease progression are only partially understood, but clearly include the following: selective decline in specialized T-cell subsets, blockade in HIV-specific T-cell function, rate of decline in total T-cell count, levels of immune activation, plasma and cell associated viral load, prevalence of drug-resistance genotypes, host chemokine receptor tropism, viral genetic subtype, race and ethnicity, and various host genetic factors including age, gender, mode of transmission, psychosocial factors, resource limitations and body mass index as a marker for nutritional status (for review see Ref. 27). Although, current therapeutic guidelines take many of these into account, improvements in the identification of prognostic cellular or plasma biomarkers would be useful in patient management and in the design of treatment guidelines.

3. HOST VARIATION 3.1 Host Factors Influencing HIV Infection and Disease Progression Relatively few studies have examined the impact of HIV infection on a genome-wide level to characterize the role of host factor gene expression in response to infection and in identifying pathways for distinct disease progression trajectories. Preliminary studies to date tend to focus on a small number of candidate genes, with the notable exception of a recent study.28 There is a clear advantage in the genomic survey approach. With the use of gene-expression profiling, particularly in conjunction with the genome wide associations (GWA), human single-nucleotide polymorphisms (SNPs)

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can be used to correlate genomic variation with phenotypic variation. There are no data correlating genome-wide sequence data with gene expression profiling data to link SNPs with phenotypic variation. Gene expression profiles have been primarily used to characterize HIV infection for subjects in the United States.29,30 To date, there are no comparative data among distinct populations, particularly among African subjects, which account for most of the infections worldwide. HIV infections have been previously associated with a complex set of host-virus interactions that variably contribute to influence overall host response.31,32 The considerable genetic variation present within the human population allows for the possibility of differential host effects on viral replication and immune response.33–35 To date, many polymorphic genes, again predominantly within US based populations, have been described that influence HIV disease progression and infection (termed ARGs: AIDS restriction genes) and they include those genes that affect infection: CCR5,36 CCL2/MCP-1, CCL7/MCP-3, CCL11/Eotaxin,37 IL-10;38–40 that accelerate AIDS progression: CCR5,41 IL-10,38 CCL5/RANTES,42 IFN-γ ,43 HLA,44–46 APOBEC3G,47 Cyclophilin A48,49 and that delay AIDS progression: CCR5,36 CCR2,50,51 CXCL12/SDF-1,35,52 KIR3DS1.53,54 See Ref. 28 and reviews by O’Brien.55,56 There is also a growing literature on the role of human genetic variation and response to antiretroviral treatment.57,58

4. HOST RESPONSE TO HIV INFECTION: BIOLOGICAL CATEGORIES AND SIGNATURES With the aforementioned as a preamble, we will now describe data that represent our analysis of host gene-expression profiles among populations of cross-sectional HIV-infected subjects within the US and in Africa (Botswana). The goal was to determine whether the expression differences identified between HIV infected subjects and their age and location matched controls in Botswana displayed any shared features with HIV infected subjects in the US population. We have organized our analysis in this section to answer the following questions: Are there differences in host gene expression associated with HIV infection? Do differences in gene expression fall into distinct biological categories? Are the enriched biological categories shared in different populations? Are

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the enriched biological categories shared in different infections? And finally, is there a minimum predictive gene set for HIV infection? What follows is a discussion of our results based on considerations of each of these questions.

4.1 Are There Differences in Host Gene Expression Associated with HIV Infection? To begin to answer this question, we chose full genome arrays from studies in our laboratory designed to evaluate host response to HIV infection in Botswana. The array data sets were analyzed for differential expression based on HIV status (seronegative, seropositive) and using BADGE (Bayesian Analysis of Differential Gene Expression) version 1.0, a computer program implementing a Bayesian approach to identify differentially expressed genes across experimental conditions59–61, and is available from (http://people.bu.edu/sebas/software.htm). The differential expression of each gene in the two conditions (infected, noninfected) is estimated by the fold change and the statistical significance is measured by the probability that the fold change exceeds a fixed threshold, which is conditional on the data. To compute this probability, BADGE uses model averaging to gain robustness over model misspecifications. The current implementation of BADGE uses two models for the gene expression data: log-normal and gamma distributions. By combining both models, BADGE gains robustness and reproducibility over simpler analyses. In the analysis, we used an expected false positive rate of one percent, and chose those genes that changed expression by at least one and a half fold.

4.2 Do Differences in Gene Expression Fall into Distinct Biological Categories? Once a list of differentially expressed genes were identified by BADGE, biologically enriched categories were identified, as recently described,19 by implementing a stand-alone version of the EASE statistical software.62 This program computes a modified Fisher’s exact probability score for observing the number of genes from the list that are assigned

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to each biological category, compared with the likelihood of seeing that category by chance, given the total number of gene probes in the whole array that are annotated as belonging to the category. The list of categories to be tested is created by annotating genes within a given system of annotation, for example specific molecular functions or cellular components defined by Gene Ontology, or specific pathways defined by KEGG or GeneMap. An adjusted score is then reported representing the upper bound of the distribution of Jackknife Fisher exact probabilities for observing an enriched biological category. For more details, see Ref. 62. The differences in host profiles based on infection status were characterized specifically by enriched gene sets and were associated with immune response and RNA categories (mRNA metabolism including processing and editing). The immune response categories included many genes that overlapped with other significant categories including antiviral and interferon. Based on the significance score for biological categories identified in the HIV+ and HIV− comparison, profiles for all differentially expressed gene sets were systematically converted into heatmaps. Because immune response and RNA categories were prominent in the HIV+ vs. HIV− subjects, representative heatmaps for these selected gene sets (immune response, interferon and mRNA metabolism) were generated, as shown in Figures 3(A)–(C). Many genes within the immune response and interferon categories were upregulated, whereas most genes within the mRNA metabolism category were downregulated in association with HIV-1 infection.

4.3 Are the Enriched Biological Categories Shared in Different Populations? How generalizable are these profiles? To address this concern, we were interested in determining whether the HIV signatures among PBMCs in the Botswana population dataset were comparable to other PBMC based datasets for HIV infection. To this end, we identified a study comparing the expression profiles of HIV-1 infection in PBMCs from 22 HIV positive subjects versus 12 healthy controls from an US Army cohort,29 using web-based data available from GEO (GDS1449). Because the US study evaluated a smaller focused chip (Affymetrix

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GeneChip Human HG-Focus Target Array with 8793 gene probes) than the chip used in our Botswana study (22 777 gene probes); both datasets (our HIV infection dataset (set 1) and a US Army HIV infection dataset (set 2)) were re-analyzed using the list of 8793 gene probes

Figure 3. Comparative gene expression (HIV negative vs HIV positive). Heatmaps for Gene Ontology (GO) categories: (A) Immune response; (B) Interferon; (C) mRNA metabolism. Note that in heatmaps, the color red represents downregulated and the color green represents upregulated.

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that were common to both sets. BADGE and EASE analysis were conducted on each infection and control series. The top 20 categories representing enriched gene sets are shown in Figure 4. Interestingly, immune response categories for HIV infection (both in the Botswana and the US Army datasets) included the same top 4 categories (top 20 are shown), emphasizing the similarity between expression patterns of biological categories (although not necessarily the same gene probes). This is intriguing and may indicate that category analysis holds promise for infection profiling.

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Figure 4. Enriched category validation in an independent HIV-1 dataset. Shown are the top 20 biological categories enriched in two independent HIV infection datasets. Overlapping gene probes (8793) were identified for our HIV-positive/negative Botswana dataset, a US Army HIV-positive/negative dataset. The top 20 categories representing enriched gene sets are shown for the Botswana dataset (set 1: 25 HIV positive vs. 20 negative controls), the US Army dataset (set 2: 22 HIV positive vs. 12 negative controls, GEO series 2171). Note that the use of a 8793 focus chip in set 2 represents a subset of the 22777 gene-probes initially evaluated in set 1.

4.4 Are the Enriched Biological Categories Shared in Different Infections? In an independent comparison, we evaluated host response profiles in PBMCs from individuals infected with an unrelated pathogen, i.e., severe acute respiratory virus (SARS).63 Although the HIV retrovirus and the SARS coronavirus are both plus-strand RNA viruses, their mode of infection, viral life cycle and pathogenic sequelae are distinct (for review see Refs. 64, 65). We compared the expression profiles of PBMCs from eight adult patients with SARS with the expression profiles of four adult controls. The data are available from GEO (GDS1028) and the expression profiles were measured with the same Affymetrix GeneChip Human HGFocus Target Array described above. Both datasets were analyzed using the exact same overlapping gene list (8793 gene probes); that is, for our

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Figure 5. Distinct category enrichment in HIV versus SARS datasets. Shown are the top 20 biological categories enriched in our HIV infection dataset and in a SARS infection dataset. Overlapping gene probes (8793) were identified for our HIV-positive/negative Botswana dataset and a SARS-positive/negative dataset. The top 20 categories representing enriched gene sets are shown for the Botswana dataset (set 1: 25 HIV positive vs. 20 negative controls) and the SARS dataset (set 3: eight SARS positive vs. four negative controls, GEO series GSE1739). Note that the use of a 8793 focus chip in set 3 represents a subset of the 22 777 gene probes initially evaluated in set 1.

HIV infection dataset (set 1) and the SARS infection dataset (set 3). As shown in Figure 5, there was a sharp contrast in the top 20 gene categories (i.e., the presence of immune response categories for HIV infection in the Botswana and the US Army HIV datasets shown in Figure 4) compared with the RNA associated categories that were predominant in the SARS infection dataset. This supports the view that HIV infection elicits distinct host response gene sets in contrast to host response to SARS infection.

4.5 Is There a Minimum Predictive Gene Set for HIV Infection? To address this intriguing concern, we were interested in refining our analysis of differential gene expression in the HIV datasets to find a predictive signature between HIV-negative and HIV-positive subjects in distinct

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populations, as a preliminary attempt to identify biomarkers for HIV infection in disparate populations that only share the state of HIV infection status. Our methodological approach and objective was to find the smallest set of genes that were able to distinguish between negative and positive subjects while maintaining the largest accuracy. To identify the signature, we used a Bayesian classification rule and searched for the smallest set of genes that yields the largest accuracy in leave-one-out cross validation. This is a standard validation technique in which each microarray sample in the dataset uses all the remaining samples to identify the gene expression signature that is used to classify the sample left out. We present in Figure 6 our preliminary results from four predictive models based on the use of 20 HIV− and 25 HIV+ Botswana array datasets produced in our laboratory and 12 HIV− and 22 HIV+ US Army datasets downloaded from the Gene Ontology database, described in Ref. 29. The results are as follows: (1) Seven-gene set using our approach on the Botswana HIV+/HIV− dataset had a 98% accuracy in leave-one-out cross validation and a 70% accuracy to distinguish US HIV+/HIV− array datasets.

+/HIV− − datasets in Figure 6. Predictive gene sets derived from an analysis of HIV+ Botswana and US Army. Shown are a seven-gene set (upper) that accurately predicts infection in the Botswana dataset and a ten-gene set (lower) that accurately predicts a US Army dataset. Note that diagnostic genes do not overlap. The prediction is based on a Bayesian model that extends the voting algorithm.59

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(2) Ten-gene set using our approach with the US dataset had a 100% accuracy in leave-one-out cross validation of the US HIV+/HIV− array datasets and a 56% accuracy with our dataset. (3) Ten-gene set using the published US Army approach and dataset had a 100% accuracy in leave-one-out cross validation of the US HIV+/HIV− array datasets and had a 56% accuracy on our Botswana dataset. (4) Six-gene set using an overlapping gene-probe approach predicted 100% of US and 56% of the Botswana microarray profiles. Thus, in this analysis, we observed a seven-gene set that accurately predicted infection status in the Botswana dataset and a ten-gene set that accurately predicted infection status in a US Army dataset. More importantly, we also noted that diagnostic genes did not overlap. This raises the possibility that carefully controlled cohorts may indeed allow for the identification of biomarker signatures for infection, but those signatures may not be reflected in distinct populations. A limitation in our analysis may be that the prediction was based on a simple Bayesian classification rule and a more sophisticated model may be necessary.

5. DISCUSSION OF BIOMARKERS FOR HIV INFECTION Overall, in our genomic analysis, we identified patterns of expression for specific gene sets that were related to infection status. In this exercise, HIV-1 infection appeared to be associated with the differential expression of multiple gene sets representing a broad innate response, characterized by an activation of TLR, interferon and antiviral RNA response pathways. These response pathways are functionally related.66,67 Recent studies indicate that TLRs trigger interferon associated genes (e.g., IFN-α /β ) to initiate adaptive immunity by providing a link between the innate and adaptive immune response to infection,68,69 with subsequent influence on the expression of co-stimulatory molecules (e.g., CD80/86, class II),70 CTL/CD8+ T-cell effector activity71–73 and antigen presentation.74 Inspection of the data indicate that HIV-1 infection in Botswana was associated with a differential expression of innate response genes in the TLR pathway,

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including IL-1A, MYD88, RIP2/RIPK2, IRAK3/IRAKM, TRIF/TICAM1, NFKB2 and IP-10/CXCL10. Upregulation of the adaptor protein MYD88 and TRIF suggested that both MYD88-dependent and MYD88-independent (TRIF-mediated) pathways are engaged in HIV-1 infection. Members of the TLR family of receptors mediate the innate immune response to a broad range of microbial ligands via activation of members of the REL, IRF and STAT transcription factor families and their respective target genes. MYD88-dependent effectors include proinflammatory and chemotactic cytokines, whereas the MYD88-independent effectors are associated with type I/II interferons and stimulation of the JAK-STAT pathway. Studies in vitro have shown that HIV-1 RNA can activate TLR signaling75 and that microbial TLR engagement can activate HIV-1 transcription76,77 and proinflammatory chemokine release.78 HIV-1 infection was also associated with the differential expression of interferon-stimulated genes (e.g., STAT1, TRIM22, MX1, ISGF3G, IRF2, IRF7, IFI27, CXCR3 and PRKR. Interferons are a family of proteins produced in response to viral infection (notably RNA viruses) and/or microbial activation through TLRs and various other cytokine signaling pathways, including RNA degradation and editing responses. Interferon ligand-receptor interactions stimulate JAK-STAT signaling that induces various IRFs, that in turn upregulate host chemotactic effector genes (e.g., IP-10, MIG, I-TAC, MCP-1) and multiple antiviral RNA response effectors.79 STATs are activated by multiple cytokines and interferons (e.g., IFN-α /β, IFN-γ ).80 Multiple STATs are activated by HIV-1 infection in vitro81 and chronic HIV1 infection in vivo.82 Mechanistic studies in vitro also implicate a role for IRFs in HIV-1 expression through the HIV-1 long terminal repeat (LTR)83–85 and the HIV-1 transactivator protein pTAT;86 suggesting that host induction of interferon and antiviral RNA response may be beneficial to the virus by influencing replication. Interferon stimulated genes in the peripheral blood have also been detected in acute infection using an SIV/HIV-1 chimeric virus, SHIV89.687 and have been detected in lymph node biopsies from HIV-1 infected subjects.30 Our data also indicated a differential expression of interferon associated antiviral RNA response genes (e.g., MX1, PKR, OAS, ADAR, APOBEC3G). Many of these genes are associated with type I/II interferon response (for review see Ref. 88) and may influence transmission.89 Activation of these genes are often associated with

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interferon-induced response to RNA viral infection. An examination of the distribution of predicted regulatory elements within the promoter region for APOBEC3G suggests the presence of multiple IRF binding sites (data not shown). Initial in vitro studies of HIV-1-infected cell lines did not show activation of APOBEC3G expression, potentially suggesting differences between cell types or in vitro/in vivo differences. Interestingly, APOBEC3G has been associated with G-to-A hypermutations of coding sequence for viral and cellular genes and restricts viral replication, in the absence of the HIV-1 vif gene.90 Hypermutation of transmitted HIV sequences implicating RNA editing activity has been noted among newborns in Tanzania.91 The activation of APOBEC3G and other antiviral RNA response genes may in part represent an ancient innate response to invading viral RNA that is engaged in addition to adaptive immunity.92 The observed presence of common features of host response induced during HIV-1 infection in different settings, despite ethnographic, gender and viral subtype differences, and in contradistinction with other infection, seems promising (see Figures 4–5). These data raise enthusiasm for the potential of utilizing gene and biological category profiling to detect and characterize pathogen-specific host responses. Direct evaluation of specific gene role(s) in local infection, and expression monitoring of these genes in at-risk subjects, may help augment efforts to both understand pathogen-specific host response and intervene in viral-host mechanisms engaged during HIV infection. As shown in Figures 4–5, we describe an approach that uses gene set categories (which contain 200–300 genes) that identified broad similarities between HIV infection datasets in distinct populations and may be a useful primer for further defining smaller gene sets that accurately describe HIV infection in distinct populations worldwide or in populations with diverse treatment backgrounds. The exploratory attempts at identifying biomarker signatures (presented in Figure 6) indicate that a diagnostic approach that attempts to maximize accuracy with a minimal gene list (< 20) can weaken the cross validation accuracy in distinct populations. We speculate that a biologically meaningful gene signature, if it can be determined, particularly in disparate populations, will likely be composed of an intermediate number of genes (e.g., 20–200). Most genome-wide studies have been limited to evaluating specimens obtained from a single location, therefore attempts intended to

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identify a gene expression profile that represents a host signature for HIV infection may be of limited relevance beyond the population evaluated. In this analysis, we demonstrated the limitations of such an analysis by evaluating HIV-signatures identified in a distinct published HIV infection dataset,29 and we observed that the signatures derived did not overlap with signatures that were identified in a single population within Botswana, or vice versa. Although unlikely, this may be an outcome of the statistical method used — in the case of signature discovery: a computational search implemented to find the smallest number of genes with the highest diagnostic accuracy for discrimination between HIV+ and HIV−. Ultimately, our results underscore the need to evaluate multiple infected populations and control subjects to define signatures that have broad relevance. By evaluating multiple populations, we hope to improve the capacity to define an HIV signature that may have broad relevance.

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34. O’Brien SJ, Nelson GW, Winkler CA, Smith MW (2000). Ann Rev Genet 34, 563–591. 35. Carrington M, O’Brien SJ (2003). Ann Rev Med 54, 535–551. 36. Dean M, Jacobson LP, McFarlane G, Margolick JB, Jenkins FJ, Howard OM, Dong HF, Goedert JJ, Buchbinder S, Gomperts E, et al. (1999). Cancer Res 59, 3561–3564. 37. Modi WS, Goedert JJ, Strathdee S, Buchbinder S, Detels R, Donfield S, O’Brien SJ, Winkler C (2003). AIDS 17, 2357–2365. 38. Shin HD, Winkler C, Stephens JC, Bream J, Young H, Goedert JJ, O’Brien TR, Vlahov D, Buchbinder S, Giorgi J, et al. (2000). Proc Natl Acad Sci USA 97, 14467–14472. 39. Javanbakht H, An P, Gold B, Petersen DC, O’Huigin C, Nelson GW, O’Brien SJ, Kirk GD, Detels R, Buchbinder S, et al. (2006). Virology 354, 15–27. 40. Martin MP, Lederman MM, Hutcheson HB, Goedert JJ, Nelson GW, van Kooyk Y, Detels R, Buchbinder S, Hoots K, Vlahov D, et al. (2004). J Virol 78, 14053–14056. 41. Martin MP, Dean M, Smith MW, Winkler C, Gerrard B, Michael NL, Lee B, Doms RW, Margolick J, Buchbinder S, et al. (1998). Science 282, 1907–1911. 42. An P, Nelson GW, Wang L, Donfield S, Goedert JJ, Phair J, Vlahov D, Buchbinder S, Farrar WL, Modi W, et al. (2002). Proc Natl Acad Sci USA 99, 10002–10007. 43. An P, Vlahov D, Margolick JB, Phair J, O’Brien TR, Lautenberger J, O’Brien SJ, Winkler CA (2003). J Infect Dis 188, 228–231. 44. Carrington M, Nelson GW, Martin MP, Kissner T, Vlahov D, Goedert JJ, Kaslow R, Buchbinder S, Hoots K, O’Brien SJ (1999). Science 283, 1748–1752. 45. Tang J, Costello C, Keet IP, Rivers C, Leblanc S, Karita E, Allen S, Kaslow RA (1999). AIDS Res Hum Retrov 15, 317–324. 46. Gao X, Nelson GW, Karacki P, Martin MP, Phair J, Kaslow R, Goedert JJ, Buchbinder S, Hoots K, Vlahov D, et al. (2001). New Engl J Med 344, 1668–1675. 47. An P, Bleiber G, Duggal P, Nelson G, May M, Mangeat B, Alobwede I, Trono D, Vlahov D, Donfield S, et al. (2004). J Virol 78, 11070–11076. 48. An P, Wang LH, Hutcheson-Dilks H, Nelson G, Donfield S, Goedert JJ, Rinaldo CR, Buchbinder S, Kirk GD, O’Brien S J, et al. (2007). PLoS Pathog 3, e88.

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49. An P, Duggal P, Wang LH, O’Brien SJ, Donfield S, Goedert JJ, Phair J, Buchbinder S, Kirk GD, Winkler CA (2007). PLoS Genet 3, e19. 50. Smith MW, Dean M, Carrington M, Winkler C, Huttley GA, Lomb DA, Goeder, JJ, O’Brien TR, Jacobson LP, Kaslow R, et al. (1997). Science 277, 959–965. 51. Anzala AO, Ball TB, Rostron T, O’Brien SJ, Plummer FA, Rowland-Jones SL (1998). Lancet 351, 1632–1633. 52. Winkler C, Modi W, Smith MW, Nelson GW, Wu X, Carrington M, Dean M, Honjo T, Tashiro K, Yabe D, et al. (1998). Science 279, 389–393. 53. Martin MP, Gao X, Lee JH, Nelson GW, Detels R, Goedert JJ, Buchbinder S, Hoots K, Vlahov D, Trowsdale J, et al. (2002). Nat Genet 31, 429–434. 54. Martin MP, Qi Y, Gao X, Yamada E, Martin JN, Pereyra F, Colombo S, Brown EE, Shupert WL, Phair J, et al. (2007). Nat Genet 39, 733–740. 55. O’Brien SJ, Nelson GW (2004). Nat Genet 36, 565–574. 56. Lama J, Planelles V (2007). Retrovirology 4, 52. 57. Fox J, Boffito M, Winston A (2006). Pharmacogenomics 7, 587–596. 58. Haas DW (2006). Curr HIV/AIDS Rep 3, 53–58. 59. Klings ES, Safaya S. Adewoye AH, Odhiambo A, Frampton G, Lenburg M, Gerry N, Sebastiani P, Steinberg MH, Farber HW (2005). Physiol Genomics 21, 293–298. 60. Sebastiani P, Jeneralczuk J, Ramoni M (2005). Design and Analysis of Screening Experiments with Microarrays. Springer Verlag, NY. 61. Sebastiani P, Ramoni M, Kohane I (2003). In The Analysis of Gene Expression Data: Methods and Software, eds. Parmigiani G, Garrett ES, Irizarry R, Zeger S, Springer Verlag, NY, pp. 400–427. 62. Hosack DA, Dennis G Jr, Sherman BT, Lane HC, Lempicki RA (2003). Genome Biol 4, R70. 63. Reghunathan R, Jayapal M, Hsu LY, Chng HH, Tai D, Leung BP, Melendez AJ (2005). BMC Immunol 6, 2. 64. Montano M, Williamson C (2002). In AIDS in Africa, ed. Essex M. Kluwer, Academic, New York, pp. 11–34. 65. Ziebuhr J (2004). Curr Opin Microbiol 7, 412–419. 66. Moynagh N (2005). Trends Immunol 26, 469–476. 67. Doyle S, Vaidya S, O’Connell R, Dadgostar H, Dempsey P, Wu T, Rao G, Sun R, Haberland M, Modlin R, et al. (2002). Immunity 17, 251–263. 68. Le Bon A, Tough DF (2002). Curr Opin Immunol 14, 432–436.

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69. Honda K, Yanai H, Takaoka A, Taniguchi T (2005). Int Immunol 17, 1367–1378. 70. Beutler B, Hoebe K, Georgel P, Tabeta K, Du X (2005). Adv Exp Med Biol 560, 29–39. 71. Tough DF, Borrow P, Sprent J (1996). Science 272, 1947–1950. 72. Le Bon A, Etchart N, Rossmann C, Ashton M, Hou S, Gewert D, Borrow P, Tough DF (2003). Nat Immunol 4, 1009–1015. 73. Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF, Barth RJ, Vasilakos JP, Noelle RJ, Kedl RM (2004). J Exp Med 199, 775–784. 74. Blander JM, Medzhitov R (2004). Science 304, 1014–1018. 75. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S (2004). Science 303, 1526–1529. 76. Bafica A, Scanga CA, Equils O, Sher A (2004). J Leukocyte Biol 75, 460–466. 77. Equils O, Madak Z, Liu C, Michelsen KS, Bulut Y, Lu D (2004). J Immunol 172, 7642–7646. 78. Heggelund L, Damas JK, Yndestad A, Holm AM, Muller F, Lien E, Espevik T, Aukrust P, Froland SS (2004). Clin Expl Immunol 138, 116–121. 79. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N (2001). Ann Rev Immunol 19, 623–655. 80. Leonard WJ, O’Shea JJ (1998). Ann Rev Immunol 16, 293–322. 81. Kohler JJ, Tuttle DL, Coberley CR, Sleasman JW, Goodenow MM (2003). J Leukocyte Biol 73, 407–416. 82. Bovolenta C, Camorali L, Lorini AL, Ghezzi S, Vicenzi E, Lazzarin A, Poli G (1999). Blood 94, 4202–4209. 83. Battistini A, Marsili G, Sgarbanti M, Ensoli B, Hiscott J (2002). J Interferon Cytokine Res 22, 27–37. 84. Marsili G, Borsetti A, Sgarbanti M, Remoli AL, Ridolfi B, Stellacci E, Ensoli B, Battistini A (2003). Ann NY Acad Sci 1010, 29–42. 85. Sgarbanti M, Borsetti A, Moscufo N, Bellocchi MC, Ridolfi B, Nappi F, Marsili G, Marziali G, Coccia E M, Ensoli B, et al. (2002). J Exp Med 195, 1359–1370. 86. Izmailova E, Bertley FM. Huang Q, Makori N, Miller CJ, Young RA, Aldovini A (2003). Nat Med 9, 191–197. 87. Bosinger SE, Hosiawa KA, Cameron MJ, Persad D, Ran L, Xu L, Boulassel MR, Parenteau M, Fournier J, Rud EW, et al. (2004). J Immunol 173, 6858–6863.

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88. Samuel E (2001). Clin Microbiol Rev 14, 778–809. 89. Lee BN, Hammill H, Popek EJ, Cron S, Kozinetz C, Paul M, Shearer WT, Reuben JM (2001). Infect Dis Obstet Gynecol 9, 95–104. 90. Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002). Nature 418, 646–650. 91. Koulinska IN, Chaplin B, Mwakagile D, Essex M, Renjifo B (2003). AIDS Res Hum Retroviruses 19, 1115–1123. 92. Bagasra O, Prilliman KR (2004). J Mol Histol 35, 545–553.

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CHAPTER 2

Signaling Pathways Activated by HIV and Their Impact on Immune Responses Katrina Gee, Sasmita Mishra, Wei Ma, Marko Kryworuchko & Ashok Kumar

ABSTRACT The progressive loss of general and HIV-specific cellular immunity due to infection, subsequent depletion of CD4+ T cells, and the formation of viral reservoirs constitute the major hallmarks of HIV immunopathogenesis and disease progression. Therefore, the molecular mechanism by which HIV causes cellular immune dysfunction is critically important in understanding HIV immunopathogenesis. HIV contains three structural proteins (Gag, Pol and Env) and several non-structural regulatory (Tat and Rev) and accessory proteins (Nef, Vpr, Vif and Vpu). These proteins target the host cell signaling pathways resulting in the modulation of gene expression especially in virus replication, T-cell apoptosis, and cytokine and chemokine expression, leading to immune suppression and the formation of viral reservoirs. Among these proteins, Tat, Nef and Vpr are expressed intracellularly as well as secreted into the serum and cerebrospinal fluids of HIV patients. The molecular mechanisms and the signaling pathways by which these proteins target infected and uninfected bystander cells to modulate HIV replication and host gene expression are not well understood. This review will primarily discuss the role of Tat, Nef and Vpr, the best studied HIV proteins, in viral replication, apoptosis, and cytokine expression.

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1. INTRODUCTION In order for the replication and propagation of retroviruses to occur, the virus must take advantage of existing host gene regulatory pathways. In HIV infection, specific host factors are intricately involved in this process. These include host transcription factors recruited for viral transcription and cellular signaling pathways that are either activated or disrupted as a result of viral protein expression and function. These host factors and the viral components are all linked together in a battle for survival at the cellular level. The HIV virus expresses structural (Gag, Pol, and Env) proteins as well as non-structural regulatory (Tat and Rev) and accessory proteins (Nef, Vpr, Vif, Vpu) (reviewed in Ref. 1). The Tat and Rev genes encode regulatory proteins that are essential for virus replication. On the other hand, the Nef, Vif, Vpr, Vpx, and Vpu proteins are dispensable for virus growth in in vitro systems, but are essential for effective viral replication and pathogenesis in vivo.1 These regulatory and accessory proteins are responsible for the aberrant regulation of signaling pathways observed in HIV infection.2–9 Among these proteins, Tat, Nef, and Vpr are known to be secreted by HIV-infected cells and thus have been shown to affect uninfected cells as well.10–14 These three proteins not only have specific functions with respect to viral replication, but also have significant effects on the host cell regulatory pathways, resulting in deviant regulation of the intracellular signaling cascades in immune cells, altered cytokine expression as well as an altered propensity for apoptosis or cell survival.4,15–18 This chapter will discuss primarily the role of Tat, Nef and Vpr, the best studied HIV proteins, in viral replication, apoptosis, and cytokine expression.

2. HIV-TAT AND TAT-MEDIATED BIOLOGICAL EFFECTS 2.1 HIV-Tat HIV-Tat is a multifunctional regulatory protein that plays a critical role in viral replication and HIV pathogenesis. HIV-Tat is composed of 101 amino acids (aa) in clinical isolates and 86 aa in laboratory strains.19

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The Tat gene contains two exons each with distinct domains. Exon 1 contains an acidic region, a cysteine-rich region, the core region and a basic region.13 The basic region has been implicated in secretion of Tat from the infected cells and is involved in lipid association, vascular endothelial growth factor (VEGF) receptor binding, nuclear localization, and transactivation responsive region (TAR) binding. Exon 2 contains an RGD tripeptide responsible for integrin binding.19 Tat plays a key role in virus replication by acting as a transactivator protein through its ability to recruit host transcription factors, stabilizing viral transcription as well as playing a role in the elongation of viral transcripts.20 Tat is secreted by HIV-infected cells and intracellular as well as extracellular Tat is known to impair the regulation of immune responses by affecting cytokine production, apoptosis and cell survival.8,13,14,18 However, the mechanism by which Tat mediates its biological functions is not well understood. Tat can be internalized through endocytosis and is also known to interact with various surface receptors including integrins and VEGF receptors.19 It has also been shown to play a critical role in neurotoxicity and AIDS-related dementia.11 The mechanism of Tat-induced signal transduction pathways that affect viral replication, cytokine production, apoptosis, and neurotoxicity are discussed below.

2.2 HIV Replication Intracellular Tat expressed in HIV-infected cells as well as extracellular Tat are known to mediate initiation and elongation of viral transcription. The signal transduction pathways responsible for the initiation of viral transcription are complex. Tat functions as an adaptor protein and recruits cellular factors to the viral RNA and also promotes the processing of RNA polymerase-II.21 In the absence of Tat, short non-polyadenylated RNA are detected whereas in the presence of Tat, longer polyadenylated RNAs predominate.22 The critical steps involved in the regulation of Tat-mediated initiation of viral replication include the binding of Tat to TAR, an unusual stem-loop RNA structure essential for Tat binding. Once Tat binds to TAR, the host elongation factor, positive transcriptional elongation factor (P-TEFβ ),19,20,23 which mediates phosphorylation of the carboxy-terminus of RNA polymerase II, is recruited. In addition, Tat plays a role in the

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chromatin remodeling of the transcription start site by interacting with cellular histone acetylases, which results in the acetylation and the enhancement of Tat-induced transcription of the LTR.24 Several acetylases such as p300 and p300/CBP-associating factor (PCAF), and hGCN5 as well as components of the chromatin remodeling complex SWI/SNF have been shown to be involved in this process.24–26 The SWI/SNF complex subunits INI-1 and BRG-1 interact with HIV promoter and synergize with p300 acetyltransferase activity to activate the HIV promoter.25 Tat has also been shown to regulate HIV co-receptor expression and thus affects HIV infection and replication. Expression of chemokine coreceptors CCR5 and CXCR4 were shown to be differentially regulated by Tat; Tat induced CXCR4 expression on lymphocytes as well as monocytic cells whereas CCR5 expression was only induced on monocytic cells.27 Additionally, the HIV-1 Tat protein was shown to be a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in PBMCs. It was proposed that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.28 Recently, extracellular Tat was shown to induce CD4+ T-cell proliferation and IFN-γ secretion, upregulation of T-bet expression which is implicated in generating TH1 type of immune responses, and inhibition of HIV replication. Thus, apart from its transactivation activity, extracellular Tat acts as a co-stimulatory molecule that affects viral replication by modulating host immune response possibly through induction of T-bet expression and IFN-γ secretion.29 Moreover, Tat peptides have also been used to inhibit HIV replication and can act as a potential therapy for HIV infection.30

2.3 Cytokine Regulation Tat has been shown to regulate the expression of several cytokines in HIVinfected and uninfected bystander cells possibly by altering the intracellular signaling pathways. For example, Tat treatment of monocytes upregulated matrix metalloproteinase (MMP-9) and β 2 integrin expression through upregulation of cytokines such as IL-1β, IL-6, IL-8, TNF-α but not IL-3, GM-CSF, FGF, or MIP1α.31 Inhibition of both IL-1β and TNF-α blocked MMP-9 upregulation, indicating a role for these cytokines

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in the Tat-induced activation of monocytes.31 In addition, Tat directly induced MMP-9 expression that was dependent on NFκ B activation.32 HIV infection upregulated the expression of IL-10 and TNF-α in activated monocytes. The mechanism underlying HIV-induced upregulation of IL-10 and TNF-α is not well understood. A role for protein kinase C (PKC) isoforms β -II and δ was demonstrated in IL-10 induction by Tat in primary monocytes.4,33 Recently, mitogen-activated protein kinases (MAPKs) have been shown to regulate IL-10 production in primary monocytes.8,18 Initially, using THP-1 cells infected with a retroviral construct expressing Tat, we demonstrated a role for p42/44 extracellular signal regulated kinase (ERK) MAPK and cAMP response element binding (CREB-1) in IL-10 production.8 However, IL-10 production induced by exogenous Tat required the activation of intracellular calcium signaling through calmodulindependent protein kinase (CaMK-II). Additionally, we showed that IL-10 production in response to recombinant Tat selectively required p38 MAPK that was dependent on the upstream activation of Calcium/CaMK-II.18 We and others also demonstrated a role for MAPKs and transcription factors including Sp-1, Ets-1, and CREB-1 in human monocytes.8,18,34 A role for calcium signaling, PKC and NFκ B have also been shown in Tat-induced TNF-α expression in primary human monocytes.35 Taken together, these observations highlight the complexity of the signals required for Tatinduced IL-10 expression in monocytic cells.

2.4 Apoptosis and Cell Survival Tat can regulate apoptosis and hence it may play a key role in the generation and maintenance of viral reservoirs. It was suggested to play a protective role by inhibiting apoptosis via an increase in Bcl-2 expression in Jurkat, epithelial and neuronal cells, and primary human macrophages.36 However, Tat was also shown to induce apoptosis in T cells.37,38 Subsequently, exongenous Tat was shown to induce apoptosis, whereas the endogenous Tat protected infected T cells from apoptosis.39 The glutamine-rich, cysteine-rich, and C-terminal domains of Tat were found to be involved in the induction of apoptosis.13,40 The molecular mechanisms regulating Tat’s ability to either induce or protect cells from apoptosis have been attributed to different signaling

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pathways. Tat-induced apoptosis in Jurkat T cells, primary T cells, and microvascular endothelial cells involved the upregulation of caspase-8 and caspase-3 activity, respectively, which was independent of Fas-FasL interactions.41,42 In Jurkat T cells, Tat-induced apoptosis was found to be dependent on p56lck which in turn activated NF-κ B, AP-1, and c-Jun N terminal kinase (JNK). Whether activation of these proteins was required for the induction of apoptosis is not clear.43 Tat has been shown to upregulate TNF-related apoptosis-inducing ligand (TRAIL) expression in primary human monocytes and macrophages that induced apoptosis in bystander CD4+ T cells.44 Tat was also shown to induce Bcl-2 expression resulting in the inhibition of TRAIL-induced apoptosis in monocytic cells.45 In Jurkat cells stably transfected with Tat, decreased levels of caspase-10 and increased levels of FLICE-like inhibitory protein (c-FLIP) were observed46. These observation suggest a possible mechanism by which Tat may mediate resistance to TRAILinduced apoptosis. There is also evidence to suggest that Tat can modulate the cytoskeleton of infected cells following its interaction with tubulin/microtubulin leading to the alteration of microtubule dynamics and activation of a mitochondria-dependent apoptotic pathway.47 The proapoptotic Bcl-2 relative, Bim, appears to be involved in this process.

2.5 Neurotoxicity HIV can be detected in the central nervous system (CNS) with a potential to induce HIV-associated encephalitis and dementia. The CNS disease is becoming more apparent in highly active antiretroviral therapy (HAART)treated patients. This disease is characterized by monocyte/macrophage infiltration into the brain along with high expression levels of inflammatory cytokines and chemokines. HIV can primarily infect and replicate in macrophages/microglia of the brain.11 Astrocytes can also be infected at low percentages. However, even though neurons are not known to be infected, they still sustain damage and cell death, most likely due to HIVTat activities. Tat has been detected in the brains of HIV-infected subjects, perhaps as a result of secretion from infected cells.11 Tat can be taken up by neuronal cells via interaction with the lowdensity lipoprotein receptor-related protein (LRP) receptor, which results

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in its internalization and translocation into the nucleus. Tat-induced apoptosis in neurons has been shown to involve multiple signaling pathways including upregulation of intracellular calcium, phospholipase C-Inositol 1,4,5-trisphosphate 3 (PLC-IP3)-mediated calcium release from intracellular stores through the glutamate receptor activity,48 caspase activation and oxidative stress49 in human and rat neurons, respectively. Tat has also been shown to block the production of cAMP, known to be protective against apoptosis.50 In primary mouse striatal neurons, Tat-induced apoptosis through JNK-mediated caspase-3 activation.51 Recently, p38 and JNK MAPK were shown to positively regulate Tat-induced apoptosis in primary rat neurons. In addition, Tat was shown to induce autophosphorylation of mixed lineage kinase (MLK) 3 in primary rat neurons which, when blocked, protected cells from undergoing apoptosis.52 In another study, endonuclease-G was implicated in Tat-induced neurotoxicity. Although Tat induced caspase-3 activation, inhibition of this activity did not abrogate Tat-induced apoptosis.53 However, blocking the activity of phosphatase and tensin homolog (PTEN) protected striatal neurons from Tat-induced apoptosis.54 One of the major players in Tat-induced neurotoxicity is the N-methyl-d-aspartic acid (NMDA) receptor.11 Tat induces the formation of a complex at the neuronal plasma membrane consisting of several proteins including NMDA, low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSID-95), NMDA receptors, and neuronal nitric oxide synthase (nNOS). The formation of this complex leads to apoptosis of neuronal cells and astrocytes.55 Interestingly, chemokines such as monocyte chemoattractant protein-1 (MCP-1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from Tat or NMDA-induced apoptosis.55,56 Interaction of Tat with the NMDA receptor has been shown to trigger intracellular calcium levels.57,58 Blocking the polyamine-sensitive site of the NMDA receptor inhibits Tatinduced calcium flux in rat hippocampus.59 In addition, Tat-NMDA interactions have been shown to activate PKC and potentiate glutamate excitotoxicity.58,60 Chemokines are another group of significant players involved in the regulation of Tat-induced neurotoxicity. Several chemokines including

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CCL2, CXCL8, CXCL10, CCL3, CCL4, and CCL5 are induced in human microglial cells in response to Tat.61 Tat-induced chemokine secretion may cause transmigration of monocytic cells into the brain and results in inflammation. Of note, CCL2, a critical chemokine responsible for transmigration of monocytes and T cells across the endothelial cell barrier, is induced by Tat through PKC activation.62 In addition, CCL2 was found to be elevated in AIDS dementia patients.62 The TGFβ -1-induced Smad 3 as well as CCAAT/enhancer-binding protein (C/EBP)-β transcription factors were also shown to be involved in Tat-induced CCL2 expression in human glial and astrocytes, respectively.63,64

3. HIV-NEF AND NEF-MEDIATED BIOLOGICAL EFFECTS 3.1 HIV-Nef Nef, an accessory protein of HIV and originally identified as a viral negative factor, plays a key role in HIV pathogenesis. It is abundantly produced in the early stages of infection by all lentiviruses and resides in the cytoplasm, plasma membranes, nucleus and nuclear membranes. It is a 27 kDa, myristoylated protein of 206 aa, while in HIV2 and SIV, it has an additional 10–30 aa C-terminal sequence. The Nef open reading frame is located at the N-terminal end of the viral genome, partially overlapping the N-terminal long-terminal repeat (LTR). It exhibits sequence polymorphism, and is translated from multiple-spliced viral mRNA.65 It can also be translated from an internal AUG, 57 bp downstream from initiating AUG, resulting in the production of a truncated, non-myristoylated 25 kDa protein. Nef is phosphorylated on both tyrosine and serine residues. It contains two major domains: the N-terminal myristoylated anchor domain (residues 1–57) and the core domain (residues 56–206).66,67 A series of sequential motifs have been identified that are highly conserved among Nef alleles of all HIV-1 subtypes. These motifs mediate a multitude of interactions in protein modification, trafficking and signaling events. The main functions of Nef can be divided into the following categories: (1) modulation of cytokines and cell surface marker expression (e.g., CD4, MHC);6,7,68–71 (2) enhancement of virion infectivity and replication;72,73 and (3) cholesterol trafficking.74,75

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3.2 Regulation of Host Cellular Genes Nef has no known catalytic function. However, it is believed to promote viral pathogenicity by altering signaling pathways in infected cells through its interactions with cellular signaling proteins such as Src tyrosine kinase family members Hck, Lck, Lyn, Fyn and c-Src.6,7,71,76–78 A N-terminal proline rich motif known as 72PxxP75 represents the main binding site for the SH3 domain of Src family kinases.76 This motif also interacts with a number of serine/threonine kinases, including p21-activated protein kinase (PAK),79 θ isoform of PKC,80 and Vav.5 The interaction of Nef with these kinases can affect multiple cellular processes, leading to dysfunction of T cells, monocytes/macrophages in terms of cytokine expression, immune evasion, and apoptosis.

3.2.1 Dysfunction of monocytes/macrophages Nef has been shown to alter monocytes/macrophages biology by modulating a variety of signaling proteins.81 For instance, Nef can interact with Hck kinase that is strongly expressed in cells of the monocytic lineage and has been correlated with high HIV replication.77,78 In addition, Nef has been shown to impair phagocytosis.82–84 Nef decreases the release of superoxide possibly through enhanced IL-10 production and its inhibitory effects on superoxide release.83 HIV-mediated impaired phagocytosis has been attributed to the loss of Fc-receptor-induced signaling involving inhibition of phosphorylation of tyrosine kinases from two different families, Hck and Syk, defective formation of Syk complexes with other tyrosine-phosphorylated proteins, and inhibition of paxillin activation.82 The disruption of immunological synapse formation is a key mechanism by which HIV exerts negative effects on host cell function.85 Recent studies indicate that Nef impairs the ability of infected lymphocytes to form immunological synapses with antigen-presenting cells and affects T-cellreceptor-mediated stimulation. Nef was shown to cause accumulation of TCR and Lck in the recycling endosomal compartment resulting in severely decreased clustering at the synapse.85,86 Concomitantly, in HIV-infected cells, tyrosine phosphorylation at the synapse and the patterns of tyrosine phosphorylated proteins were disturbed in a Nef-dependent manner.

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Alteration of endocytic and signaling networks at the immunological synapse likely impacts the function and fate of HIV-1-infected cells.85,86 It is proposed that modulating lymphocyte signaling, apoptosis and intracellular trafficking through Nef ensures efficient spread of the virus in the hostile environment of the immune system. In addition, Nef expression in monocytic cells has been shown to modulate the expression of various cytokines and chemokines.87,88

3.2.2 T cell activation In chronic infection, HIV-1 persists in T cells, which causes hyperactivation and redundant cycles of T cell proliferation and eventually T cell exhaustion and cell death.89 HIV-Nef induces a state of TCR hyperresponsiveness, resulting in accumulation of tyrosine phosphorylation and activation of NFκ B and NFAT transcriptional response.90 Xu et al. also observed that binding of HIV-Nef to TCR induced Fas ligand upregulation, causing massive T cell death and progression to AIDS.91 Furthermore, Nef-mediated upregulation of Fas ligand expression was found to be dependent on p38 MAPK and AP-1 activation.92 In contrast, SIV-Nef can block TCR/CD3 signal by binding to CD3-ξ chain of CD3 complex followed by endocytosis,93 thus preventing T cell activation and apoptosis.

3.2.3 Downregulation of CD4 Nef disrupts T cell activation by the downregulation of CD4 and CD28 co-stimulatory molecules. 94,95 Five sequence motifs within the N-terminal and core domains have been implicated in CD4 downregulation. The first motif is the myristoylation signaling sequence that anchors Nef to the membrane where it binds directly to the cytoplasmic tail of CD4 through the second motif, centered on residues 57–59, targeting CD4 into clathrin coated pits (CCPs). Thereafter, three other binding motifs in the loop region of the core domain continually direct the Nef/CD4 complex into the endocytic pathway. The first one, a di-Leu-based internalization motif (164LL165) in the middle of the loop connects Nef with clathrin-associated adaptor protein complexes and recruits CD4 into CCPs. Simultaneously, Nef interacts with a subunit

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of the v-ATPase via a second diacidic motif, 174DD175, located at the C-terminal end of the loop that may facilitate AP2 recruitment. This is followed by the association of Nef with β -COP-1 coatomers in the endosome through a third diacidic motif, 154EE155, located at the N-terminal end of the loop. Together, this directs CD4 to lysosomes where it is degraded.94,96

3.2.4 Downregulation of HLA-A and B Three Nef motifs, namely acidic cluster 62EEEE65, 72PxxP75, and M20 have been identified to be specifically required for HLA-A and B downregulation in an endocytosis/trans-Golgi network (TGN) pathway through a Nef/PACS-1/ARF6/ PI3K axis.97,98 This process is initiated by binding of Nef to the cytoplasmic tail of HLA-A and B under the regulation of PI3K whose activation requires the M20 motif at the N-terminus of Nef. Subsequently, Nef links HLA-A and B to the trafficking proteins AP-1 (adaptor protein-1) and PACS-1 (phosphofurin acidic cluster sorting protein-1) through its acidic cluster 62EEEE65. This is followed by 72PxxP75-mediated activation of ARF6 (ADP ribosylation factor 6), the activated ARF6 triggers transport of HLA-A and B from the cell surface to TGN through the ARF6 compartment in vesicles. In TGN, HLA-A and B is degraded by endocytosis and Nef is recycled back into cytoplasm.9

3.2.5 Downregulation of CD1d Four Nef motifs are known to be involved in downregulation of a nonclassical MHC-I-like molecule, CD1d by TGN internalization in a distinct but shared pathway with MHC-I and CD4 downregulation. This effect depends on a tyrosine-based motif present in the CD1d cytoplasmic tail as well as the actions of four Nef motifs including 62EEEE65, 72PxxP75, 164 LL165 and 174DD175,69,99 the motifs involved in MHC-I and CD4 downregulation.9,97,98 Downregulation of CD1d by Nef could affect NKT cell recognition, which may lead viruses to directly inhibit CD1-mediated immune responses, and avoid any virus-associated lipid antigen becoming a target for CD1-restricted NKT cells.

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3.3 HIV-1 Pathogenicity The role of Nef in HIV-1 pathogenesis and AIDS progression has been demonstrated with the help of Nef-deleted or Nef-mutant HIV and SIV variants in mice and rhesus macaque models.100–103 Recently, Nef was shown to protect macrophages from HIV-1-induced apoptosis and to favour in vivo establishment of the virus reservoirs.104 Nef can also increase clustering of dendritic cells (DCs) with T cells thereby promoting dissemination of virus to lymphocytes.105 Additionally, Nef was shown to block immunoglobulin class switching by inhibiting NFκ B and STAT transcription factors in B cells.106 As a virion protein, Nef can increase viral infectivity by enhancing biosynthesis of lipid rafts and coupling of newly synthesized cholesterol to lipid rafts and virus particles, leading to increased release of the core protein into the cytoplasm.75 Moreover, Nef augments efficiency of reverse transcription and virus infectivity.72,73 Nef also increases proviral DNA synthesis via interaction with Tat and inhibition of p53, which regulates HIV-1 gene expression by suppressing transcriptional activation of LTR.107 Nef may contribute to immune evasion through the downregulation of MHC-I molecules, thereby protecting the virus-infected cells from CTLand NK-mediated killing.108 The Nef-induced MHC-I downregulation was shown to be mediated by PI3K intracellular pathways.9,109 Nef was also shown to inhibit both DC’s maturation and antigen presentation via activation of PAK2.110 Although HLA-A and HLA-B are downregulated, the expression of HLA-C and E, the major ligands for the NK cell inhibitory receptors (CD94/NKG2A) are not affected, thereby reducing the susceptibility of HIV-infected cells to NK cell cytotoxicity.111 Nef is believed to disrupt antigen presentation by targeting early forms of MHC-I molecules in the endoplasmic reticulum by preferentially binding hypophosphorylated cytoplasmic tails. The Nef-MHC-I complex migrates normally into the Golgi apparatus but subsequently fails to arrive at the cell surface and become phosphorylated following interaction with the adaptor protein 1 (AP-1).112 AP-1 was also implicated in Nef-induced impairment in antigen trafficking.113 Moreover, Nef downregulates CD4 and CD28 expression to limit superinfection,95 enhances virion release and envelope incorporation.114 Secondly, Nef inhibits Fas/TNFα-induced death in virus-infected

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cells by binding and blocking the functions of apoptosis signal regulating kinase-1 (ASK1), a key signaling intermediate in the Fas/TNFα-death signaling pathway.16,115 Alternatively, Nef can block death signals in virusinfected cells by inactivation of Bad through PAK and PI3K phosphorylation.17,116 Finally, through the TCR/CD3 signaling pathway, Nef induces the expression of FasL in infected cells, which interacts with Fas on the surface of bystander cells including CTL, leading to apoptosis.91 Nef also inhibits p53-dependent apoptosis within the infected cells.117

3.4 Cytokine Expression During HIV-1 infection, Th1 cytokines are generally downregulated, while Th2 cytokines are upregulated leading to impaired antiviral immune responses and disease progression.118 Nef has been shown to decrease Th1 cytokines including IL-2 and IFNγ expression in activated T cells.119 Paradoxically, Nef has also been shown to induce IL-2 production via PI3K activation in Jurkat T cells120 and regulated IL-15-mediated PBMC proliferation.121 Nef has also been shown to induce the expression of inflammatory cytokines (IL-6, TNF-α, IL-1β ) and chemokines (MIP-1α, MIP-1β ) in macrophages, DCs and PBMC.88,122 In human glial cells, Nef expression decreases TNF-α-induced apoptosis via JNK activation.115 Nef also induces the anti-inflammatory cytokine IL-10 via a calcium-dependent pathway.123 However, we did not observe IL-10 induction in monocytic cells following treatment with soluble124 or intracellular Nef (unpublished observations). More recent data indicates that exogenous Nef inhibits the release of IL-18, a co-inducer of IFNγ with IL-12, in THP-1 promonocytic cells.125 Recently, we have demonstrated that retroviral expression of Nef downregulates LPSinduced IL-12 production through JNK-activated NFκ B (unpublished data).

4. HIV-VPR AND VPR-MEDIATED BIOLOGICAL EFFECTS 4.1 HIV Viral Protein R (Vpr) Vpr is a 14 kDa, 96 amino acid, multifunctional regulatory protein that is highly conserved in HIV-1, HIV-2, and in SIV.126 This protein is packaged

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in large quantities into the virion core through specific interactions with the p6 domain of p55gag precursor protein and aids in proviral DNA transportation into the nucleus.127 Vpr is expressed late in infection suggesting its importance throughout the viral life cycle.128 This protein is also found in the sera and cerebrospinal fluid of AIDS patients.10 Mapping studies performed on lymphocytes or isolated mitochondria revealed that the Nterminal 1–51 aa Vpr protein is required for virion incorporation and nuclear localization, whereas the C-terminal domain encompassing 52–96 aa is essential for protein stability, induction of cell cycle arrest and apoptosis.129–133 The signaling mechanisms underlying Vpr-mediated functions will be discussed in this section.

4.2 Virus Replication In HIV infection, an important target for viral replication is the nonreplicating T cell population and macrophages.134 HIV has evolved multiple redundant karyophilic proteins to facilitate nuclear uptake of preintegration complex (PIC).135 Proteins that are targeted for transport through nuclear pore complex (NPC) possess a nuclear localization signal (NLS). Although HIV-Vpr is characterized as a nuclear protein, it possesses no discernable NLS.136 However, Vpr has been reported to contain multiple and diffuse nuclear entry signals. Vpr also causes disruption in the nuclear envelope, thus facilitating the entry of PIC into the nucleus.126 Besides transportation of HIV-PIC from the cytoplasm into the nucleus of a target cell, Vpr also influences the fidelity of the reverse transcription process by interacting with the nuclear form of uracil DNA glycosylase.137 HIV-Vpr has also been shown to have transcriptional activity not only on the viral LTR promoter but also on host cell promoters.138 Vprinduced transactivation of HIV is mediated through cis-acting elements present on the LTR (NFκ B and Sp1). Host cell genes such as NFκ B, NFIL-6, p21waf and survivin are also found to be regulated by HIV-Vpr.139,140 It is believed that Vpr does not bind to any specific nucleotide sequence directly; however, it activates HIV-LTR after binding with cellular factors such as Sp1 and p300/CBP acting as transcriptional co-activators.141 Recently, the Vpr peptide was shown to activate JNK MAPK and was

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implicated in enhanced HIV-1 replication in chronically-infected U1 promonocytic cells142 and monocyte apoptosis.129

4.3 Cell Cycle Arrest Vpr inhibits cell proliferation and causes cell cycle arrest at the G2/M phase in many human cells including primary CD4+ T cells and T cell lines resulting in enhanced HIV transcription. The cyclin-dependent kinase (CDK1), the yeast homologue cell division cycle 2 (Cdc2), and its complex with cyclin B1 are the key regulators for the check point between G2 and M phase. This process is regulated mainly by Wee1 kinase and Cdc25 phosphatase. During interphase, Wee1 kinase and CDK1/Cdc2 are localized in the nucleus, whereas Cdc25 phosphatase and cyclin B1 are in the cytoplasm. In general, during that time, CDK1/Cdc2 is hyperphosphorylated by Wee1 kinase and transported to the cytoplasm where it complexes with cyclin B1.143 This complex is a target for Cdc25 phosphatase before it enters into the nucleus. Cdc25 normally dephosphorylates Cdc2/CDK1 to promote mitosis; whereas Wee1 kinase phosphorylates Cdc2/CDK1 to prevent entry of mitosis. Vpr induces G2/M cell cycle arrest by promoting phosphorylation of CDK1/Cdc2143–145 by binding and inactivating Cdc25 phosphatase,126,146–148 as well as by activating Wee1 kinase.146,149 Vpr promotes CDK1/Cdc2 phosphorylation through its actions on the upstream kinases/proteins involved in the regulation of Cdc25 or Wee1 kinase. Fission yeast Wos2, a human p23 homologue and a Wee1 inhibitor has been shown to be a multicopy Vpr suppressor.146 Furthermore, Vpr binds to 14.3.3, the yeast homologue of Cdc25 inhibitor rad24, and prevents entry of Cdc25 into the nucleus resulting in subsequent G2 arrest.146,148,150,151 There is also evidence to suggest that Vpr may mediate cell cycle arrest by activating the ATM- and Rad3-related protein (ATR) DNA damage response pathway.152 The ATR kinase is a member of the PI3K related kinase family.153 Activation of the ATR pathway initiates a cascade of phosphorylation events including activation of Chk1 effector kinase. The Chk1 kinase phosphorylates and inactivates Cdc25 phosphatase, ultimately resulting in the persistence of hyperphosphorylated CDK1/Cdc2 and G2 arrest.154 The ATR pathway mediates Vpr-induced

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cell cycle arrest through phosphorylation and activation of Chk1.152 The Rad17 and Hus1 proteins involved in the ATR pathway are required for Vpr-induced cell cycle arrest as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and formation of nuclear foci containing H2AX and breast cancer susceptibility protein 1.155 Recently, Vprinduced Cdc25 phosphorylation and cell cycle G2 arrest were shown to be mediated at least in part through another Srk1/MK2-mediated regulatory pathway.156 In addition, Belzile et al. (2007) demonstrated the interaction of Vpr with E3 ubiquitin ligase complex, which consists of the damaged DNA binding protein 1 (DDB1), the E3 ubiquitin ligase scaffold protein cullin 4A (CUL4A), and DDB1-CUL4A–associated factor 1 (DCAF1) and claimed that the complex could be involved in activation of ATR pathway subsequently leading to cell cycle arrest.157 Recently, the role of protein phosphatase 2A (PP2A) has been shown in Vpr-mediated cell cycle arrest.158 PP2A is one of the major Ser/Thr phosphatases implicated in the regulation of many cellular processes including regulation of signal transduction pathways, cell cycle progression, DNA replication, gene transcription, and protein translation.159 There is evidence for a role of PP2A in Vpr-induced G2 arrest in fission yeast.146,160 In mammalian cells, Li et al. demonstrated that Cβ and Aα but not the Cα subunits of PP2A are required for the Vpr-induced and ATR-dependent Chk1 phosphorylation.158 In addition, Vpr-induced cell cycle arrest has also been associated with the downregulation of the ERK MAPK pathway in 293 epithelial cells.161

4.4 Apoptosis Vpr-induced apoptosis was first demonstrated by Stewart et al. (1997) in human fibroblasts, T cells, and peripheral blood lymphocytes.162 They reported that although the extent of Vpr-induced G2 arrest correlated with apoptosis, induction of G2 arrest but not its continued maintenance is necessary for apoptosis to occur. The mechanism underlying Vpr-induced apoptosis in various cell types has been investigated. There is strong evidence to suggest that mitochondria play a key role in Vpr-induced apoptosis. However, Vpr under certain conditions can negatively influence apoptosis.

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4.4.1 Role of mitochondria in Vpr-induced apoptosis Vpr not only causes apoptosis in HIV-infected cells but it also induces apoptosis when added extracellularly in a variety of cell types, including T cells and monocytes.129,132,163 There is strong evidence to suggest the involvement of mitochondria as the major pathway for Vpr-induced apoptosis. The intact Vpr protein (1–96 aa) as well as its C terminal (52–96 aa) peptide targets the mitochondrial apoptotic pathway. When Vpr is added to the intact cell or purified mitochondria, it causes loss of mitochondrial membrane potential (MMP) (∆ψm), leading to release of Cytochrome C (cyt-c) and apoptosisinducing factor (AIF). Finally, synthetic Vpr was shown to interact with adenine nucleotide translocase (ANT) of mitochondria resulting in induction of MMP, rapid dissipation of ∆ψm, and release of apoptogenic factors such as cyt-c and AIF.132,133,164 Vpr induced release of cyt-c interacts with Apaf-1 and procaspase-9 to create an apoptosome, the caspase activation complex that causes activation of other effector caspases such as caspase-3, 6 and 7 and downstream effects of apoptosis.163,165 In addition, Vpr also activated caspase-8 in NT2 neuronal and Jurkat T cells.166,167 Interestingly, Vprinduced apoptosis has also been shown to be caspase-independent.168 Very little is known regarding the signaling pathways involved in Vpr-induced apoptosis. We have recently shown the involvement of JNK MAPK in Vprinduced apoptosis in monocytic cells.129

4.4.2 Indirect role of Vpr in apoptosis Vpr has been shown to indirectly influence other signaling molecules to induce apoptosis. For example, Vpr enhances the expression of procaspase-9/caspase-9 in adCMV-Vpr infected cells.165,169 It also suppresses NFκ B activity,3,170 which is involved in transcriptional modification of several survival factors such as TRAF1, TRAF2, c-IAP1, c-IAP2, and XIAP170 as well as the Bcl2 family of anti-apoptotic proteins such as Bcl2 and BclXL.171 The suppressive effect of Vpr on NFκ B is mediated following interaction of Vpr with glucocorticoid receptor (GR) through Vpr interacting protein (VIP-1).3 Recently, we have demonstrated that Vpr inhibits c-IAP-2 and Bcl2 induction by downregulating NFκ B and CREB1 transcription factors.129

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4.4.3 Vpr as a negative regulator of apoptosis Vpr has also been shown to inhibit apoptosis depending on cell conditions and concentration of Vpr. For example, low levels of Vpr were shown to protect T cells from apoptosis induced by either cyclohexamide, TNF-α, anti-Fas antibody, or serum starvation through Bcl2 upregulation or Bax downregulation.15,128 Vpr has also been shown to induce the expression of survivin, one of the IAPs, resulting in the inhibition of apoptosis.140 These observations suggest that during early stages of infection, when Vpr is expressed at low levels, it protects infected T cells from apoptosis and HIV-induced cell death.15,128 However, at later stages when high levels of Vpr are expressed, Vpr induces apoptosis in the infected cells.3,163

4.5 Neurotoxicity Besides Tat and gp120, Vpr also plays a critical role in AIDS dementia as a result of its proapoptotic activity. The presence of soluble Vpr in serum and CSF of AIDS dementia patients suggests its involvement in HIVinduced neurological disorders.10 Vpr stimulation of rat and human neurons and rat astrocytes exhibited necrotic death.166,172,173 Vpr is believed to cause neurotoxicity by activating caspase-8, effector caspase-9 and 3 and possibly by forming ion channels on the cell surface.166,174

4.6 Cytokine Production HIV-Vpr interferes with the host inflammatory responses by inhibiting the production of proinflammatory cytokines such as IL-2, TNF-α and IL-12 and chemokines such as RANTES, MIP-1α and MIP-1β.3 Mirani et al. (2002) also reported that extracellular administration of Vpr inhibited LPS induced IL-12p35 and IL-12p70, but not IL-12p40 production by human monocytes/macrophages.175 HIV-Vpr also inhibits the production of IL-4 and IL-10; however, the production of IL-7 remains unchanged.3 Rafaeili et al. reported that Vpr interacts with GRII and one of the cytoplasmic proteins, RIP-1, which leads to nuclear translocation of the whole complex.170 Since Vpr binds to GRII, it acts like glucocorticoids and downregulates proinflammatory cytokines and chemokines. Besides binding to GR, Vpr inhibits

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the activation of NFκ B by inducing Iκ B.3,170 On the contrary, Roux et al. (2000) demonstrated an increased IL-6, IL-8 and IL-10 expression in monocytic cells, and TNF-α in primary T cells by activating NFκ B and NF-IL6.139

5. CONCLUSIONS AND FUTURE PROSPECTS The evolving nature of HIV and its escape from the host immune system highlights the need for a deeper understanding of the effects that this virus exerts on the immune cells. The HIV proteins Tat, Nef, and Vpr play an important role in viral infection, replication, and latency by hijacking infected and bystander cells’ intracellular signaling cascades to ensure HIV propagation and immune dysregulation. Therefore, understanding the molecular mechanisms and the signaling pathways underlying HIVinduced immune dysfunction may potentially lead to the development of therapies capable of protecting the immune system from the adverse effects of HIV infection. Further studies are necessary to understand the roles played by Tat and Vpr in the induction of HIV neurotoxicity. Moreover, how Tat, Nef, and Vpr regulate the signaling pathways, affecting cytokine expression will also be critical in the development of effective vaccines and improvements in treatment modalities.

ACKNOWLEDGMENTS This work was supported by grants from the CIHR (A. Kumar). S. Mishra was supported by scholarships from the OGSST and OGS. W. Ma was supported by a fellowship from the Natural Sciences and Engineering Research Council of Canada. K. Gee was supported by a fellowship from the Ontario HIV Treatment Networks and the Canadian Institute of Health Research. A. Kumar is a recipient of the Career Scientist Award from the OHTN.

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CHAPTER 3

Host Immune Responses in HIV Infection Robert D. Allison & Shyam Kottilil

ABSTRACT More than 40 million people worldwide are living with HIV infection and approximately three million die with AIDS each year. Profound immunodeficiency resulting from the progressive decline of CD4+ helper T lymphocytes is the hallmark of HIV disease. Robust innate and adaptive host immune responses are activated by HIV infection; however, HIV almost invariably establishes chronic infection. Moreover, immune activation seen in HIV infected individuals often contributes to the immunopathogenesis of HIV infection. Although introduction of highly effective antiretroviral therapy has resulted in significant reduction in the morbidity and mortality of individuals infected with HIV, these therapies have not been shown to eradicate HIV from chronically infected individuals and limited access to treatment in the developing world has further limited its efficacy. Development of an effective preventive vaccine is still the most promising approach for controlling the spread of HIV infection worldwide. Increasing our understanding of innate and adaptive host immune system activation against HIV and of mechanisms of viral immune escape will be paramount in the design of an effective preventive vaccine. This review highlights the pathogenesis of HIV infection, the nature of innate and adaptive immune responses to HIV in the control of HIV replication and strategies to develop protective immunity in susceptible individuals. 59

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1. PATHOGENESIS OF HIV INFECTION The trademark of HIV disease is a profound immunodeficiency resulting primarily from a progressive quantitative and qualitative deficiency of CD4+ T lymphocytes. Although several mechanisms responsible for immune dysfunction of CD4+ T cells have been demonstrated in vitro, the mechanisms primarily responsible for their progressive depletion and functional impairment in vivo remains unclear. When the number of CD4+ T cells declines below a critical level, the patient is at high risk of developing a variety of opportunistic infections and neoplasms. The combination of viral and immunopathogenic events that occurs during the course of HIV disease from the moment of initial infection to the development of advanced stage disease is complex. The pathogenic mechanisms responsible for the clinical manifestations associated with HIV infection are multifactorial and diverse at different stages of the disease and this review focuses on the role of host immune responses in the control of HIV replication and the pathogenesis of progressive immunodeficiency in an untreated HIV-infected individual.

1.1 Acute Viral Infection The natural history of HIV begins with a primary infection that is characterized by a high plasma viremia, an acute HIV syndrome, wide viral dissemination, and viral seeding of lymphoid organs. A robust immune response follows acute infection but fails to control viral replication adequately, resulting in a chronic, persistent and progressive viral infection. The acute phase of HIV infection is followed by a clinical latency period for a median of ten years in most untreated patients before development of the profound immunosuppression of AIDS. For fusion and entry into host cells, HIV primarily requires the presence of the CD4 molecule along with one of two major co-receptors, CCR5 or CXCR4.1 Viruses that preferentially use CCR5 (R5 viruses) predominate in the early stages of infection, whereas in later stages, viruses using CXCR4 (R4 viruses) are detected in approximately half of the patients.2 HIV is likely to become established by infecting local CCR5+ resting memory T cells, which is the most abundant CD4+ T cell population in extra-lymphoid mucosal sites.

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Though resting memory T cells have a limited viral replicative capacity, they represent a dense network that facilitates an efficient direct cell-tocell spread via the virological synapse.3 Studies of SIV in rhesus macaques have shown that within ten days of infection, most mucosal CD4+CCR5+ memory T cells have been infected or have been induced to undergo apoptosis. During the process of acute infection, HIV is thought to become established by infecting the largest collection of lymphoid tissue in the body, the gut-associated lymphoid tissue (GALT).4–6 Massive infection of the CD4+ memory T cell pool results in a profound depletion of memory T cells, most notably in GALT where about 60% of the CD4+ T cells reside.7,8 In response, surviving memory T cells significantly increase their proliferative activity and central memory and naive T cells are recruited to stabilize immune function at extra-lymphoid sites but may not fully replenish depleted memory cells.9 Dissemination of HIV throughout the mucosa and to distal sites is assisted by HIV replication in activated CD4+ cells, in macrophages and by migration of dendritic cells. The mechanisms for CD4+ memory T cell depletion during acute HIV infection are not clear. One recent study using the SIV model suggested that the high rate of CD4+ memory T cell infection observed ten days after challenge and the disappearance of 80% of these cells four days later could be accounted for by direct cytopathology.8 Several other studies have suggested that the death of infected and uninfected bystander T cells during acute infection is probably mediated by apoptotic mechanisms.7,10 Mucosal memory T cells naturally express Fas ligand and may be maintained in a state that makes them particularly vulnerable to Fas-Fas ligandmediated apoptosis.11 Loss of mucosa-associated CD4+ memory T cells during acute infection correlates with a massive increase of plasma viremia.12,13 This burst of viremia is associated with clinical symptoms of an HIV syndrome in up to 70% of individuals during the acute phase of HIV, typically three to six weeks from the primary infection. Clinical symptoms have been compared to acute infectious mononucleosis and are typical for acute viral illnesses including fever, skin rash, pharyngitis and myalgia. Most individuals recover spontaneously from this syndrome within one to several weeks with a mildly depressed or normal CD4+ T cell count. It appears that the initial level of plasma viremia in primary HIV

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infection does not necessarily determine the rate of disease progression. However, the set point of the level of steady-state plasma viremia after six months to one year does seem to correlate with the rapidity of disease progression.14

1.2 Chronic Persistent Viral Infection Individuals with acute HIV develop robust, virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses against HIV core proteins and the appearance of HIV-specific CTLs correlates with reduction in the plasma viremia.15 Following the robust CTL-mediated adaptive immune responses against HIV infection, a steady-state of viremia, called the viral set point, is reached at approximately six months to one year post-infection, and has important prognostic implications for the progression to AIDS.14 HIV-infected individuals with higher viral set points progress more rapidly to clinical HIV disease than those with low viral set points.14 Despite the robust cellular and humoral immune responses that are mounted following acute infection, the virus succeeds in escaping immune-mediated clearance and is never eliminated completely from the body. This leads to a chronic infection that persists with varying degrees of virus replication in the untreated patient for a median of ten years before the patient develops opportunistic infections or AIDS. Throughout the often protracted course of chronic infection, virus replication can be detected in untreated patients by highly sensitive assays. Lymphoid tissues are the main anatomic sites for the establishment of HIV during primary infection and also the main sources of viral replication and plasma viremia throughout the chronic disease phase. During the early stages of HIV disease, the architecture of the lymph node germinal centers is generally preserved or may even be hyperplastic owing to in situ proliferation and recruitment of a number of cell types (B cells, CD4+ and CD8+ T cells). The trapping of antigen is a physiologically normal function for follicular dendritic cells (FDC), which present antigens to B cells and contribute to the generation of B cell memory.16 However, in the case of HIV, the trapped virions serve as a persistent source of cellular activation, resulting in the secretion of proinflammatory cytokines such as interleukin (IL) 1β , tumor necrosis factor (TNF)-α , and IL-6,

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which can upregulate viral replication in infected cells. Furthermore, although trapped virus is coated by neutralizing antibodies (Nab), it has been demonstrated that these virions remain infectious for CD4+ T cells while attached to the processes of the FDCs.16 CD4+ T cells that migrate into germinal centers to provide help to B cells in the generation of an HIV-specific immune response are susceptible to infection by these trapped virions. Thus, in HIV infection, a normal physiological function of the immune system, which contributes to the clearance of virus and the generation of a specific immune response, also serves deleterious consequences. As HIV progresses to an advanced stage, there is complete disruption of germinal center architecture accompanied by dissolution of the FDC network. This destruction of lymphoid tissue compounds the immunodeficiency of HIV disease and contributes to both the inability to control HIV replication and the inability to mount adequate immune responses against opportunistic pathogens. During primary infection, HIV establishes a pool of latently infected resting CD4+ T cells that serves as a persistent reservoir of virus. Such cells manifest postintegration latency in which the HIV proviral DNA integrates into the genome of a resting CD4+ memory T cell or an activated CD4+ T cell that then reverts to a quiescent state.17 HIV can then remain in this state, potentially for many years, until an activation signal drives the expression of HIV transcripts and ultimately to replicationcompetent virus.18 Despite the suppression of plasma viremia to < 50 copies of HIV RNA per milliliter by antiretroviral therapy (ART) for as long as five years, the pool of latently infected cells persists and can give rise to replication-competent virus.19,20 Modeling studies built on projections of decay curves have estimated that in a setting of prolonged suppression of plasma viremia to < 50 copies of HIV RNA per milliliter by ART, it would require from seven to 70 years for the pool of latently infected cells to be completely eliminated.21,22 Furthermore, the reservoir of latently infected cells is replenished during minor rebounds of virus replication that may occur intermittently, even in patients who for the most part are treated successfully, and certainly during major rebounds of viremia in patients whose therapy is interrupted for a period of weeks or longer.23 Thus, this persistent pool of latently infected cells is a major obstacle to any goal of eradicating the virus from

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infected individuals, despite the favorable clinical outcomes that have resulted from ART. The natural history of the disease course of untreated patients is quite predictable. When there is a progressive decline in the CD4+ T cell count to below a critical level (< 200/µL), the patients become highly susceptible to opportunistic infections and neoplasia. The depletion of CD4+ T cells continues in this phase. In this regard, control of plasma viremia by ART, even in individuals with extremely low CD4+ T cell counts, has increased survival. Ultimately, patients who progress to this severest form of immunodeficiency usually succumb to opportunistic infections or neoplasms.

1.3 Slow Progressors and Long-Term Non-Progressors The median time from HIV infection to the development of clinical AIDS is approximately ten years, but a small proportion of HIV infected individuals (< 10%) show slow progression of HIV-related immunosuppression and disease.24 In the absence of antiretroviral therapy, long-term non-progressors (LTNPs) remain asymptomatic, retain normal CD4+ T cell levels (> 500) and have low or undetectable viral loads. Slow progressors have measurable but delayed T cell decline despite long-term untreated HIV infection. The cause of slow progression in these patients is unclear, although a complex interaction between host and viral factors including host immune responses, host genetics and HIV virulence, is thought to play an important role in determining the course of HIV disease. Disease progression appears to be influenced by HIV-specific host immune responses and evidence suggests a crucial role of CD8+ cytotoxic T lymphocytes and CD4+ T helper cells in slowing disease progression. HIV-specific responses by CTLs seem to play an important role in controlling viral replication. Studies examining the relationship between the frequency of HIV-specific CTLs and viremia or the breadth of epitopes recognized by HIV-specific CTLs and viremia have been inconclusive.25,26 However, recent studies that measured polyfunctionality of CD8+ T cell effectors suggest that there are differences in the quality of HIV-specific CD8+ T cell responses between progressors and non-progressors. For instance, the subpopulations of HIV-specific CD8+ T cells in LTNPs

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selectively maintained the ability to degranulate and to produce IFN-γ, IL-2, TNF-α and the chemokine, CCL-4.27,28 Recently, in rare untreated LTNPs with normal CD4+ T cell counts and viral RNA < 50 copies per milliliter who have been infected for ≥ 20 years, strong associations with HLA B*5701 or HLA B*2705 alleles have been observed.29 In addition, the HIV-specific CD8+ T cell response in these patients is highly focused on B*5701-restricted peptides, suggesting that the B*5701 molecule plays a direct role in restriction of virus replication in these individuals, although the precise mechanisms of this effect remain unclear.29 A number of other host genetic factors may exert more modest effects on restriction of HIV replication, but may also be associated with slower progression of disease. These include heterozygosity for the CCR5-∆32 deletion, heterozygosity for the CCR2-64I mutation, homozygosity for the SDF1-3′A mutation, and heterozygosity for the RANTES-28G mutation. Since CCR5 is the major co-receptor for R5 strains of HIV and since individuals who are homozygous for the CCR5-∆32 deletion are, with rare exceptions, protected against HIV infection, the potential mechanism for slow progression in heterozygotes is clear. In addition, certain single nucleotide polymorphisms in the CCR5 promoter have been shown to be associated with slower progression of disease. The reason for the slowing of HIV disease progression in heterozygotes for the CCR2-64I mutation is less clear; however, it has been demonstrated that CXCR4 can dimerize with the CCR2-64I mutant but not with wild-type CCR2.30 This dimerization may reduce the amount of CXCR4 on the cell surface and as a result inhibit infection by X4 viruses. Homozygosity for the SDF1-3′A mutation may upregulate the SDF1 gene, enabling SDF-1, the natural ligand for CXCR4, to compete more effectively with X4 virus for the CXCR4 co-receptor. The RANTES-28G mutation increases CCL-5 expression, which is the natural ligand for CCR5 and may thus inhibit infection by R5 viruses. Several reports have suggested that infection with an attenuated HIV strain with defects in the nef gene is associated with slow progression of disease.31 In a cluster of transfusion-infected patients from the Sydney Blood Bank Cohort, deletions in the nef/long-terminal repeat (LTR) region of HIV-1 seem to contribute to viral attenuation in LTNPs.32 Originally thought to be LTNPs, subsequent follow-up has revealed slow

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progression in some members.33 Mutations in other HIV-1 genes such as env, gag, rev, vif, vpr and vpu have also been associated with slow or nonprogression.34 Additionally, compared to HIV-1, the related human retrovirus, HIV-2, has been associated with less virulence.35,36 Although LTNPs have robust HIV-specific immune responses and competent CD8+ T cell suppressors of HIV replication, it is unclear whether these factors are directly responsible for the state of nonprogression. A substantial proportion of HIV-infected individuals manifest comparable immune responses early in the course of their disease and still experience disease progression. As noted above, the lack of disease progression may be explained in some by a variety of host factors, including recognized and as yet unrecognized genetic factors; in others by a defect in the virus; and in others by a combination of both.

2. HOST IMMUNE RESPONSES TO HIV Following the initial burst of viremia during primary infection, HIVinfected individuals mount a robust immune response that is likely to contribute to delaying the ultimate development of clinically apparent disease for a median of ten years. This immune response is directed against multiple antigenic determinants of HIV virions. Although a great deal of investigation has been directed toward delineating the components of this immune response, it remains unclear which of these phenomena are most important in delaying progression of infection and which, if any, play a role in the pathogenesis of HIV disease.

2.1 Innate Immune Responses Viral infection spurs innate immune responses that serve as first line of defense prior to the emergence of adaptive responses. Innate responses to HIV can be mediated by secreted soluble anti-HIV factors or components of innate immunity, such as Natural Killer cells (NK) which are capable of eliminating virally infected cells. In order to escape from cytotoxic T cell-mediated lysis, HIV is known to downregulate MHC class I molecules on the surface of infected cells in vitro, but rendering them susceptible to NK cell-mediated lysis.37

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However, recent studies have shown that HIV-infected target cells have significantly downregulated expression of MHC class I molecules but surprisingly were resistant to lysis by NK cells.38 This may result from the fact that HIV selectively downregulates cell-surface expression of HLA-A and HLA-B molecules, while preserving the expression of HLA-C and HLA-E molecules, thereby evading both CD8+ T cell responses and NK cell-mediated lysis.39,40 In this regard, HIV viremic states have been shown to be associated with an increment of the NK cell subset expressing HLA-C-specific iNKRs (inhibitory NK receptors), thereby increasing the fraction of NK cells that are unable to kill HIV-infected CD4+ T cells.41 A recent study demonstrated that HIV infection induces the NKG2D ligands ULBP-1, -2, and -3 on infected cells.41 These ligands are involved in triggering NK cells to kill autologous HIV-infected cells, mediated by NKG2D. Thus, ligands for NK cell receptors are modulated during the course of HIV infection, which may greatly alter NK cells’ ability to kill infected cells. However, the in vivo implications of these findings are being investigated. NK cells produce high levels of CCL-3, CCL-4 and CCL-5, which are ligands for HIV co-receptor, CCR5, and have been shown to suppress HIV replication in vitro by inhibiting CCR5-dependent entry of HIV into target cells.42 Similar to CD8+ T cells, NK cells can suppress endogenous HIV replication by cell-cell contact as well as by soluble factors.43 The exact mechanism of cell-contact-mediated suppression by NK cells has not yet been determined, and further studies are required to delineate the precise mechanisms of NK cell suppression of HIV replication in vivo. As NK cells can lyse targets before the recruitment of the adaptive immune response, they could play a major role in protecting a host from initially acquiring HIV infection. Recently, NK cell functions of Vietnamese intravenous drug users (IDUs), who seemed to remain HIV seronegative despite several years of high-risk exposure to HIV, were compared with those of HIV-infected patients and normal controls.44 NK cells from the exposed uninfected individuals exhibited increased lytic activity against various targets when compared to those from individuals who were either HIV-infected or HIV seronegative. Moreover, NK cells from the exposed uninfected subjects produced significantly higher levels of the chemokines CCL-3, CCL-4 and CCL-5, and the cytokines

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IFN-γ and TNF-α when compared to those of HIV-positive individuals and HIV-negative volunteers. NK cell activities, both lytic and secretory, were lower both before and after seroconversion among IDUs who seroconverted during the study, when compared with individuals who remained uninfected for the duration of the study. Therefore, it seems that the enhanced NK cell activity of exposed uninfected IDUs may be associated with resistance to acquiring HIV infection, supporting the hypothesis that NK cells contribute to protection from HIV infection in certain individuals. The influence of genotype on the control of HIV viremia and disease progression has been described in two recent studies. One showed that the expression of HLA-Bw4, a ligand for a KIR (killer cell immunoglobinlike receptor) on the surface of NK cells, was associated with control of HIV viremia and slower progression to AIDS.45 The second study reported that the presence of both the activating KIR allele, KIR3DS1, and the HLA-B allele, Bw4-80Ile, are associated with a delayed progression to AIDS.46 In the absence of the KIR3DS1 allele, HLA-B Bw4-80Ile allele expression did not protect against disease progression. Furthermore, occurrence of the KIR3DS1 allele in the absence of the HLA-B Bw480Ile allele was associated with a rapid progression to AIDS among HIVinfected individuals, suggesting an epistatic association between the two loci. Finally, a recent study that demonstrated KIR3DS1-expressing NK cells showed significant inhibition of HIV replication in target cells expressing HLA-B Bw4-80I compared with NK cells that did not express KIR3DS1. Furthermore, KIR3DS1+ NK cells were preferentially activated, and lysed HIV-infected target cells in an HLA-B Bw4-80I-dependent manner.47 These genetic and functional studies suggest that NK cells may be capable of controlling HIV replication in vivo and the effectiveness of NK cell activity is influenced by the variations in the KIR locus. The discovery of secondary entry chemokine receptors for HIV and their soluble ligands secreted by CD8+ T cells, CCL-3, 4, and 5, in part accounted for non-cytolytic control of HIV replication in CD4+ T cells.48 In the presence of CCL-3, 4, and 5, cellular invasion and HIV replication were limited significantly.1,49 Subsequent studies showed that CCL-3, 4 and 5 suppressed only R5 HIV strains but were inactive against X4 HIV strains.

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CAF (CD8 antiviral factor) is an unidentified soluble factor secreted by CD8+ T cells with non-cytolytic HIV suppressive activity after exclusion of other known factors (i.e. CCL-5).50 CAF appears to induce protection from HIV through the activation of STAT1 (signal transducer and activator of transcription) followed by the induction of IRF-1 (interferon regulatory factor) gene expression.51 Cellular membrane fusion events of HIV appear to rapidly induce expression of physiologically active proteins responsible for inflammatory responses in CD4+ T cells, including NF-κ B.52 Evidence exists that soluble factors in the supernatants of CD4+ T cells excluding known C-C chemokines suppressed HIV LTR-mediated gene expression.53,54 CD4+ T cells exposed to an attenuated strain of HIV-1 acquired resistance to HIV infection by a soluble factor called HRF (HIV resistance factor) and HRF positive CD4+ T cells restricted the transcription of viral genes.55 Finally, APOBEC3G (A3G) is a deoxycytidine deaminase that potentially restricts HIV replication by producing dG to dA mutation of HIV DNA formed during reverse transcription.56,57 HIV circumvents this host immune response through the actions of its gene product, Vif, that targets A3G for accelerated degradation.58

2.2 Adaptive Immune Responses 2.2.1 Humoral immune response Antibodies to HIV proteins first appear between six and twelve weeks after primary infection.59 While these early antibodies can be used for the detection of HIV, they are non-neutralizing and do not have an appreciable effect on levels of plasma viremia.59 Antibodies to HIV envelope proteins and polymerase proteins follow antibodies to gag proteins. However, it is not until after a substantial decline in viral load is observed following emergence of HIV-specific CD8+ and CD4+ T cell responses that Nab appears,60 which questions the role of such antibody responses in controlling HIV replication in vivo. Antibodies directed toward the envelope proteins of HIV may neutralize HIV directly and prevent the spread of infection to additional cells. Nab may be a component of primary HIV infection, and some

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LTNPs have been reported to have increased titers of neutralizing antibodies.60 Nabs appear to be of two forms, type-specific and groupspecific. Type-specific Nabs are generally directed to the HIV envelope V3 loop region. These antibodies neutralize only viruses of a given strain and are present in low titer in most infected individuals. Group-specific Nab are capable of neutralizing a wide variety of HIV isolates. At least two forms of group-specific antibodies have been identified: those binding to gp120 and those binding to gp41. The other major class of protective antibodies participates in antibody-dependent cell cytotoxicity (ADCC), which is actually a form of cell-mediated immunity in which NK cells that bear Fc receptors are armed with specific anti-HIV antibodies that bind to the NK cells via their Fc portion. These armed NK cells then bind to and destroy cells expressing HIV proteins. Antibodies to both gp120 and gp41 have been shown to participate in ADCCmediated killing of HIV-infected cells. Several functionally conserved regions of the Env protein are potential targets of Nab, namely the CD4 binding site and the chemokine coreceptor binding site, both on HIV-1 gp120, and regions of HIV-1 gp41 involved in viral fusion to target cells. However, only a few broadly Nabs have been isolated from infected patients over the past 20 years.61 A recent study identified several patient sera consisting of potent and broad HIV-1 neutralization.62 These investigators were able to map the neutralizing specificities of the two most broadly reactive sera to the CD4-binding region of HIV-1 gp120 using antibody adsorption and elution from selected gp120 variants.62 These data present an optimistic view of developing new approaches to present this conserved region of gp120 to the immune system that may result in improved vaccine immunogens. Structural features of the HIV envelope that result in conformational masking of receptor-binding sites may allow resistance to neutralization by host antibodies.63 Nabs are a principal component of an effective human immune response to many pathogens. A recent longitudinal follow up study of early HIV infected individuals reported the detection of autologous Nabs as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nabs resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the HIV envelope gene that did not

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map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape and the exceptional density of HIV-1 envelope glycosylation are known as an evolving “glycan shield” mechanism of neutralization escape, whereby selected changes in glycan packing prevent Nab binding but not receptor binding. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire. In addition to playing a role in host defense, HIV-specific antibodies have also been implicated in disease pathogenesis. Antibodies directed to gp41 have been shown in vitro to facilitate infection of cells through an Fc receptor-mediated mechanism known as antibody enhancement.64 Thus, the same regions of the envelope protein of HIV that give rise to antibodies which are capable of mediating ADCC also elicit the production of antibodies that can facilitate infection of cells in vitro. In addition, it has been postulated that anti-gp120 antibodies that participate in the ADCC killing of HIV-infected cells might also kill uninfected CD4+ T cells if the uninfected cells had bound free gp120, a phenomenon referred to as bystander killing.10

2.2.2 Cell-mediated immune response T cell immunity can be divided into two major categories, mediated by the CD4+ T helper cells and the CD8+ cytotoxic T cells. Several studies have focused on the potential importance of CD4+ T cell help in mounting an effective immune response to HIV-1 and SIV.65–68 In animal models, CD4+ T cell’s help is necessary to mature Nab responses to viruses.69 HIVspecific CD4+ T cells can be detected in the majority of HIV-infected patients using flow cytometry to measure single-cell IFN-γ production in response to HIV antigens, binding to MHC class II tetramers, or HIV p24 lymphocyte proliferation assays. In HIV infection, virus-specific CD4+ T cell responses are weak or undetectable in most chronically infected individuals and the loss of HIV-specific T helper function has been shown to occur early, during primary HIV infection.70,71 However, strong HIV-specific CD4+ T cell responses have been observed in LTNPs and in patients whose viral load were controlled by early intervention in acute infection with ART.68,70,72 Studies by Rosenberg et al.72 have shown that individuals

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with strong HIV-1 specific CD4+ T cell proliferative responses to HIV-1 p24 antigens are able to better control their viremia than those with diminished or absent responses. The majority of HIV-1 infected individuals, however, show poor proliferative responses to HIV-1, even after the institution of ART and control of viremia, despite the return of proliferative responses to recall antigens, indicating that the majority of HIV-1 infected individuals display CD4+ T cell anergy to HIV-1. In a recent study, low but detectable levels (0.12%) of circulating HIV-specific memory CD4+ T cells in the peripheral blood were reported.70 Recent studies have suggested that the quality of the HIV-1-specific CD4+ T cell immune response may be more predictive of viral control and lack of disease progression.73,74 These studies have suggested that the presence of IL-2-producing HIV-1-specific CD4+ T cells or HIV-1-specific CD4+ T cells that are polyfunctional secreting multiple cytokines correlates with virological control.75 A quantitative defect in HIV-specific CD4+ T cells can definitely explain the inability to develop effective CTL and antibody responses directed against the virus. In this regard, it has been shown that HIV-1 infects HIV-1 specific CD4+ T cells preferentially, rather than CD4+ T cells specific for unrelated antigens.76 These HIV-1-infected cells represented < 5% of the HIV-1-specific CD4+ T cell population, suggesting that direct infection of these cells may not be entirely responsible for the observed quantitative defect. Both HIV-specific CD8+ T cell responses and HIV-specific T helper responses may be important in the control of HIV disease. Persistence of functional CD8+ T cell responses may depend in part on the preservation of the T helper response. Patients with higher CD4+ T cell numbers and detectable CD4+ T cell proliferative responses do better clinically, which is consistent with a more effective T cell immune response and better immune control of HIV, as demonstrated by a lower viral load. Attempts to rescue the helper T cell response with early initiation of ART may enhance the CD8+ T cell response with real benefits.68 It is conceivable that CD4+ T cells could have direct antiviral effects, releasing antiviral cytokines and chemokines and killing infected cells. In summary, it is certain that CD4+ T cell function is impaired early in infection which precedes progressive decline in CD4+ T cell numbers. Failure of T cell help is a central element in the pathogenesis of HIV

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infection and could be the critical feature that ultimately undermines immune control. Strong CTL responses are elicited in HIV-infected individuals.77 In acute HIV infection, the CTL response initially follows the rise in HIV in the blood and there is an inverse relationship between CTL response and virus load.78 In the simian model of HIV infection, when the CTLs are depleted in vivo and these animals are infected with SIV, the early control of the virus fails79 and if the depletion is done during the chronic phase of infection, the virus level rises until the effects of the antibody wear off.80 These data imply that CTLs are important in control of the virus. Early analyses of the cellular immune response to HIV in acute infection showed a strong CTL response coinciding with the initial peak of viremia and preceding production of any Nab.81 Using tetramers presenting an immunodominant peptide epitope, the number of HIV-specific T cells was shown to peak just after the level of viremia begins to fall,82 suggesting a pattern similar to that observed with SIV-infected macaques.79 The central role of CTLs in controlling the virus is also emphasized by the influence of HLA type on the rate of progression of HIV infection towards AIDS.83,84 HLA types associated with slow progression of the infection, such as HLA-B27 and HLA-B57,29,83 could stimulate more effective immune responses compared with those that confer increased susceptibility, such as HLA-B35.84 In chronic HIV infection, the expanded HIV-specific oligoclonal CD8+ T cells persist at high frequencies; often 1–2% of all circulating CD8+ T cells are specific for a dominant HIV epitope.78 These CD8+ T cells probably turn over continuously, and like the acutely expanded T cells, they tend to die by apoptosis ex vivo. The high number of responding T cells is almost certainly dependent on persistent antigenic stimulation, because reduction of HIV level by ART causes a steady decline in tetramer-stained CD8+ T cells.85 Without treatment, the high number of HIV-specific CD8+ T cells often persists into late infection and they can still be detected when AIDS develops. Antigen-stimulated CD8+ T cells, even within the same clone, can be divided into two types: terminally differentiated cells and long-term memory cells. There may well be a continuum of T cells in between these extremes, in various states of differentiation.86

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Virus-specific CTLs possess a range of antiviral activities, which include the ability to kill infected cells and to produce cytokines and chemokines. It is not yet clear which functions of CTLs are most important in controlling HIV. They produce interferon-γ , which inhibits HIV replication, and tumor necrosis factor-α, which can upregulate viral replication through activation of the HIV promoter in the viral 5′ long terminal repeat (LTR).86 HIV-specific CTLs also produce the CC chemokines CCL-3, 4 and 5, which suppress HIV replication by competition for, or downregulation of CCR5.48 Cultured HIV-specific CTLs have been shown to lyse HIVinfected CD4+ T cells in vitro,87 despite the ability of the viral Nef protein to reduce expression of class I HLA molecules on their surface,88 suggesting that lysis is a potent weapon against HIV. Findings from several studies show that virus-specific CTLs taken ex vivo can have functional defects that could undermine their control of virus.89 Although tetramer staining indicates large numbers of HIV-specific T cells are present in acute or chronic HIV infection, this technique alone makes no measurement of their function. Most HIV-specific cells in chronic patients produced IFN-γ, TNF-α and CCL-4 upon contact with their cognate antigen ex vivo. However, less than 15% of HIV-specific cells contained perforin, which was reflected in poor ex vivo killing of appropriate target cells, compared with 50% of CMVspecific cells that expressed perforin and killed targets from the same donors. It is unclear whether HIV-specific CD8+ T cells in vivo may be immature rather than end stage effectors. Further studies are necessary to determine why most HIV-specific CTLs are CCR7− CD45RA−, while most of the CMV specific CTLs are CCR7− and CD45RA+.90

2.2.3 T-regulatory (T-reg) cells T-regs normally constitute a small fraction of the circulating CD4+ T cells in humans, and are identified by the expression of high cell surface levels of CD25, as well as expression of the transcription factor, FoxP3. Recently there has been emerging evidence of pathogen specific CD4+CD25+ T-regs, likely as a consequence of a previous immune response. Recent findings have shown that removal of T-regs from PBMC leads to significant increases in antigen specific cytokine production from HIV specific CD4+ and CD8+ T cells.91–93 These cells could suppress virus specific

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T cell responses, reduce general immune activation, and/or increase the risk of autoimmune diseases. HIV infection could stimulate the production of viral specific T-regs, or conversely, preferentially eliminate this subset compared to other CD4+ T cell populations. A thorough understanding of their role in HIV pathogenesis is warranted before their exact role regulating HIV immunity can be fully understood.

2.2.4 PD-1/PD-L ligand pathway The programmed death (PD-1)/PD-1 ligand (PD-L) pathway, which is part of the B7-CD28 family, consists of the PD-1 receptor and its two ligands PD-L1 and PD-L2.94 Engagement of PD-1 by its ligands inhibits immune responses, and recent work has shown that PD-1 is highly expressed on exhausted T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice.94 Blockade of this pathway reinvigorates the exhausted T cells, allowing them to expand and produce effector cytokines.94 CD8+ T cells in individuals infected with HIV have previously been shown to be dysfunctional with reduced proliferative capacity and effector function.95 Several new studies suggest a role for the PD-1-PD-L pathway in exhaustion of virus-specific CD8+ T cells during HIV infection.96–98 A large percentage of HIV-specific CD8+ T cells express PD-1 along with CD27 and CD45RO, indicating previous activation. These CD8+ T cells had lost expression of the co-stimulatory receptor CD28 and perforin and expressed only low levels of CCR7 and CD127, which are important molecules for the maintenance of memory T cells. This phenotype suggests that the T cells are poorly functional, not transiting into memory cells, and are particularly receptive to inhibitory signals. Blocking PD-L1 with a monoclonal antibody led to increased T cell proliferation and production of TNF-α, IFN-γ, and granzyme B, indicating an overall increase in effector function.

3. STRATEGIES TO AUGMENT IMMUNE RESPONSES TO HIV Development of a safe and effective vaccine to prevent HIV infection remain the best hope for preventing the spread of HIV worldwide.

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The task of developing an effective HIV vaccine is problematic for a number of reasons, including the high mutation rate of the virus, the likely need for the development of effective mucosal immunity and the fact that it has been difficult to establish the precise correlates of protective immunity against HIV infection. Some HIV-infected individuals are long-term non-progressors, and a number of individuals have been exposed to HIV multiple times but remain uninfected; these facts suggest that there are protective elements of an HIV-specific immune response. Several vaccines that induce primarily T cell-mediated immune responses are currently in clinical trials.99 These trials are evaluating whether the vaccines effectively prevent HIV infection, as well as whether the vaccines affect the viral load set point among subjects who develop HIV infection. The furthest advanced among the current phase II trials involves a combination approach using a live canarypox vector expressing one or multiple HIV epitopes given together with gp120 or using the gp120 as a boost.99–101 This approach has resulted in Nabs in virtually all recipients and HIV-specific cytolytic T cells in ∼30% of individuals at any given time during the course of the trial. Another candidate vaccine is by Merck Inc. who recently completed their study using recombinant adenovirus in combination with HIV protein immunization.99,102 In the STEP study, one of the phase II trials of Merck Inc.’s investigational HIV vaccine, V520, was not effective at either preventing infection in volunteers or at reducing viral loads in those study volunteers who became infected with HIV during the trial. Furthermore, data analyses indicated that in volunteers with pre-existing immunity to adenovirus which was used as a carrier for synthetic HIV genes in the vaccine, there were more infections in those volunteers who received the vaccine than in those who received placebo. Most of these analyses are considered exploratory in nature, and the reasons for this result are still being studied. As the efforts continue in the scientific front to develop a vaccine with high degree of efficacy, we should also focus on implementing proven methods of HIV prevention such as health education, behavior modification and counseling, usage of barrier methods of contraception, medically supervised adult circumcision, use of ART for pregnant HIV-infected females to curb vertical transmission and needle

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exchange programs for those who are likely to acquire HIV by intravenous route.

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51. Chang TL, Mosoian A, Pine R, Klotman ME, Moore JP (2002). J Virol 76, 569–581. 52. Choe W, Volsky DJ, Potash MJ (2002). J Virol 76, 5274–5277. 53. Leith JG, Copeland KF, McKay PJ, Bienzle D, Richards CD, Rosenthal KL (1999). AIDS Res Hum Retroviruses 15, 1553–1561. 54. Saha K, Volsky DJ, Matczak E (1999). J Virol 73, 7891–7898. 55. Simm M, Miller LS, Durkin HG, Allen M, Chao W, Lesner A, Potash MJ, Volsky DJ (2002). Virology 294, 1–12. 56. Harris RS, Liddament MT (2004). Nat Rev Immunol 4, 868–877. 57. Soros VB, Greene WC (2007). Curr HIV/AIDS Rep 4, 3–9. 58. Bishop KN, Holmes RK, Sheehy AM, Malim MH (2004). Science 305, 645. 59. Niu MT, Stein DS, Schnittman SM (1993). J Infect Dis 168, 1490–1501. 60. Pilgrim AK, Pantaleo G, Cohen OJ, Fink LM, Zhou JY, Zhou JT, Bolognesi DP, Fauci AS, Montefiori DC (1997). J Infect Dis 176, 924–932. 61. Haynes BF, Montefiori DC (2006). Expert Rev Vaccines 5, 347–363. 62. Li Y, Migueles SA, Welcher B, Svehla K, Phogat A, Louder MK, Wu X, Shaw GM, Connors M, Wyatt RT, et al. (2007). Nat Med 13, 1032–1034. 63. Parren PW, Moore JP, Burton DR, Sattentau QJ (1999). AIDS 13 (Suppl A), S137–S162. 64. Robinson WE, Jr., Gorny MK, Xu JY, Mitchell WM, Zolla-Pazner S (1991). J Virol 65, 4169–4176. 65. Xiong Y, Luscher MA, Altman JD, Hulsey M, Robinson HL, Ostrowski M, Barber BH, MacDonald KS (2001). J Virol 75, 3028–3033. 66. Norris PJ, Sumaroka M, Brander C, Moffett HF, Boswell SL, Nguyen T, Sykulev Y, Walker BD, Rosenberg ES (2001). J Virol 75, 9771–9779. 67. Ostrowski MA, Gu JX, Kovacs C, Freedman J, Luscher MA, MacDonald KS (2001). J Infect Dis 184, 1268–1278. 68. Rosenberg ES, Altfeld M, Poon SH, Phillips MN, Wilkes BM, Eldridge RL, Robbins GK, D’Aquila RT, Goulder PJ, Walker BD (2000). Nature 407, 523–526. 69. Oxenius A, Zinkernagel RM, Hengartner H (1998). Adv Immunol 70, 313–367. 70. Pitcher CJ, Quittner C, Peterson DM, Connors M, Koup RA, Maino VC, Picker LJ (1999). Nat Med 5, 518–525. 71. Musey LK, Krieger JN, Hughes JP, Schacker TW, Corey L, McElrath MJ (1999). J Infect Dis 180, 278–284.

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72. Rosenberg ES, Billingsley JM, Caliendo AM, Boswell SL, Sax PE, Kalams SA, Walker BD (1997). Science 278, 1447–1450. 73. Harari A, Rizzardi GP, Ellefsen K, Ciuffreda D, Champagne P, Bart PA, Kaufmann D, Telenti A, Sahli R, Tambussi G, et al. (2002). Blood 100, 1381–1387. 74. Harari A, Petitpierre S, Vallelian F, Pantaleo G (2004). Blood 103, 966–972. 75. Betts MR, Exley B, Price DA, Bansal A, Camacho ZT, Teaberry V, West SM, Ambrozak DR, Tomaras G, Roederer M, et al. (2005). Proc Natl Acad Sci USA 102, 4512–4517. 76. Douek DC, Brenchley JM, Betts MR, Ambrozak DR, Hill BJ, Okamoto Y, Casazza JP, Kuruppu J, Kunstman K, Wolinsky S, et al. (2002). Nature 417, 95–98. 77. Walker BD, Chakrabarti S, Moss B, Paradis TJ, Flynn T, Durno AG, Blumberg RS, Kaplan JC, Hirsch MS, Schooley RT (1987). Nature 328, 345–348. 78. Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V, et al. (1998). Science 279, 2103–2106. 79. Schmitz JE, Kuroda MJ, Santra S, Sasseville VG, Simon MA, Lifton MA, Racz P, Tenner-Racz K, Dalesandro M, Scallon BJ, et al. (1999). Science 283, 857–860. 80. Jin X, Bauer DE, Tuttleton SE, Lewin S, Gettie A, Blanchard J, Irwin CE, Safrit JT, Mittler J, Weinberger L, et al. (1999). J Exp Med 189, 991–998. 81. Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky W, Farthing C, Ho DD (1994). J Virol 68, 4650–4655. 82. Wilson JD, Ogg GS, Allen RL, Davis C, Shaunak S, Downie J, Dyer W, Workman C, Sullivan S, McMichael AJ, et al. (2000). AIDS 14, 225–233. 83. Kaslow RA, Carrington M, Apple R, Park L, Munoz A, Saah AJ, Goedert JJ, Winkler C, O’Brien SJ, Rinaldo C, et al. (1996). Nat Med 2, 405–411. 84. Carrington M, Nelson GW, Martin MP, Kissner T, Vlahov D, Goedert JJ, Kaslow R, Buchbinder S, Hoots K, O’Brien SJ (1999). Science 283, 1748–1752. 85. Ogg GS, Jin X, Bonhoeffer S, Moss P, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Hurley A, et al. (1999). J Virol 73, 797–800. 86. McMichael AJ, Rowland-Jones SL (2001). Nature 410, 980–987.

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87. Yang OO, Kalams SA, Trocha A, Cao H, Luster A, Johnson RP, Walker BD (1997). J Virol 71, 3120–3128. 88. Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D (1998). Nature 391, 397–401. 89. Kalams SA, Walker BD (1998). J Exp Med 188, 2199–2204. 90. Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, Appay V, Rizzardi GP, Fleury S, Lipp M, et al. (2001). Nature 410, 106–111. 91. Weiss L, Donkova-Petrini V, Caccavelli L, Balbo M, Carbonneil C, Levy Y (2004). Blood 104, 3249–3256. 92. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF (2004). J Virol 78, 2454–2459. 93. Kinter AL, Hennessey M, Bell A, Kern S, Lin Y, Daucher M, Planta M, McGlaughlin M, Jackson R, Ziegler SF, et al. (2004). J Exp Med 200, 331–343. 94. Okazaki T, Honjo T (2006). Trends Immunol 27, 195–201. 95. Migueles SA, Laborico AC, Shupert WL, Sabbaghian MS, Rabin R, Hallahan CW, Van Baarle D, Kostense S, Miedema F, McLaughlin M, et al. (2002). Nat Immunol 3, 1061–1068. 96. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, et al. (2006). Nature 443, 350–354. 97. Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, Adams WC, Precopio ML, Schacker T, Roederer M, Douek DC, et al. (2006). J Exp Med 203, 2281–2292. 98. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS, et al. (2006). Nat Med 12, 1198–1202. 99. Johnston MI, Fauci AS (2007). N Engl J Med 356, 2073–2081. 100. Nitayaphan S, Pitisuttithum P, Karnasuta C, Eamsila C, de Souza M, Morgan P, Polonis V, Benenson M, VanCott T, Ratto-Kim S, et al. (2004). J Infect Dis 190, 702–706. 101. Thongcharoen P, Suriyanon V, Paris RM, Khamboonruang C, de Souza MS, Ratto-Kim S, Karnasuta C, Polonis VR, Baglyos L, El Habib R, et al. (2007). J Acquir Immune Defic Syndr 46, 48–55 102. Xin KQ, Sekimoto Y, Takahashi T, Mizuguchi H, Ichino M, Yoshida A, Okuda K (2007). Vaccine 25, 3809–3815.

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CHAPTER 4

HIV-1 and RNA Interference — Examining a Complex System Zachary A. Klase, Kuan-Teh Jeang & Fatah Kashanchi

ABSTRACT RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from long double-stranded RNA duplexes and RNA hairpins, respectively. The siRNA or miRNA then guides an effector protein complex to a homologous mRNA sequence and regulates suppression of gene expression through one of the several mechanisms. The suppression of gene expression through these mechanisms serve to regulate endogenous cellular gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed, in the context of cellular RNAi, a suppressor of RNAi or their own viral miRNA. Emerging data support that HIV-1 may encode miRNA and that the virus could be regulated by cellular RNAi mechanisms. Here, we review complex interactions between the virus and the host cell which may impact viral replication and pathogenesis.

1. AN INTRODUCTION TO HIV-1 Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Approximately 40 million people are infected with HIV. This virus will newly infect four million people this 83

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year and will be responsible for the deaths of three million.1 Current therapies for HIV are capable of controlling the virus, but do not represent a definitive cure. HIV-1 infection is marked by a brief acute phase followed by a long chronic phase. A spike in viral load during the acute phase is followed by a plateau of viral load in the chronic phase.2,3 Patients may be asymptomatic during the chronic phase but their CD4+ T-cell counts will invariably drop in the absence of treatment. Eventually this course will lead to opportunistic infections, which often marks progression to AIDS. Left untreated, most infected persons will eventually die from AIDS.1 HIV-1 has a complex life cycle with several distinct stages.2,3 Incoming virions bind to the target cell through interaction of viral gp120 with the CD4 receptor. Recruitment of cellular co-receptor then allows a conformational change in gp120, that exposes gp41 and allows for the fusion of the viral and cellular membranes. Following fusion, the viral genome undergoes reverse transcription to a proviral DNA. This viral DNA is then transported to the nucleus through action of the preintegration complex and then integrates into the host genome.3 The viral long terminal repeat (LTR) acts as a Pol II promoter and initial levels of viral mRNA are produced at lows levels. This initially produced mRNA is doubly spliced, leading to the synthesis of the viral proteins Tat and Rev. Tat binds to an RNA structure in the LTR called the transactivation response element (TAR) and recruits cellular transcription factors such as pTEF-b (Cyclin T1/CDK9) and histone acetyl-transferases.4–7 The action of Tat elevates significantly the transcription of viral mRNAs.6 The HIV-1 genome includes nine viral genes. All the viral genes are expressed from a single promoter located in the 5′ viral LTR;2,3 three of these genes are common to all lentiviruses (Gag, Pol, Env) while the other six (Tat, Rev, Nef, Vpr, Vpu, Vif) are referred to as accessory genes. Tat, the viral transactivator, binds to the viral promoter and increases transcription.4–7 Temporal regulation of protein expression is achieved through mRNA splicing, transport, and translation regulated by the viral Rev protein.8–11 Rev serves to stabilize transcripts and leads to the nuclear export of singly spliced and unspliced mRNAs. Rev expression greatly increases viral RNA translation.12 Viral gene expression is regulated by the expression of these two viral proteins which interact with viral RNA

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and cellular factors.2,3,8 That the virus only expresses nine genes means that the virus must rely upon many cellular factors for successful completion of its life cycle. RNA interference appears to be one of the processes in the complex interplay between viral and cellular factors.

2. BRIEF OVERVIEW OF RNA INTERFERENCE RNA interference (RNAi) is a regulatory mechanism conserved in plants, nematodes, protozoan, Drosophila, and mammalian cells.13–15 Doublestranded RNA is recognized by the RNAi machinery and processed into small, 21 nucleotide small-interfering RNAs (siRNA) which are capable of suppressing gene expression. Hence, exogenously introduced doublestranded synthetic RNA (e.g. siRNA) is captured by cellular ribonuclease III enzyme Dicer and cleaved into 21 nucleotide segments. However, the physiologically more relevant arm of the RNAi pathway (e.g., the one for miRNAs) emanates from the recruitment of cellular precursor RNAs into a silencing pathway (Figure 1) which involves Drosha-mediated cleavage of precursor-RNA stem-loops in the nucleus, followed by the export of RNA into the cytoplasm by Exportin-5, and finally cleavage by Dicer to generate a small mature miRNA duplexes (miRNA).16–21 Thereafter, one strand of the mature miRNA duplex (the guide strand) is incorporated into one of the two Argonaute-containing effector complexes which can act to silence gene expression through a variety of mechanisms. In one format of Post-Transcriptional Gene Silencing (PTGS), the guide RNA associates with an RNA-induced silencing complex (RISC) and directs the complex to a complementary sequence in a target mRNA, where upon Ago2, an Argonaute family member, cleaves the mRNA.13–15,22 This can occur when the miRNA guide is fully complementary with the mRNA target. Alternatively, in a second PTGS process, the miRNA guide may direct the RISC complex to an imperfectly complementary region within the 3′UTR of a target mRNA. Amongst other fates, the association of the RISC complex with the 3′ UTR inhibits the initiation of mRNA translation and this may occur in part by shuttling the mRNA into ribosome-free P-bodies.23–30 In these latter settings, miRNARISC inhibits mRNA-translation without degradation of the target mRNA.14,31,32 Finally, in a mechanism separate from PTGS that is still

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Figure 1. A schematic summary of the synthesis and processing of primary microRNA. Precursor transcripts are cleaved by Drosha and its binding partner DGCR8 in the nucleus and then by Dicer in the cytoplasm. Dicer-cleaved miRNA is then loaded into the RISC complex (Argonaute, Dicer, TRBP and the miRNA) through the action of TRBP. Small guide RNAs accrued from miRNAs can be used in repression of transcriptional silencing (RITS) in the nucleus or interact with RNA-induced silencing complex (RISC) for post-transcriptional gene silencing (PTGS). The latter can include degradation of mRNA or inhibition of translation of mRNA.

poorly understood for vertebral cells, mi/siRNA can generate an RNAinduced initiation of transcriptional silencing (RITS) complex. In this scenario, mi/siRNA guides a protein complex, in a sequence complementarity driven manner, to chromosomal DNA and recruits factors that modify the chromatin structure to prevent initiation of transcription and thereby induces silencing.33–36 RNAi can serve several purposes. Recognition of foreign RNA and subsequent attack by RISC may provide a defense against viral infection.37 RNAi may also be a means for cells to maintain specific chromosomal

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architecture and repress transcription of retro-transposons (i.e., endogenous retroviruses).33,38,39 Additionally, cellular miRNAs may be important for embryonic development and act to regulate gene expression broadly.40–43

3. RNAi AND VIRAL INFECTION The structures of many viral RNAs resemble miRNA-precursors and may be targets for processing by the RNAi machinery. Several viruses have been identified that yield Dicer-processed small RNAs, including human cytomegalovirus, human herpesevirus 8, Epstein Barr virus and peach latent mosaic viroid.44–46 There is an attractive reason why viruses should encode miRNAs. miRNAs are non-immunogenic, and they are small in size and can plausibly fit well into the genetic repertoire of viruses with limited genome-coding capacity.47 Moreover, a single miRNA has the potential to regulate many target genes, making this a highly versatile mechanism for small viruses.48 The functions of several viral miRNA have been suggested, and they appear to regulate viral replication as well as host cell survival and evasion of the immune system (Table 1). Epstein-Barr virus encoded miRNAs have been mapped to genes involved in B-cell regulation and production of B-cell specific cytokines, which suggest a role in control of antibody-mediated immunity.49 Other viral miRNAs appear to have the capacity to be self-regulating; feeding back on certain viral transcripts to influence viral replication. This class includes miRNA produced by SV40,50 EBV,49 and KSHV.44,51 Virus-regulating viral miRNAs impact the progression of the virus life cycle by moderating viral protein synthesis;

Table 1. Selected Viral miRNA, their viral effects and identified cellular targets. Virus expressing miRNA

Viral Target

Cellular Target

Cellular Effect

Epstein-Barr Virus Simian Virus 40 KSHV HSV1

Yes Yes Yes No

Yes No Yes Yes

B-cell regulation and antibody production n/a Anti-apoptotic through THBS1 and TGF-β Anti-apoptotic through TGF-β and SMAD1

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this action in turn affects the ability of the virus to evade host immune response(s). Other viral microRNAs appear to protect host cells from apoptosis. A microRNA expressed from the HSV-1 latency associated transcript prevents apoptosis52 through downregulation of TGF-β and SMAD3.52 A related herpes virus, Kaposi’s Sarcoma associated Herpes Virus (KSHV), also blunts apoptosis through miRNA expression,53 which affects many genes including thrombospondin 1 (THBS1). THBS1 has previously been shown to be downregulated in KSHV lesions and has a role in preventing angiogenesis and triggering apoptosis through activation of TGF-β.53

4. RETROVIRUSES AND REGULATORY RNAS While extensive attention has been devoted to the therapeutic applications of RNAi in treating HIV in tissue culture, less work has been done to examine whether retroviruses generate miRNAs or how these viruses might subvert the cell’s RNAi mechanism for viral benefit.54–63 Although synthetic siRNAs have been exploited to silence experimentally genes of choice, one should keep in mind that RNAi represents a physiological pathway evolved to regulate gene expression, protect the genome integrity, and plausibly to defend cells against infecting pathogens.37,38,64,65 One view is that viruses that infect cells are subject to cellular RNAimediated restriction. Consistent with this view is the finding that several plant and mammalian viruses encode RNAi suppressors, presumably as a counter response to the cell’s restrictive action.66–70 When considering the RNAi pathway in the context of viral infection, one can imagine four possible outcomes: (a) cellular miRNAs act to regulate cellular genes in response to infection; (b) cellular miRNAs act directly on viral genomes to suppress viral genes; (c) viral RNA is processed into non-coding RNAs, and these can directly act on the viral genome to suppress viral gene expression; and (d) viral RNA is processed into non-coding RNAs that suppress cellular gene expression. Within the above outcomes, the role of cellular miRNA in controlling cellular gene expression has been well-studied;13,15,64 however, emerging evidence now support that many viruses produce RNAs that are processed into miRNAs45,46,50,71 and that viral infection can reshape the expression of

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cellular miRNAs.62,72,73 Additionally, there are findings that show that cellular miRNAs target viruses, protecting the cell from viral infection.67,74 Hence, one report has predicted that several cellular miRNAs can target HIV-1 Nef, Vif, Vpr and Vpu genes,75 and other reports have suggested that siRNA or miRNA generated from viral sequences might limit viral replication.37,64,65 That many viruses encode RNAi suppressors are consistent with the notion that viruses may be continuously restricted by cellular RNAi pressure.66,68,76 The above four potential interactions may be further categorized in two ways — what the cell is doing to combat the virus, and what counter strategies the virus is employing to maintain replication. Synthesis by the cell of miRNAs that target the virus, or processing by the cell of non-coding RNAs derived from viral sequence that repress viral replication may be considered a part of the first category. The second category would include alterations of cellular gene expression by viral miRNAs. Some illustrations of how these two categories of interactions exist for HIV infection will be discussed below.

5. THE CELL’S ACTION ON THE VIRUS RNAi has been hypothesized to act as a component of the molecular innate immune system. In yeasts and plants, which lack an adaptive immune system, it appears that RNAi plays a major role in host cell defense. The proteins central to the function of RNAi — Dicer, Drosha and the Argonaute family, are conserved from prokaryotes to yeast, plants and mammals. The RNAi defense functions seen in lower organisms may also be conserved in mammals, and while this has been debated, some experimental findings are compatible with such an interpretation.

5.1 Targeting of Virus by Cellular miRNA The human genome encodes cellular restriction factors that limit the replication of certain viruses. For HIV, some of these factors include APOBEC3G and TRIM5∝.77–80 These restriction factors limit the ability of HIV to replicate, and the virus carries measures which can counteract these proteins. Is miRNA an additional restriction factor used by the cell

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against HIV? Interestingly, recent bioinformatics studies predict that several human miRNAs possess complementarity to HIV-1 viral genes. Specifically miR-29a and 29b were predicted to target the Nef gene, miR149 to target the Vpr gene, miR-378 to pair with Env, and miR-324-5p to target the Vif gene.75 Although direct experiments have yet been done to validate these targets, analysis of various viral isolates of HIV-1 suggest that the targets for each of these miRNA are conserved.75 Separate work by Bagasra et al. examined a possible role for cellular miRNA in preintegration latency.81 They examined the ability of cellular miRNA to form intramolecular triplexes with the polypurine tract contained in the DNA of lentiviral preintegration complexes. Immuno-staining followed by confocal microscopy revealed the presence of RNA/DNA triplexes in the cytoplasm of latently infected cells. The presence of these triplexes was inversely proportionate to viral production, suggesting that the formation of the complexes blocks integration and subsequent transcription.81 Stimulation of latently infected cells was shown to cause a loss of triplexes associated with progression through mitosis. Stimulated cells thus became productively infected.81 The authors speculate that the endogenous siRNA capable of targeting the HIV-1 polypurine tract may derive from non-coding endogenous transposons and retroelements. These two studies suggest a role for cellular RNAi in regulation of the viral life cycle. In a third study, human miRNAs were found to be causative of HIV-1 latency in infected cells.74 Finally, a fourth approach examined the rate of viral replication after the knockdown of Dicer and Drosha.62 Collectively, these works suggest that cellular RNAi serves to regulate viral replication in human cells. Although cellular miRNA may act directly upon the virus, it is also possible that cellular miRNA may alter the expression of cellular genes important to viral replication. The Triboulet paper makes a strong case for this mechanism. The authors narrowed down the effect of knocking out Dicer and Drosha to cellular miRNAs expressed as part of the miR1792 cluster, which produces miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b-1, miR-20a and miR-92. Sequence analysis indicated that these miRNAs do not target viral genes.62 Interestingly miR-17 and miR-20 are predicted to target the cellular histone acetyl transferase, PCAF. As PCAF is an important co-factor to Tat-activated transcription, the expression of

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miR-17 and miR-20 may be detrimental to the virus. The authors demonstrated that blocking miR-17 and miR-20 increased viral replication, overexpressing these miRNA blocked replication, and that transfection with a plasmid containing a mutant PCAF gene, which lacks target sites for miR-17 and miR-20, overcame this effect.62 A well-known cellular response to viral infection is the induction of the interferon pathway.82–85 Indeed, triggering an interferon response leads to the execution of specific programs within the cell that result in cell death, heightened immune response, and decreased viral replication. Interestingly, the interferon pathways appear to be connected to RNAi. For example, studies have examined the expression of cellular miRNA after exposure to IFN-β as well as TLR receptor ligands86,87 and revealed the upregulation of three miRNA in response to interferon induction; miR132, miR-146 and miR-155. Another study shows the involvement of the interferon response in inducing miRNA expression specific for the hepatitis C virus.88 Conversely, a human protein TAR RNA binding protein (TRBP), which was discovered because of its avid binding to the HIV-1 TAR RNA element, is a known inhibitor of the interferon effector, PKR.89–91 Intriguingly, TRBP has also been shown to be a crucial factor for the processing and recruitment of the miRNA guide strand into the RISC complex.73,89,92–94 Thus TRBP appears to be a nexus which connects the antiviral responses of the interferon pathway, RNAi and HIV-1. Added support for the regulatory function of RNA interference in retroviruses comes from findings on endogenous retroelements called long interspaced elements (LINEs). Retroelements are examples of endogenous retroviruses. Most LINE elements are inactive, but the human genome contains approximately 100 active LINE elements. Interestingly, transcription of these elements is mediated by a 5′ UTR, which has antisense promoter activity. This antisense activity gives rise to the production of negative strand RNA that can hybridize with the positive strand to form dsRNA. This dsRNA is acted upon by Dicer to produce siRNA that represses LINE activity.95,96 Proof of the involvement of RNAi in controlling transposons also comes from work in yeast showing that after Dicer knockout, transposon expression increases.97,98 Various sequence analyses in a variety of organisms have identified miRNA that correspond to many repetitive elements including: LTR and non-LTR

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retrotransposons, transposons and heterochromatic regions.96,99–105 Indeed a LINE derived siRNA may account for the triplex forming small RNA described by Bagasra et al.81

5.2 Cleavage of Viral RNA by Cellular Dicer or Drosha Dicer and Drosha are both capable of binding and directly cleaving dsRNA. Dicer can recognize any stretch of dsRNA that possesses a 3′ overhang, while Drosha requires the presence of secondary structure containing at least 30 bases of dsRNA with a terminal loop of eight to ten bases. For HIV-1, a computer modeling predicts the presence of at least five stem and loop structures in viral RNA that could be candidates for Dicer processing. In principle, cleavage of viral genomes by Dicer would inactivate the virus; however, it may be that in many instances viral genomes are shielded by cellular RNA-binding proteins that prevent Dicer recognition. Indeed, a study has examined whether protective shielding of infecting RNA genomes can occur.106 Using a lentiviral vector containing a Nef gene and a GFP gene driven by different promoters, this study measured the ability of a siRNA to target an infecting lentivirus genome. Thus, by performing lentiviral infection in the presence of a siRNA that targets Nef, but does not affect GFP and using GFP as a readout, it was shown that siRNA that should target HIV-1 Nef in the incoming sequence apparently was ineffective and failed to influence virus infection.106 The same results were achieved regardless of whether the incoming genome was packaged within a VSV-G pseudotype or using HIV-1 Env.106 Although this work specifically addresses an RNA-genome coated in RNA-binding proteins within an infecting viral particle, the finding provides proof-of-concept that not all theoretically RNAi accessible sites are vulnerable to restriction, and that RNA-shielding can occur inside cells. Although the incoming HIV-viral genome appears to be shielded from access by RNAi or cleavage by Dicer and Drosha, computation predictions suggest that portions of HIV-1 transcripts if not shielded by RNA-binding proteins, are amenable to processing.107,108 Indeed, recent work has shown that the HIV-1 TAR can be processed into a miRNA.109

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HIV-1 TAR is a regulatory RNA element consisting of a 52 base pair stem and loop structure. TAR is found at the 5′ and the 3′ ends of every viral mRNA transcript. Indeed, TAR is a structure predicted to be a potential Dicer target.107 The ability of Dicer to cleave TAR structure has implications for the involvement of RNAi in the viral life cycle. Interaction between the Tat protein and TAR is required for activated transcription and production of viral protein, and cleavage of TAR could lead to loss of transactivation and degradation of viral mRNA. On the other hand, a TAR miRNA could direct a RISC/RITS complex to the integrated provirus,109 and participate in conferring viral latency (see below).

5.3 RNAi regulation of HIV-1 Replication A TAR miRNA could potentially act to suppress HIV-1 replication and be associated with viral latency.110–112 Indeed, latently infected resting T-cells express short transcripts that contain HIV-1 TAR but are otherwise incapable of coding for any protein.110,113,114 It has been shown that these short TAR containing transcripts in latently infected cells are processed into a viral miRNA.109 RNase protection assay performed on a panel of latently infected cell lines and primary T-cells infected de novo reveal that TAR miRNA is, in fact, produced in both latent and productively infected cells. Moreover, TAR miRNA appears to be capable of repressing transcription of the integrated provirus through chromatin remodeling. Indeed, ChIP assays indicate that in the presence of TAR miRNA, HDAC-1, a repressive modifier of chromatin, is recruited to the proviral promoter.109 These findings implicate that the cellular RNAi machinery through TAR miRNA could be a contributing factor to transcriptional latency. Could retroviruses such as HIV produce long stretches of dsRNA that can be acted upon by Dicer? This is not clear, but there is some evidence that this might be possible. First, retroviral LTRs can act as bidirectional promoters. There is a minus strand transcript associated with HTLV.115–118 Indeed this mRNA encodes for a protein called HBZ.115,117 Additionally, endogenous retroviral elements called LINEs also have LTRs with bidirectional promoter activity. Hence, it can be imagined that there are regions of retrovirus transcriptions that can produce sense and

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antisense transcripts leading to the production of dsRNAs which could be substrates for Dicer to produce siRNAs. Second, there is evidence which suggests that an antisense transcript is produced by HIV-1.119–122 A decade ago, Bukrinsky et al. showed the presence of antisense transcripts in the early stages of infection which might be translated into protein.119–121 This antisense transcription may form the basis for siRNAs reportedly derived from the viral Nef sequence,123,124 which may influence viral replication. Interestingly, some of the original research performed on antisense transcripts indicated a role for Tat in repressing this transcript. It appears that Tat driven transcription of the sense transcript blocks the reverse promoter activity, suggesting that in the presence of Tat, these siRNA may not be produced. Therefore, in this respect, Tat acts indirectly to suppress the potential for virus-derived antisense RNAi.

5.4 RNA Silencing Suppressors If RNA interference is an ancient mechanism for altering gene expression and protecting the cell from pathogens, then viruses may have evolved methods to suppress RNAi, including proteins and RNAs that block RNAi, termed RNA silencing suppressors (RSS). Examples of RSS are common in plant viruses, including tombusvirus P19, turnip crinkle virus P83, P1-HcPro of turnip mosaic virus, the P25 protein of potato virus X, and the P15 protein of peanut clump virus.47,125 Examples are also found in human viruses. Ebola virus P35 appears to block RNAi.126 The influenza virus neuraminidase inhibits Dicer function.127,128 Additionally, RNAi can be blocked by the vaccinia virus E3L, hepatitis C virus Core and foamy virus type 1 Tas.67,129,130 Adenovirus blocks the action of RNAi by flooding the cellular RNAi system with an excess of viral miRNA derived from a non-coding region of the viral genome.76,129,131 Intriguingly, all of the known mammalian viral proteins capable of suppressing RNAi also modulate the interferon response.125,127,132–139 HIV-1 appears to carry two RNAi suppressing functions, the first is in its RNA-binding Tat protein.107,126 How well Tat works as an RNAi suppressor has not been queried,140 and further studies are needed to

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sort out the different observations. However, in keeping with the observation that viral RSS proteins modulate the interferon response, Tat has also been shown to block the antiviral mechanisms of the interferon pathway.141–143 A second HIV RNAi suppressor appears to be its TAR RNA. This function apparently parallels a similar ability by the Adenovirus VA1 RNA to block miRNA biogenesis.76,131 The human TAR RNA binding protein (TRBP) is essential for loading of Dicer generated siRNA/ miRNA into the RISC complex.73,92 This protein was originally discovered due to its ability to bind to the HIV-1 TAR.93 In HIV-1 infection, TAR apparently can sequester TRBP and thereby prevent the processing and function of cellular miRNA.70,73 The observations that TAR is a potential Dicer target and that TRBP, the partner of Dicer, is already present on the structure suggest that TAR RNA may also serve to squelch Dicer.60,73 Indeed, findings related to a TAR derived miRNA support this model. The TAR derived miRNA can be detected in latently infected cells and expression of the miRNA doubles upon activation.109 However, during Tat activated transcription, the production of virus increases 500-fold.144–146 Tat-mediated activation and reversal of the silenced chromatin state likely helps the virus avoid the detrimental effects of the TAR miRNA in this situation. During activated transcription, it is possible that excess TAR RNA is used as a decoy to bind Dicer and TRBP, thus inhibits RNAi. Such a mechanism would allow the virus to avoid the possible detrimental effects of cellular miRNAs and the action of Dicer upon secondary structures within the coding regions of the viral mRNAs.

6. THE VIRUS’ ACTION ON THE HOST CELL Finally, one needs to consider what pressures the virus exerts on cellular RNAi pathways. As mentioned above, many viruses express viral miRNA(s). Some of these have been shown to regulate viral gene expression and progression through the viral life cycle. However, others have been shown to target cellular genes. In the case of HSV-1 and KSHV, the viral miRNA acts to prevent apoptosis,52,53 while EBV miRNA acts to avoid detection by the immune system.49 Alternatively, the action of viral

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RSS proteins and RNA decoys may alter the expression of cellular miRNA, thus altering other cellular processes.

6.1 Changes in Cellular miRNA in Virus-Infected Cells The ability of HIV-1 Tat to modulate Dicer function and the possibility that the TAR RNA structure sequesters TRBP means that HIV infection could affect the expression of cellular miRNAs.70,107 Currently, there are limited findings which address this hypothesis. First, the above mentioned study by Triboulet et al.62 used microarray technology to survey the expression of miRNA before and during infection of the Jurkat T-cell line.62 Eleven miRNA were found to be upregulated including: miR-122, miR-370, miR-373* and miR-297 that were detected only after infection. The polycistronic miRNA cluster miR-17/92 was shown to be downregulated upon infection.62 Second, a study by Yeung et al. found that many human cellular miRNAs were downregulated when HeLa cells were transfected with an infectious HIV-1 molecular clone.72 How these changes in miRNA expression contribute to the metabolism of the virus and the cell during infection remains to be further investigated. Although it is possible to imagine that Tat action on Dicer or sequestration of TRBP lead to a general downregulation of RNAi, it does not explain how specific miRNA are regulated. The ability of viral infection to activate the IFN response and thereby trigger miRNA expression may explain the observed upregulation of specific IFN-responsive miRNA.86,87 Alternatively, certain miRNA promoters may be acted upon by Tat, leading to increased expression. Total miRNA expression in HIV-1 infection might be suppressed due to Tat inhibition of Dicer or TRBP sequestration. However the ability of HIV to repress specific cellular miRNA may be more complex. Along these lines, the ability of Tat to block Dicer function is dependent upon binding to RNA.147 Therefore it is possible that the kinetics of the Tat-DicerRNA interaction may favor the suppression of certain miRNA more than others due to levels of pre-miRNA available and their ability to bind to Tat.

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6.2 Changes in Cellular Gene Expression Caused by Viral miRNA The ability of the virus to produce a miRNA from the defined structure of TAR is interesting when considering alteration in cellular gene expression.109 The processing of TAR into a miRNA will generate the same miRNA sequence each time. This will allow a TAR miRNA to target cellular genes. Research in progress suggests that expression of the TAR miRNA is associated with a decrease in apoptosis in response to cellular stress. In keeping with this, several genes related to apoptosis and cellular replication were found to be downregulated by TAR miRNA: IER3 — an immediate early gene shown to be important in inducing apoptosis in HeLa cells; ERCC1 — an excision repair gene; PIASγ — a suppressor of STAT-1 signaling and GIT2 — a protein involved in G-protein signaling. Taken together, suppression of these genes appears to decrease apoptosis and increase survival signals. As TAR miRNA is detectable in both latent and active infection, this suggests that the miRNA may be increasing the life of the latent cell and protecting the actively infected cell from apoptosis.109 These two actions would increase the life of the latent pool, thus making clearance of infection more difficult, and keeping the active cell alive longer.

7. MODELING A COMPLEX INTERACTION In examining the many interfaces between the cell and virus that are related to RNAi, it becomes apparent that no simple paradigm can fully explain all extant observations. Although the effects of viral and cellular miRNA on cellular/viral gene expression are important, there appears to be several complex overlapping pathways (Figure 2). For instance, relevant pathways include restriction of virus by cellular miRNA, suppression of miRNA expression by viral proteins, repression of RNAi action by viral decoy RNAs and virus-encoded suppressors, expression of interferon response by the cell, and suppression of interferon by the virus. At the big picture level, these networks of events reflect what the virus and the cell are doing to each other in order to modulate the complex and opposing processes of viral replication and cellular proliferation. Future studies are needed to

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Figure 2. A model of the complex interactions between viruses and cellular RNAi pathways. Interferon is shown here to have miRNA-independent and miRNA-dependent effects on viral replication. Separately, emerging evidence indicates that cellular miRNAs serve to restrict replication of viruses and that viruses in response have evolved ways to suppress and evade these restrictions.

tease out in detail how these myriad interactions integrate into a coherent picture of cellular immunity and viral pathogenesis.

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94. Gatignol A, Buckler C, Jeang KT (1993). Mol Cell Biol 13, 2193–2202. 95. Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T, Livingston DM, Rajewsky K (2005). Genes Dev 19, 489–501. 96. Yang N, Kazazian HH, Jr. (2006). Nat Struct Mol Biol 13, 763–771. 97. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999). Cell 99, 123–132. 98. Schramke V, Allshire R (2003). Science 301, 1069–1074. 99. Aravin AA, Lagos-Quintana M, Yalcin A, Zavolan M, Marks D, Snyder B, Gaasterland T, Meyer J, Tuschl T (2003). Dev Cell 5, 337–350. 100. Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, Siomi H, Siomi MC (2006). Genes Dev 20, 2214–2222. 101. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD (2006). Science 313, 320–324. 102. Djikeng A, Shi H, Tschudi C, Ullu E (2001). RNA 7, 1522–1530. 103. Grivna ST, Pyhtila B, Lin H (2006). Proc Natl Acad Sci USA 103, 13415–13420. 104. Grivna ST, Beyret E, Wang Z, Lin H (2006). Genes Dev 20, 1709–1714. 105. Watanabe T, Takeda A, Tsukiyama T, Mise K, Okuno T, Sasaki H, Minami N, Imai H (2006). Genes Dev 20, 1732–1743. 106. Westerhout EM, ter Brake O, Berkhout B (2006). Retrovirology 3, 57. 107. Bennasser Y, Le SY, Benkirane M, Jeang KT (2005). Immunity 22, 607–619. 108. Bennasser Y, Le SY, Yeung ML, Jeang KT (2004). Retrovirology 1, 43. 109. Klase Z, Kale P, Winograd R, Gupta MV, Heydarian M, Berro R, McCaffrey T, Kashanchi F (2007). BMC Mol Biol 8, 63. 110. Lassen KG, Bailey JR, Siliciano RF (2004). J Virol 78, 9105–9114. 111. Lassen K, Han Y, Zhou Y, Siliciano J, Siliciano RF (2004). Trends Mol Med 10, 525–531. 112. Han Y, Lassen K, Monie D, Sedaghat AR, Shimoji S, Liu X, Pierson TC, Margolick JB, Siliciano RF, Siliciano JD (2004). J Virol 78, 6122–6133. 113. Lin X, Irwin D, Kanazawa S, Huang L, Romeo J, Yen TS, Peterlin BM (2003). J Virol 77, 8227–8236. 114. Adams M, Sharmeen L, Kimpton J, Romeo JM, Garcia JV, Peterlin BM, Groudine M, Emerman M (1994). Proc Natl Acad Sci USA 91, 3862–3866. 115. Mesnard JM, Barbeau B, Devaux C (2006). Blood 108, 3979–3982.

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116. Cavanagh MH, Landry S, Audet B, Arpin-Andre C, Hivin P, Pare ME, Thete J, Wattel E, Marriott SJ, Mesnard JM, et al. (2006). Retrovirology 3, 15. 117. Gaudray G, Gachon F, Basbous J, Biard-Piechaczyk M, Devaux C, Mesnard JM (2002). J Virol 76, 12813–12822. 118. Larocca D, Chao LA, Seto MH, Brunck TK (1989). Biochem Biophys Res Commun 163, 1006–1013. 119. Miller RH (1988). Science 239, 1420–1422. 120. Bukrinsky MI, Etkin AF (1990). AIDS Res Hum Retroviruses 6, 425–426. 121. Ludwig LB, Ambrus JL Jr., Krawczyk KA, Sharma S, Brooks S, Hsiao CB, Schwartz SA (2006). Retrovirology 3, 80. 122. Landry S, Halin M, Lefort S, Audet B, Vaquero C, Mesnard JM, Barbeau B (2007). Retrovirology 4, 71. 123. Omoto S, Ito M, Tsutsumi Y, Ichikawa Y, Okuyama H, Brisibe EA, Saksena NK, Fujii YR (2004). Retrovirology 1, 44. 124. Omoto S, Fujii YR (2005). J Gen Virol 86, 751–755. 125. Dunoyer P, Lecellier CH, Parizotto EA, Himber C, Voinnet O (2004). Plant Cell 16, 1235–1250. 126. Haasnoot J, de Vries W, Geutjes EJ, Prins M, de Haan P, Berkhout B (2007). PLoS Pathog 3, e86. 127. Li WX, Li H, Lu R, Li F, DusM, AtkinsonP, BrydonEW, Johnson KL, Garcia-Sastre A, Ball LA, et al. (2004). Proc Natl Acad Sci USA 101, 1350–1355. 128. Bucher E, Hemmes H, de Haan P, Goldbach R, Prins M (2004). J Gen Virol 85, 983–991. 129. Hutvagner G, Simard MJ, Mello CC, Zamore PD (2004). PLoS Biol 2, E98. 130. Wang Y, Kato N, Jazag A, Dharel N, Otsuka M, Taniguchi H, Kawabe T, Omata, M (2006). Gastroenterology 130, 883–892. 131. Lu S, Cullen BR (2004). J Virol 78, 12868–12876. 132. Dave RS, McGettigan JP, Qureshi T, Schnell MJ, Nunnari G, Pomerantz RJ (2006). Virology 348, 489–497. 133. Langland JO, Kash JC, Carter V, Thomas MJ, Katze MG, Jacobs BL (2006). J Virol 80, 10083–10095. 134. Basler CF, Mikulasova A, Martinez-Sobrido L, Paragas J, Muhlberger E, Bray M, Klenk HD, Palese P, Garcia-Sastre A (2003). J Virol 77, 7945–7956.

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135. Basler CF, Wang X, Muhlberger E, Volchkov V, Paragas J, Klenk HD, Garcia-Sastre A, Palese P (2000). Proc Natl Acad Sci USA 97, 12289–12294. 136. Shors ST, Beattie E, Paoletti E, Tartaglia J, Jacobs BL (1998). J Interferon Cytokine Res 18, 721–729. 137. Beattie E, Paoletti E, Tartaglia J (1995). Virology 210, 254–263. 138. Dolganiuc A, Chang S, Kodys K, Mandrekar P, Bakis G, CormierM, Szabo, G (2006). J Immunol 177, 6758–6768. 139. Paul S, Tabassum S, Islam MN (2004). Bangladesh Med Res Counc Bull 30, 9–15. 140. Lin J, Cullen BR (2007). J Virol. 141. McMillan NA, Chun RF, Siderovski DP, Galabru J, Toone WM, Samuel CE, Mak TW, Hovanessian AG, Jeang KT, Williams BR (1995). Virology 213, 413–424. 142. Shirazi Y, Popik W, Pitha PM (1994). J Interferon Res 14, 259–263. 143. Roy S, Katze MG, Parkin NT, Edery I, Hovanessian AG, Sonenberg N (1990). Science 247, 1216–1219. 144. Rana TM, Jeang KT (1999). Arch Biochem Biophys 365, 175–185. 145. Brady J, Kashanchi F (2005). Retrovirology 2, 69. 146. Sharp PA, Marciniak RA (1989). Cell 59, 229–230. 147. Bennasser Y, Jeang KT (2006). Retrovirology 3, 95.

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SECTION II

Negative Single-Stranded RNA Virus

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CHAPTER 5

Signaling Pathways Induced by Influenza Viruses Stephan Pleschka, Stephan Ludwig, Oliver Planz & Thorsten Wolff

ABSTRACT Influenza viruses remain a continuous and severe global health threat to man as well as to many other mammals and birds. The re-emerging disease causes thousands of deaths and significant economic losses each year. The devastating results of recent outbreaks of avian influenza in Europe and South East Asia demonstrate this imminent danger. Influenza viruses are small RNA viruses. Due to their limited coding capacity, they manipulate host-cell functions extensively to support their replication. The infected cell simultaneously induces a wide range of defense mechanisms to fight the invader. These are mediated by a variety of intracellular signaling cascades. This chapter provides an overview on the current knowledge of functional kinase signaling and apoptotic events in influenza virus-infected cells and summarizes how these viruses have learned to misuse these cellular responses for efficient replication.

1. INTRODUCTION Influenza is a highly contagious, acute respiratory disease with global significance that affects all age groups and can occur repeatedly in any individual. The etiological agent of the disease, influenza virus, is responsible for an average of between three and five millions cases of severe influenza 109

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leading to about 250 000–500 000 mortalities annually in the industrialized world, according to WHO estimates. Compared to an otherwise healthy person, death rates in risk groups such as the elderly or the immune compromised are 50–100-fold higher. Waterfowl represents the natural reservoir for influenza-A-viruses1–4 that can infect many other animal species. Therefore the eradication of the virus is impossible and a constant re-emergence of the disease will occur. Epidemics appear almost annually and are due to an antigenic change of the viral surface glycoproteins (Figure 1). Additionally, highly pathogenic strains of influenza-A-virus have emerged in recent history, causing pandemic outbreaks like the “Spanish-Flu” that was responsible for the death of 20–40 millions people worldwide.2,5 The measures to fight this foe are restricted due the high genetic variability of the virus leading to the rapid generation of new variants that can escape vaccination-induced immunity or show resistance to antiviral

Figure 1. The influenza-A-virus particle. Schematic representation of the spherical influenza-A-virus particle that has a diameter of about 100 nm. The eight viral RNA segments were separated by urea-polyacrylamide gel electrophoresis and visualized by silver staining (left). The corresponding gene products and their presumed location in the virus particle are indicated (right). NS1 is not a structural part of the mature virion. For details see text.

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agents. Therefore the range of admitted measures to fight acute infection is very limited. Nevertheless, same as all viruses, influenza viruses interact intensively with cellular factors during their replication in order to support their own propagation. In this respect, intracellular signaling cascades activated by the virus, in particular MAPK pathways, have recently come into focus.6,7 Cell fate decisions in response to extracellular agents, including pathogenic invaders, are commonly mediated by intracellular signaling cascades that transduce signals into stimulus-specific actions, e.g., changes in gene expression patterns, alterations in the metabolic state of the cell or induction of programmed cell death (apoptosis). Thus, these signaling molecules are at the bottleneck of the control of cellular responses. Many DNA viruses and retroviruses are known to induce cellular signaling mainly to drive cells into a proliferative state. The reason is quite obvious since these pathogens partly employ the DNA synthesis machinery for their replication. The consequences of signaling induced by RNA viruses, including influenza viruses, were less clear since this area was not a focus of research for a long time. This chapter gives an overview on the current knowledge of signaling pathways induced by influenza virus that affect its replication in one way or another. As influenza viruses, like every virus, depend on its host cell, the understanding of cellular functions such as signaling pathways that are essential for viral replication may be suitable to define targets for antiviral therapy and pave the way towards effective drugs against viral functions or essential cellular activities supporting viral replication.

2. THE VIRUS AND ITS REPLICATION 2.1 Viral Components Influenza viruses belong to the order of Orthomyxoviridae. They possess a segmented, single-stranded RNA-genome with negative orientation. They are divided into three types: A, B and C, based on genetic and antigenic differences. Among the three types, influenza-A-viruses are clinically the most important pathogens since they have been responsible for

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severe epidemics in humans and domestic animals in the past. Thus the focus of this chapter will be on type-A influenza viruses. A detailed description of the viral proteins and the replication cycle of influenzaA-viruses can be found elsewhere.1,4 Therefore we will only give a brief overview on these topics without referring to individual references. The influenza-A-virus particle is composed of a lipid envelope derived from the host cell and of nine to ten structural virus proteins (Figure 1 and Table 1). The components of the RNA-dependent RNApolymerase complex (RDRP), PB2, PB1 and PA are associated with the ribonucleoprotein complex (RNP) and are encoded by the vRNA segments 1–3. The PB1 segment of many, but not all influenza-A-virus strains also contains a +1-reading frame encoding the recently discovered

Table 1. Influenza-A-Virus Genome (Strain A/PR/8/34). Segment

vRNA

Protein

AA

Function(s)

1

2341

PB2

759

Subunit of viral RNA polymerase; cap-binding.

2

2341

PB1 PB1-F2

757 87

Catalytic subunit of viral RNA polymerase. Proapoptotic activity.

3

2233

PA

716

Subunit of viral RNA polymerase.

4

1778

HA

566

Surface glycoprotein; receptor binding, membrane fusion.

5

1565

NP

498

Nucleoprotein; encapsidation of viral genomic and anti-genomic RNA.

6

1413

NA

454

Neuraminidase.

7

1027

Ml M2

252 97

Matrix protein. Ion channel activity, protecting HA conformation.

8

890

NS1

230

Regulator of viral RDRP activity. Interferon antagonist; enhancer of viral mRNA translation; inhibitor of (i) pre-mRNA splicing, (ii) cellular mRNA-polyadenylation, (iii) PKR activity. Activator of PI-3-kinase.

NEP

121

Nuclear export factor.

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PB1-F2 protein.8 The viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are expressed from vRNA segments 4 and 6, respectively. The nucleoprotein (NP) is encoded by segment 5 and associates with the vRNA segments. It is the major component of the RNPs. The two smallest vRNA segments each code for two proteins. The matrix protein (M1) is a colinear translate from the mRNA of segment 7 and forms an inner layer within the virion. A spliced version of the mRNA gives rise to a third viral transmembrane component, the M2 protein, which functions as a pH-dependent ion channel. Employing a similar coding strategy, segment 8 harbors the sequence information for the non-structural NS1 protein and the nuclear export protein NEP. NEP is a minor component of the virion and can associate with the M1 protein. Table 1 summarizes details of the genome segments, the encoded viral proteins and their respective functions.

2.2 The Influenza Replication Cycle The viral replication cycle is initiated by binding of the HA to sialic-acid (neuraminic acid) containing cellular receptors and subsequent endocytosis of the virus (Figure 2).9,10 The active HA molecule consists of two subunits (HA1/HA2) derived from the uncleaved precursor HA0, which becomes proteolytically processed after the release of the virion by extracellular proteases. This cleavage is absolutely essential for HA-function and cell infection. Virus disassembly occurs in the acidic environment of late endosomal vesicles and involves two crucial events. First, the conformation of the HA is changed to a low-pH form, which results in exposure of a fusion active protein sequence within the HA2. This fusion peptide is thought to contact the endosomal membrane and initiate fusion with the viral envelope. Second, the low pH in the endosomes activates the viral M2 ion channel protein, resulting in a flow of protons into the interior of the virion. Acidification facilitates dissociation of the RNPs from the M1 protein. The RNPs are subsequently released into the cytoplasm and rapidly imported into the nucleus through the nuclear pore complexes. The viral genomic segments are replicated and transcribed by the viral RDRP associated with the RNPs in the nucleus of the infected cell. The vRNA is directly transcribed to mRNA and it serves as a template for a

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Figure 2. The replication cycle of influenza viruses. The virion attaches to the cellular receptor determinant. The receptor-bound particle enters the cell via endocytosis. After fusion of the viral and the endosomal membrane the viral genome is released into the cytoplasm. The RNPs are transported into the nucleus where replication and transcription of the viral RNA segments occurs. The mRNAs are exported into the cytoplasm and are translated into viral proteins. The viral glycoproteins enter the exocytotic transport pathway to the cell surface. Replicative viral proteins enter the nucleus to amplify the viral genome. In the late stage of the infection cycle, newly synthesized RNPs are exported from the nucleus and assembled into progeny virions that bud from the cell surface.

complementary copy (cRNA), which is the template for new vRNA. In the late phase of infection, newly synthesized viral RNPs are exported to the cytoplasm. NS1 protein functions as a regulatory factor in the virus infected cell (see below). The NA, the M2 and the precursor HA (HA0) proteins follow the exocytotic transport pathway from the rER via the Golgi complex and the trans Golgi network. The mature HA and NA glycoproteins and the non-glycosylated M2 are finally integrated into the plasma membrane as trimers (HA) and tetramers (NA, M2), respectively.

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M1 assembles in patches at the cell membrane. It is thought to associate with the glycoproteins (HA and NA) and to recruit the RNPs to the plasma membrane in the late phase of the replication cycle. Finally the viral RNPs become enveloped by a cellular lipid bilayer carrying the HA, NA and M2 proteins resulting in budding of new virus particles from the apical cell surface. With the receptor destroying neuraminidase activity of the NA, the progeny virions are able to detach from the cell surface, to which they would otherwise be reattached by the HA activity.

3. CELLULAR SIGNALING THAT RESTRICTS INFLUENZA VIRUS REPLICATION AND SPREAD 3.1 Components of the Antiviral Cellular Defense Vertebrate cells have evolved powerful innate defense reactions that effectively block the propagation of many viral pathogens without the requirement for a prior contact. A prominent part of this resistance is the type I interferon (IFN) system which limits the replication and spread of viral pathogens including influenza viruses.11–13 The gearing up of this antiviral reaction is tightly controlled and necessitates the detection of the invading virus by cellular sensor(s) and the subsequent conversion of this information into changes in gene expression. In the following, the recently identified intracellular signaling module governing type I IFN induction and the antiviral response to an influenza virus infection are discussed, and emphasis is also put on the induced host proteins exhibiting antiviral effector activities. Furthermore, the mechanisms by which influenza viruses still succeed to multiply in the face of this hardwired defense are addressed. Many different cell types are capable of producing type I IFN (mainly IFN-α and IFN-β in humans), allowing them to respond instantly towards an invading virus and to prepare neighboring cells for the imminent attack of a pathogen.14 Secreted IFN-α /β bind to a common IFN-α /β receptor, which by signaling through the JAK-STAT pathway leads to the formation of the trimeric transcription factor ISGF3 that in turn upregulates more than 100 latent host genes, many of which encode proteins with antiviral activities (see below).12 As a result, IFN-treated cells establish an antiviral state in which many viruses cannot efficiently replicate.

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Although interferon activity was described for the first time in 1957,15 it was not until recently that cellular factors mediating the induction of these cytokines were described. Several studies showed that fibroblastoid and epithelial cells respond to viral nucleic acids produced during virus infection such as dsRNA or single-stranded RNAs carrying 5′-triphosphate via the homologous RNA helicases MDA5 or RIG-I.16–18 Upon RNA recognition, these two proteins trigger a signaling module that leads to the activation of type I IFN genes by the latent transcription factors IRF3/-7, NF-κ B and ATF-2/c-Jun.19 The two RNA helicases interact via two caspase recruitment domains (CARD) with the mitochondrial interferon-β promoter stimulator 1 (IPS-1) protein.20,21 This complex formation is thought to mediate activation of the transcription factors IRF-3 and IRF-7 by phosphorylation of the Iκ -B kinase family members, TBK-1 and/or IKK-ε after recruitment by a complex consisting of TANK and the NF-κ B modulator NEMO.19,22 IPS-1 also activates NF-κ B that participates in the induction of IFN-β and proinflammatory cytokine genes.23 Interestingly, influenza viruses and several other negative strand RNA viruses are recognized in epithelial cells selectively by RIG-I and not MDA-5,16,24,25 most likely by virtue of their genomic RNAs carrying a 5′ triphosphate group.17 The molecular basis of this selectivity for RIG-I versus MDA5 is currently under investigation. The expression of RIG-I is upregulated by type I IFN indicating the presence of a positive feedback loop.26 RIG-I appears to be dispensable for the detection of influenza viruses in plasmacytoid dendritic cells, in which single-stranded RNA viruses are recognized within an endosomal compartment by Toll-like receptors.27 However, influenza viruses usually do not propagate in these cells and influenza virus-infected mice were capable of clearing the infection in the absence of TLR signaling.28,29

3.2 Suppression of Type I IFN Induction and IFN-Inducible Gene Products is a Prerequisite of Influenza-A-Virus Pathogenicity Influenza virus propagation is sensitive to IFN activities and therefore these viruses like other viral pathogens not only induce type I IFN, but at the same time also antagonize the production and effects of these

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cytokines.30 For influenza-A and B-viruses, this is accomplished through their non-structural NS1 proteins that are structurally related polypeptides of 26 kDa (A/NS1) and 32 kDa (B/NS1) and are abundantly expressed in infected cells.31 Thus, influenza viruses of human, porcine, equine and avian origin expressing truncated or deleted NS1 genes were shown to be much stronger IFN inducers compared to wild-type and this correlated with a pronounced attenuation in animal experiments.32–36 In fact, mutant influenza viruses with engineered NS1 genes have been proposed to be suitable for the development of novel attenuated live vaccines.37 Recent molecular works indicated that the NS1 proteins function both on the level of inhibiting the expression of type I IFN genes as well as blocking the antiviral effects of some IFN-inducible proteins. Concerning IFN induction, it was recently shown that the viral NS1 protein forms a complex with RIG-I17,38 and inhibits RIG-I-dependent IFN-β promoter activation.24,25 Recombinant influenza viruses expressing dsRNA-binding defective NS1 proteins were shown to suppress IFN induction, at least to some extent.39,40 Collectively, these findings suggest that the NS1 proteins regulate IFN production in infected cells by obstructing RIG-I-dependent signaling through interaction with cellular factor(s) rather than by a sequestration of RNAs generated during virus replication. Some NS1 proteins were also described to inhibit the maturation of cellular pre-mRNAs, raising the possibility that this activity additionally reduces the production of IFN-α /β in infected cells.41–43 The inhibition of IFN induction by the NS1 protein is not absolute and therefore influenza virus-infected cells will produce some IFN and, hence, express IFN-stimulated genes (ISGs). Many ISGs have strong antiviral activities, although there appear to be specific sensitivities of virus families towards certain ISGs. Several IFN-inducible proteins have been identified, which appear to be particularly active in the anti-influenza defense and provoked the evolvement of pathogen countermeasures that are mediated by the viral dsRNA-binding NS1 protein. Among those host proteins are the enzymes protein kinase R (PKR) and the 2′-5′ oligoadenylate synthetases (OAS) that both require dsRNA as a cofactor. Activation of PKR leads to hyperphosphorylation of the translation initiation factor eIF2α, causing a sustained arrest of cellular translation.12 An NS1-deleted mutant

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Figure 3. Model for induction and blockade of type I IFN genes by influenza virus. The genomic RNAs of influenza virus (vRNA) are exported to the cytoplasm in the late phase of infection. They are recognized by the cytosolic RNA helicase RIG-I via their unmodified 5′-triphosphate group, which leads to the exposure of two N-terminal CARD domains of RIG-I, which mediate an interaction with the mitochondrial IPS-1 protein. The IPS-1 / RIG-I interaction triggers activation of the kinases TBK-1/IKKε and of the IKK complex that in turn signal for the activation of the transcription factors IRF3 and IRF7, and NF-κ B, respectively resulting in the induction of type I IFN genes. However, IFN induction by influenza wild-type virus is generally low due to the activity of the viral NS1 protein in complex formation with the RIG-I protein.

influenza-A-virus replicated poorly and behaved benignly in wild-type mice, but was capable to replicate and kill PKR-deficient mice, indicating that PKR confers substantial protection against influenza viruses.44 Further experiments with influenza viruses expressing dsRNA-binding deficient NS1 proteins showed a direct correlation of PKR activation with attenuated viral growth, indicating that dsRNA binding plays an important role in blocking PKR activation.39,45 The dsRNA binding activity of the NS1 protein also inhibits activation of 2′-5′ OAS that generates 2′-5′ oligo (A) chains, thereby activating the latent RNase L that degrades single-stranded RNA.46 Another effector with anti-influenza activity is the ubiquitin-like molecule ISG15 that is conjugated to many host proteins in IFN-treated cells, but unlike ubiquitin, it is not involved in protein degradation.47 The precise

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benefit of protein ISGylation for an infected cell or organism remains to be determined, but mice lacking ISG15 genes were more sensitive towards lethal challenges by influenza-A- and B-viruses.48 An important role of this modification in antiviral defense is suggested by the finding that the NS1 protein of influenza-B-virus binds directly to ISG15 and prevents its conjugation to cellular target proteins.49 Finally, the presence or absence of the IFN-inducible Mx1 gene is a superior determinant for survival in mice strains.50 Murine Mx1 was the first gene known to confer a strong resistance towards an infection with influenza viruses and belongs to the dynamin superfamily of GTPases, but no conclusive mechanism of its antiviral activity has been described yet.

4. CELLULAR SIGNALING THAT HAS BEEN CONQUERED BY INFLUENZA VIRUSES 4.1 Phosphatidylinositol-3-Kinase (PI3K) and Influenza Virus Infection PI3K consists of a regulatory (p85) and an enzymatic subunit (p110) and exhibits both a protein kinase and a lipid kinase activity.51 The kinase regulates various cellular processes, such as cell metabolism, proliferation, and survival.52 The consequence of PI3K activation is the generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2) in the membrane, which functions as a second messenger to recruit pleckstrin homology (PH) domain-containing proteins, such as the kinase Akt/PKB and phosphoinositide-dependent kinase 1 (PDK-1).52 Akt/PKB is a major PI3K effector and gets further activated by phosphorylation at Thr308 and at Ser473. In the context of viral infections the role of the PI3K/Akt pathway kinase has mainly been discussed due to its function to suppress apoptosis.53 Recently it was shown that in addition to this function, PI3K is also involved in phosphorylation and activation of IRF-3 in response to TLR3 engagement.54 Interestingly, this occurred independent of TBK-1 or IKK-ε and is most likely mediated via phosphorylation of a different target phosphorylation site on IRF-3.54 After first indications that this pathway is also activated in influenza virus-infected cells,55 it has finally been shown that

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inhibition of PI3K or blockage of its effector PIP3 results in a misphosphorylation of IRF-3 and impaired transcriptional activation of the IFN-β promoter upon influenza virus infection.56 However, besides this clear-cut antiviral activity, PI3K also exhibits virus-supportive functions at several levels of the viral replication cycle. Initial data revealed that PI3K and PIP3 seem to regulate a very early step in the virus life cycle, namely the early uptake of virus particles.56 More recently, several laboratories have shown that viral activation of PI3K and Akt serves to suppress premature apoptosis later in the infection cycle.57,58 Most strikingly, these and other studies revealed that the late PI3K activation is induced by the viral NS1 protein, which so far has only been described as a signaling suppressor. While first indications of this novel NS1 function were presented in a report by Ehrhardt et al.56 only a few months later, three independent studies from different laboratories demonstrated that the activation of PI3K is mediated by direct binding of NS1 to p85, the regulatory subunit of the kinase.57,59,60 This interaction appears to be unique for NS1 proteins of influenza-A-viruses since p85 binding and NS1-mediated PI3K activation was not observed upon infection with influenza-B-viruses or expression of the B/NS1 protein.57 Several results have been obtained with regard to the identification of the effector site in the influenza-A-virus NS1. While the integrity of amino acids both in the RNA binding domain as well as in a C-terminal stretch between amino acid 181–185 have been shown to interfere with PI3K activation,57 other studies favored a SH2-like interaction involving Y89 of NS1.59,60 A more recent study further identified a polyproline PXXP motif at amino acid 164–167 of NS1 as a potential binding site that may interact with the SH3 domain of p85.61 While a functional role of the NS1-PI3K interaction in the control of the apoptotic response has been confirmed in this report, there may be still other functions of the kinase pathway to control viral replication.62 Thus, both the mode of interaction as well as the complete functional consequences of the NS1-PI3K binding are not fully understood and will deserve further examinations.

4.2 Influenza Virus and the IKK/NF-κ B-Pathway The nuclear factor kappaB (NF-κ B) family comprises seven structurally related transcription factors that fulfill a central role in the cellular stress

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response and in inflammation by controlling a network of gene expression.63 Moreover, NF-κ B is commonly activated upon virus infections resulting in the expression of an array of cytokine and chemokine genes.64 Although the NF-κ B subunits are ubiquitously expressed, their actions are regulated in a cell type- and stimulus-specific manner, allowing for a diverse range of effects. Recent molecular dissection of NF-κ B activation has shown that NF-κ B can be induced by the so-called “canonical (classical) and “non-canonical” (alternative) signaling pathways, leading to distinct patterns in the individual NF-κ B subunits that are activated and consequently in the induced downstream genetic responses.65 The canonical mechanism of NF-κ B activation includes activation of the Iκ B-kinase ß (IKKß) that phosphorylates the inhibitor of NF-κ B (Iκ B) and thereby targets this protein for subsequent degradation. This consequently leads to the release and translocation of NF-κ B in the form of p65 (also known as Rel-A; a subunit of NF-κ B) and p50 (also known as NFκ B1) dimers.66 The IKK-complex consists of at least three isozymes of IKK: (I) IKK1/IKKα, (II) IKK2/IKKβ and (III) NEMO/IKK-γ. The most important isozyme for NF-κ B-activation via the degradation of Iκ B is IKK2.66 Although identified as one target of the suppressive action of the viral NS1 proteins, influenza virus infection still leads to a significant activation of IKK and NF-κ B as well as induction of NF-κ B-dependent gene expression (reviewed in Refs. 31 and 67). One explanation for this observation might be that influenza viral NF-κ B-activation is not only achieved by accumulation of viral nucleic acids but also by overexpression of the viral HA, NP or M1 proteins,68 a process that most likely cannot be blocked by the NS1. Due to their role in antiviral gene expression, NF-κ B and IKK were regarded for a long time as bona fide components of the innate immune response to virus infections. In support of that view it has been shown that influenza virus-induced IFNβ -promoter activity is impaired in cells expressing transdominant negative (dn) mutants of IKK2/IKKβ or Iκ Bα.69,70 Furthermore influenza virus titers are slightly enhanced in infected wild-type cells treated with an anti-IFN-α /β receptor antibody while this is not the case in cells expressing dnIκ Bα, indicating that NF-κ B mediates influenza virus induced IFN expression.70 It was thus surprising when two independent studies demonstrated that influenza viruses replicated much better in cells where NF-κ B was

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pre-activated.70,71 Conversely, progeny virus titers were reduced when grown in host cells in which NF-κ B-signaling was impaired by means of specific inhibitors or dominant-negative mutants.70,71 Thus, influenza virus appears to be capable of turning the antiviral activity of NF-κ B into a virus-supportive one. On a molecular basis this was shown to be due to the NF-κ B-dependent expression of factors, such as TNF-related apoptosis inducing ligand (TRAIL) or FasL,70 that are known activators of a cell death program, including activation of caspases. Accordingly, it was shown that influenza virus propagation was also strongly impaired in the presence of caspase inhibitors or siRNA against caspase 3.72 Mechanistically, the block in virus propagation appeared to be due to a nuclear retention of viral ribonucleoprotein (RNP) complexes preventing formation of progeny virus particles, an effect that could be shown both in the presence of NF-κ B- or caspase-inhibitors.72 Taken together, this delineates a scenario in which influenza virus is reprogramming NF-κ B action by suppression of an antiviral activity and recruitment of the factor for a virus-supportive function, namely activation of caspase-mediated RNP export from the nucleus. Besides this surprising activity of NF-κ B in the context of an influenza-virus infection, it has recently been demonstrated that IFNinduced gene expression is also negatively regulated by NF-κ B.73 Thus, the factor may even negatively interfere with other levels of the antiviral response. NF-κ B influences the pathogenesis after influenza virus infection by a direct effect on the virus life cycle. Moreover, excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells (also known as cytokine burst) is considered an important factor in disease pathogenesis, where influenza-A-virus induces IKKβ activity in human airway epithelial cells, resulting in persistent activation of NF-κ B.74,75

4.3 Influenza Virus and the Mitogenic Raf/MEK/ERK Signaling Cascade Mitogen activated protein kinase (MAPK)-cascades regulate numerous cellular decision processes, such as proliferation and differentiation, but also cell activation and immune responses.76 Four different members of

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the MAPK-family that are organized in separate cascades have been identified so far: ERK (extracellular signal regulated kinase), JNK (Jun-N-terminal kinase), p38 and ERK5/BMK-1 (Big MAP kinase).76 ERK and its upstream activators, the serine-threonine kinase Raf that activates the dual specificity kinase MEK (MAP kinase kinase/ERK kinase) form the prototype module of a MAPK pathway and comprise the classical mitogenic cascade. While all four so far defined MAPK family members are activated upon an influenza virus infection (reviewed in Ref. 31), the ERK-signaling pathway appears to serve a mechanism that is particularly beneficial for virus replication.77 Blockade of the pathway by specific inhibitors of MEK or dominant-negative mutants of ERK or Raf resulted in a strongly impaired growth of both influenza-A and B-viruses.77,78 Conversely, virus titers are enhanced in cells expressing active mutants of Raf or MEK.78,79 This has not only been demonstrated in cell culture but also in vivo in infected mice expressing a constitutively active form of the Raf kinase in the alveolar epithelial cells of the lung.79 This indicates that activation of the Raf/MEK/ERK pathway is required for efficient virus growth, most likely by regulating nuclear export of the viral RNPcomplexes that are readily retained in the nucleus upon blockade of the signaling pathway.77,78 In contrast to the passive caspase mediated process described above, Raf/MEK/ERK activity appears to affect the active export machinery most likely by interference with the activity of the viral NEP.77 This again indicates that RNP export is a signaling-induced rather than a constitutive event, and appears to involve both active ERK-mediated step and passive caspase-mediated step, which may replace the active process at later stages of the viral life cycle. With respect to activation, it is striking to note that the Raf/MEK/ ERK cascade is one of a few signaling pathways not activated by dsRNA or suppressed by the viral NS1 protein.80 In contrast, it has recently been shown that ERK activation correlates with expression of the viral HA and requires proper assembly of the HA into lipid raft-like domains in the cell membrane. Since raft accumulation of the HA correlated with ERK activation and subsequent RNP export,80 it can be hypothesized that the pathway gets activated late in infection at a time-point when newly synthesized RNPs are ready to be packaged into new virus particles.

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Figure 4. The PI3K/Akt pathway is multifunctional in influenza virus-infected cells. The PI3K/Akt pathway is found to be activated during influenza virus infection in a biphasic manner. Early activation is most likely due to virus binding at cellular receptors and is required for efficient virus uptake. The signaling pathway is also activated by accumulating RNA. Productive influenza virus infection results in accumulation of RNA, that may appear in the form of ss or dsRNA or as uncapped 5′ triphosphate RNA (5´PPPRNA). Later in the infection the pathway targets phosphorylation of IRF-3 that is needed for full activation of an antiviral IFN response. Finally, PI3K is also directly activated by the viral NS1 protein via binding to the regulatory PI3K subunit p85. This event takes place late in the infection cycle and is required for suppression of premature apoptosis.

5. CONCLUSION Our current understanding of influenza virus-induced cellular signaling events demonstrates that these virus/host-interactions are numerous and multifaceted. We are just beginning to understand the complexity of this relation. Cellular signaling represents a regulatory principle that enables the cell to respond to the extracellular enviroment and invading pathogens. In the case of the induction of the type I IFN-expression and the activation of the antiviral state, the cell reacts towards the intruder in order to defend itself as well as to alert other cells. Nevertheless, influenza viruses have learned to cope with this obstacle by downregulating the effectiveness of

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the signaling events involved. Surprisingly the virus has not only acquired the capability to suppress this cellular defense mechanism, but it also appears to misuse the residual activity left in order to further promote its own replication, which seems to be the case for NF-κ B dependent activation of caspases. On top of that, influenza viruses even seem to selectively induce certain signaling pathway to their advantage. This is clearly the case for the virus-induced activation of the Raf/MEK/ERK cascade or NS1mediated activation of the PI3K/Akt pathway. Again, they are able to balance this activity in a way that promotes virus replication. The work on influenza virus-induced signaling has not only provided us with new insight into the molecular mechanisms that influenza virus use for their propagation, but it has opened the door to new innovative antiviral approaches. Blocking virus-induced or -used cellular signaling by highly specific inhibitors at non-toxic concentration allows for the countering of the virus with little chance for the virus to adapt to this selective pressure. This might prove to become highly important in regards to the limited measures that we currently have in the treatment of the influenza and with regard to the imminent threat by new pandemic strains.

REFERENCES 1. Palese P, Shaw ML (2007). In Fields Virology, eds. Knipe, DM, Howley PM, Lippincott Williams & Wilkins, Philadelphia, pp. 1647–1689. 2. Webster RG (1999). Proc Natl Acad Sci USA 96, 1164–1166. 3. Wilschut J, McElhaney JF (2005). Influenza, Mosby Elsvier Limited. 4. Wright PF, Naumann G, Kawaoka Y (2007). In Fields Virology, eds. Knipe DM, Howley PM, Lippincott Williams & Wilkins, Philadelphia, pp. 1691–1740. 5. Taubenberger JK, Reid AH, Fanning TG (2000). Virology 274, 241–245. 6. Ludwig S, Planz O, Pleschka S, Wolff T (2003). Trends Mol Med 9, 46–52. 7. Ludwig S, Pleschka S, Wolff T (1999). Viral Immunol 12, 175–196. 8. Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, Basta S, O’Neill R, Schickli J, Palese P, Henklein P, Bennink JR, Yewdell JW (2001). Nat Med 7, 1306–1312. 9. Lamb RA, Krug RM (2001). In Fields Virology, eds. Knipe DM, Howley PM, Griffin DE, Lippincott Williams & Williams, pp. 1487–1531.

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10. Wright PF, Webster RG (2001). In Fields Virology, eds. Knipe DM, Howley PM, Griffin DE, Lippincott Williams & Williams, pp. 1533–1579. 11. Portnoy J and Merigan TC (1971). J Infect Dis 124, 545–252. 12. Samuel CE (2001). Clin Microbiol Rev 14, 778–809, table of contents. 13. Strube M, Bodo G, Jungwirth C (1985). Mol Biol Rep 10, 237–243. 14. Biron CA, Sen GC (2001). eds. Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin, MA, Roizman B Straus SE, Lippincott Williams & Wilkins, Philadelphia, pp. 321–351. 15. Isaacs A and Lindenmann J (1957). Proc R Soc Lond B Biol Sci 147, 258–267. 16. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S (2006). Nature 441, 101–115. 17. Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F, Reis e Sousa C (2006). Science 314, 997–1001. 18. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira, K, Akira S, Fujita T (2004). Nat Immunol 5, 730–737. 19. Yoneyama M and Fujita T (2007). Cytokine Growth Factor Rev 18, 545–551. 20. Kawai T, Takahashi K, Sato S, Coba C, Kumar H, Kato H, Ishii KJ, Takeuchi O and Akira S (2005). Nat Immunol 6, 981–988. 21. Kumar H, Kawai T, Kato H, Sato S, Takahashi K, Coban C, Yamamoto M, Uematsu S, Ishii KJ, Takeuchi O, Akira S (2006). J Exp Med 203, 1795–1803. 22. Zhao T, Yang L, Sun Q, Arguello M, Ballard DW, Hiscott J, Lin R (2007). Nat Immunol 8, 592–600. 23. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB (2005). Mol Cell 19, 727–740. 24. Guo Z, Chen LM, Zeng H, Gomez JA, Plowden J, Fujita T, Katz JM, Donis RO and Sambhara S (2007). Am J Respir Cell Mol Biol 36, 263–269. 25. Opitz B, Rejaibi A, Dauber B, Eckhard J, Vinzing M, Schmeck B, Hippenstiel S, Suttorp N, Wolff T (2007). Cell Microbiol 9, 930–938. 26. Matikainen S, Siren J, Tissari J, Veckman V, Pirhonen J, Severa M, Sun Q, Lin R, Meri S, Uze G, Hiscott J, Julkunen I (2006). J Virol 80, 3515–3522. 27. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, Matsui K, Tsujimura T, Takeda K, Fujita T, Takeuchi O, Akira S (2005). Immunity 23, 19–28.

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28. Le Goffic R, Balloy V, Lagranderie M, Alexopoulou L, Escriou N, Flavell R, Chignard M, Si-Tahar M (2006). PLoS Pathog 2, e53. 29. Lopez CB, Yount JS, Moran TM (2006). J Virol 80, 3128–3134. 30. Garcia-Sastre A, Biron CA (2006). Science 312, 879–882. 31. Ludwig S, Pleschka S, Planz O, Wolff T (2006). Cell Microbiol 8, 375–386. 32. Cauthen AN, Swayne DE, Sekellick MJ, Marcus PI, Suarez DL (2007). J Virol 81, 1838–1847. 33. Dauber B, Heins G, Wolff T (2004). J Virol 78, 1865–1872. 34. Garcia-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, Durbin JE, Palese P, Muster T (1998). Virology 252, 324–330. 35. Quinlivan M, Zamarin D, Garcia-Sastre A, Cullinane A, Chambers T, Palese P (2005). J Virol 79, 8431–8439. 36. Solorzano A, Webby RJ, Lager KM, Janke BH, Garcia-Sastre A, Richt JA (2005). J Virol 79, 7535–7543. 37. Talon J, Salvatore M, O’Neill RE, Nakaya Y, Zheng H, Muster T, GarciaSastre A, Palese P (2000). Proc Natl Acad Sci USA 97, 4309–4314. 38. Mibayashi M, Martinez-Sobrido L, Loo YM, Cardenas WB, Gale M, Jr., Garcia-Sastre A (2007). J Virol 81, 514–524. 39. Dauber B, Schneider J, Wolff T (2006). J Virol 80, 11667–11677. 40. Donelan NR, Basler CF, Garcia-Sastre A (2003). J Virol 77, 13257–13266. 41. Fortes P, Beloso A, Ortin J (1994). Embo J 13, 704–712. 42. Kochs G, Garcia-Sastre A, Martinez-Sobrido L (2007). J Virol 81, 7011–7021. 43. Noah DL, Twu KY, Krug RM (2003). Virology 307, 386–395. 44. Bergmann M, Garcia-Sastre A, Carnero E, Pehamberger H, Wolff K, Palese P, Muster T (2000). J Virol 74, 6203–6206. 45. Hatada E, Saito S, Fukuda R (1999). J Virol 73, 2425–2433. 46. Min JY, Krug RM (2006). Proc Natl Acad Sci USA 103, 7100–7105. 47. Giannakopoulos NV, Luo JK, Papov V, Zou W, Lenschow DJ, Jacobs BS, Borden EC, Li J, Virgin HW, Zhang DE (2005). Biochem Biophys Res Commun 336, 496–506. 48. Lenschow DJ, Lai C, Frias-Staheli N, Giannakopoulos NV, Lutz A, Wolff T, Osiak A, Levine B, Schmidt RE, Garcia-Sastre A, Leib DA, Pekosz A, Knobeloch KP, Horak I, Virgin HW (2007). Proc Natl Acad Sci USA 104, 1371–1376. 49. Yuan W, Krug RM (2001). Embo J 20, 362–371.

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50. Haller O, Kochs G, Weber F (2007). Cytokine Growth Factor Rev 18, 425–433. 51. Dhand R, Hiles I, Panayotou G, Roche S, Fry MJ, Gout I, Totty NF, Truong O, Vicendo P, Yonezawa K, et al. (1994). Embo J 13, 522–533. 52. Neri LM, Borgatti P, Capitani S, Martelli AM (2002). Biochim Biophys Acta 1584, 73–80. 53. Cooray S (2004). J Gen Virol 85, 1065–1076. 54. Sarkar SN, Peters KL, Elco CP, Sakamoto S, Pal S, Sen GC (2004). Nat Struct Mol Biol 11, 1060–1067. 55. Guillot L, Le Goffic R, Bloch S, Escriou N, Akira S, Chignard M, Si-Tahar M (2005). J Biol Chem 280, 5571–5580. 56. Ehrhardt C, Marjuki H, Wolff T, Nurnberg B, Planz O, Pleschka S, Ludwig S (2006). Cell Microbiol 8, 1336–1348. 57. Ehrhardt C, Wolff T, Pleschka S, Planz O, Beermann W, Bode JG, Schmolke M, Ludwig S (2007). J Virol 81, 3058–3067. 58. Zhirnov OP, Klenk HD (2007). Apoptosis 12, 1419–1432. 59. Hale BG, Jackson D, Chen YH, Lamb RA, Randall RE (2006). Proc Natl Acad Sci USA 103, 14194–14199. 60. Shin YK, Liu Q, Tikoo SK, Babiuk LA, Zhou Y (2007). J Gen Virol 88, 13–18. 61. Shin YK, Li Y, Liu Q, Anderson DH, Babiuk LA, Zhou Y (2007). J Virol 81, 12730–12739. 62. Shin YK, Liu Q, Tikoo SK, Babiuk LA, Zhou Y (2007). J Gen Virol 88, 942–950. 63. Hayden MS, Ghosh S (2004). Genes Dev 18, 2195–2224. 64. Hiscott J, Kwon H, Genin P (2001). J Clin Invest 107, 143–151. 65. Bonizzi G, Karin M (2004). Trends Immunol 25, 280–288. 66. Karin M, Delhase M (2000). Semin Immunol 12, 85–98. 67. Ludwig S, Planz O, Pleschka S, Wolff T (2003). Trends Mol. Med. 9, 46–51. 68. Flory E, Kunz M, Scheller C, Jassoy C, Stauber R, Rap, UR, Ludwig S (2000). J Biol Chem 275, 8307–8314. 69. Wang X, Li M, Zheng H, Muster T, Palese P, Beg AA, Garcia-Sastre A (2000). J Virol 74, 11566–11573. 70. Wurzer WJ, Ehrhardt C, Pleschka S, Berberich-Siebelt F, Wolff T, Walczak H, Planz O, Ludwig S (2004). J Biol Chem 279, 30931–30937.

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71. Nimmerjahn F, Dudziak D, Dirmeier U, Hobom G, Riedel A, Schlee M, Staudt LM, Rosenwald A, Behrends U, Bornkamm GW, Mautner J (2004). J Gen Virol 85, 2347–2356. 72. Wurzer WJ, Planz O, Ehrhardt C, Giner M, Silberzahn T, Pleschka S, Ludwig S (2003). Embo J 22, 2717–2728. 73. Wei L, Sandbulte MR, Thomas PG, Webby RJ, Homayouni R, Pfeffer LM (2006). J Biol Chem 281, 11678–11684. 74. Bernasconi D, Amici C, La Frazia S, Ianaro A, Santoro MG (2005). J Biol Chem 280, 24127–24134. 75. Peiris JS, de Jong MD, Guan Y (2007). Clin Microbiol Rev 20, 243–267. 76. Widmann C, Gibson S, Jarpe MB, Johnson GL (1999). Physiol Rev 79, 143–180. 77. Pleschka S, Wolff T, Ehrhardt C, Hobom G, Planz O, Rapp, UR, Ludwig S (2001). Nat Cell Biol 3, 301–305. 78. Ludwig S, Wolff T, Ehrhardt C, Wurzer WJ, Reinhardt J, Planz O, Pleschka S (2004). FEBS Lett 561, 37–43. 79. Olschlager V, Pleschka S, Fischer T, Rziha HJ, Wurzer W, Stitz L, Rapp UR, Ludwig S, Planz O (2004). Oncogene 23, 6639–6646. 80. Marjuki H, Alam MI, Ehrhardt C, Wagner R, Planz O, Klenk HD, Ludwig S, Pleschka S (2006). J Biol Chem 281, 16707–16715.

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CHAPTER 6

Host Immune Response to Influenza Virus Thomas M. Moran & Carolina B. López

ABSTRACT Influenza virus causes yearly epidemics and sporadic pandemics worldwide. Influenza virus is predominantly spread by the respiratory route through aerosol particles. Infection of the lungs depends upon the virus’ ability to evade innate immune mechanisms such as defensins, natural killer cells, and most importantly, type I interferons (IFN). The virus is carried from the lungs to draining lymph nodes by dendritic cells (DCs) that present peptides from viral proteins to B and T cells leading to their expansion and differentiation. The T cells mediate clearance of virus from infected tissue and the B cells generate humoral immunity that protects against reinfection with the same virus subtype. In this chapter, we will discuss the host immune response to influenza virus based on current data obtained from in vitro and in vivo systems. The interaction of the virus with innate immune mechanisms will be discussed, with a focus on the mechanisms of influenza evasion. The transition between innate and adaptive antiviral immunity and the role of DCs will be emphasized. Finally, the generation of CD4+ and CD8+ T cells, antibodies, heterosubtypic immunity and the immune elements involved in efficient protection from infection will be discussed.

1. INTRODUCTION Seasonal influenza virus causes a mild to severe illness in humans, characterized by the sudden onset of chills, fever, nasal congestion, aches and 131

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pains throughout the body. Pneumonia and other complications which may result in death, are most common in infants, the elderly, and immunocompromised patients.1 The symptoms observed during an influenza virus infection are largely attributed to the immune response mounted against the virus. Our current knowledge of the immune response against influenza virus is mostly based on studies using mice as a model system. The prevalence of quality reagents available, as well as the convenience of their size and manageability has encouraged the use of this animal model for the study of immunity. A number of studies performed in humans after community acquisition or controlled inoculation with virus have largely confirmed the viability of the mouse model with regard to the speed of replication, the release of inflammatory cytokines, and the rate of recovery.2–4 Nevertheless, mice are not natural hosts for influenza virus, therefore a number of factors should be considered when analyzing their response to infection. For example, influenza virus is not transmitted between mice as occurs following infection of its natural hosts. In addition, mouse-adapted strains of influenza virus are used to infect inbred mouse strains that usually lack the myxovirus resistance-1 (MX1) gene, an important innate antiviral mechanism. In mice, the disease induced by influenza virus is characterized by an acute pneumonia with virus clearance by day 10.5 Ferrets, guinea pigs, and cotton rats represent some of the alternative models for influenza virus infection, but their use is currently limited by the lack of reagents.6–8 The first line of defense against invading pathogens is provided by elements of the innate immune system. Successful pathogens must evade innate immunity to establish an infection. Virtually all pathogens devote some portion of their genome to immune antagonism. Influenza is no exception as it codes for the NS1 protein that inhibits innate immunity and is essential for successful infection. Nevertheless, once the infection is established, the adaptive immune response is alerted and cellular immunity is activated. The transition between innate and adaptive immunity is mediated by dendritic cells (DCs) which function as sentinels to identify microbial invaders in the respiratory tract. Two different populations of DCs play important roles in antiviral immunity. Conventional DCs (cDCs) populate peripheral tissues and survey for the presence of

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invading pathogens. Upon encountering virus, cDCs become activated by recognition of invariant patterns on the microbe leading to their migration to local draining lymph nodes where they present viral peptides to T cells and initiate adaptive immunity. Plasmacytoid DCs are specialized cells that secrete large amounts of type I IFNs in response to virus infection. Type I IFNs responsive genes are the primary innate antiviral mechanism. Only through adaptive immune effector mechanisms can viral clearance be achieved. Influenza infection triggers a potent humoral response that protects against reinfection with the same viral subtype. However, humoral population immunity exerts selective pressure on the virus selecting for changes in antigenic structures (antigenic drift) that allow the virus to reinfect. Three times in the last century a new influenza virus was generated by reassortment (antigenic shift) with a virus from an animal reservoir leading to a worldwide pandemic. The complete lack of humoral memory to these viruses renders them particularly lethal to the human population. A description of the multiple elements of immunity that participate in the response to influenza virus infection is the focus of this chapter.

2. INNATE IMMUNITY AND VIRAL ANTAGONISM Innate immunity represents the initial barrier to the establishment of a viral infection. It encompasses mucociliary movement, phagocytic cells, and soluble mediators such as defensins, interferons (IFNs), natural antibodies, and complement (Figure 1). In addition, variation in physiological characteristics (e.g. receptor glycosylation) represents a barrier to infection when a virus is transmitted from one animal species to another as occurred in the recent human infections by avian influenza virus.9 The innate immune system also communicates information collected during its interaction with the virus to the adaptive immune system. This function is directed by cDCs that pick up the virus, are activated through pattern recognition receptors, and present representative viral peptides to virusspecific T cells. The absence of any of the important innate immune functions predisposes an individual to infection and increases morbidity and mortality.

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Figure 1. Components of innate immunity in the airways. Scheme of an alveolus and the different immune elements associated to it in both the luminal cavity and the interstitial tissue.

2.1 The First Line of Defense: Mucus, Cilia, and Alveolar Fluid The upper respiratory tract is lined with a layer of mucus that works in concert with ciliated epithelial cells to capture and extrude invaders or particulate matter that enters the respiratory tract. Ciliary movement is a coordinated process that pushes particles out toward the pharynx by passing it from one ciliated area to another.10 Suboptimal mucociliary

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clearance increases the risk of virus infection.10,11 Mucus contains more than 100 proteins, many of which function to kill, opsonize, or inactivate microbial invaders.12 Proteins such as lysozyme and lactoferrin found in mucus are of particular importance in preventing bacterial infections but probably have a lesser role in the prevention of viral infections. Defensins, complement, and antibodies in mucus and throughout the respiratory tract represent important primary barriers to virus infection. The fluid that bathes the alveolar surfaces similarly contains many of the same important antimicrobial substances including the surfactant proteins SP-A and SP-D, and mannose binding lectin (MBL) that are members of the collectin family of proteins which function to capture and opsonize invading microbes.13

2.2 The Phagocytes Both neutrophils and macrophages control infection by influenza virus. Diminished phagocytic activity through the depletion of either neutrophils or alveolar macrophages correlates with a rise in virus titers and an increase in mortality in mouse models.14–17 In addition to ingesting opsonized virus, macrophages have been shown to phagocytize cells made apoptotic by influenza virus infection.14,17 Alveolar macrophages that are rapidly triggered into apoptosis by influenza virus are phagocytized by DCs that migrate to lymph nodes and activate adaptive immunity. This event represents an important vehicle for initiation of adaptive immunity through the cross-presentation of viral antigens.18,19

2.3 Natural Antibodies and Complement Natural antibodies are a component of the first response to pathogen invasion. These antibodies, produced by B-1 B cells, are generated in the absence of antigen stimulation.20 Thus, serum from naive mice contains antibodies, primarily IgMs, capable of neutralizing influenza virus to a limited degree. The effectiveness of these antibodies depends upon the activation of the classical complement pathway as neutralization of influenza virus depends upon the presence of serum and is

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lost if the serum is collected from animals deficient in the C3, C1q, or C4 component of the complement cascade. The neutralization observed with natural antibodies and complement does not depend upon lysis of the virus since serum from C5 deficient animals maintain inhibitory activity even though it is unable to activate the membrane attack complex.21

2.4 Defensins and Collectins, Small Multifunctional Proteins Defensins are cationic cysteine-rich peptides that function to control infectious agents through a number of mechanisms.22 Defensin production is triggered by infection through TLR signaling, or in response to cytokines produced during inflammation. Some defensins like human beta defensin 1 (HBD1) are constitutively produced by epithelial cells.23 Defensins can act directly on viruses by disrupting the viral envelope or by crosslinking the surface glycoproteins. During influenza virus infection the retrocyclin-2 defensin prevents endosomal fusion by blocking the glycoprotein conformational change essential for this process to occur. Retrocyclin-2 is a carbohydrate-binding lectin24 and may also aggregate virus, increasing opsonization and phagocytosis by neutrophils.25 Human alpha defensin 1 (HNP-1) inhibits influenza virus infection by targeting the infected cell through the suppression of protein kinase C activation.26 Defensins also function to induce or enhance the release of IL-8 from airway epithelial cells.27–30 and to chemoattract monocytes and immature DCs.31 It has been reported that β -defensin 2 can function as a ligand for TLR4 and trigger the maturation of immature DCs.32 Surfactant Protein A (SP-A), Surfactant Protein D (SP-D) and Mannan Binding Lectin (MBL) comprise the collectins of the respiratory tract.33,34 They are C-type lectins that recognize microbes through conserved surface carbohydrates. They primarily function by agglutinating and opsonizing microbes leading to enhanced phagocytosis by alveolar macrophages. In addition, MBL is able to activate complement, augmenting its opsoninzing ability and enhancing monocyte activation and phagocytosis.34 Animals deficient in SP-D show a decreased clearance of influenza virus and an increased inflammatory response.35

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2.5 NK Cells, Innate Cellular Effectors NK cells express a constellation of activating and inhibiting receptors that determine their activation or suppression. Binding of activating receptors can result in direct killing of target cells or the release of various cytokines such as IFN-γ . Two of the natural cytotoxic receptors NKp44 and NKp46 expressed on the surface of NK cells can interact directly with the influenza virus hemagglutinin and activate NK cells.36–38 Elimination of the mouse equivalent of these receptors renders mice much more susceptible to death induced by influenza virus.39 Substantial evidence exists for a role of DCs in the activation of NK cells.40,41 NKp46 and NKG2D receptor interaction with influenza-infected DCs has been shown to lead to NK cell activation that presumably functions to help eliminate flu infected cells in vivo.42

3. INNATE ACTIVATING RECEPTORS AND TYPE I IFN PRODUCTION The innate immune response identifies the presence of microbial invaders and signals for the recruitment of both innate and adaptive immune elements. Most studies of initiation of immunity in response to influenza virus infection focus on the release of type I IFNs from infected cells, a hallmark of antiviral responses. Type I IFNs, including IFN-β and the different IFN-α types are produced by most cell types in response to virus infection. However, the specialized pDCs release larger quantities of IFN-α in response to influenza virus infection.43–45 Type I IFNs are produced in response to conserved structures found in many microbes by one of the two mechanisms: toll-like receptor (TLR) signaling in endosomal sites, or stimulation of retinoid acid inducible gene-I-like receptors (RLR) in the cytoplasm of all types of cells. A discussion on these mechanisms follows.

3.1 TLRs TLRs represent one class of pattern recognition receptors (PRR) specifically activated by invariant structures present in many infectious agents

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know as pathogen-associated molecular patterns (PAMPs). The identification of this family of receptors influenced the perception of how the immune system interacts with invading microbes.46 In the respiratory tract, TLRs are expressed on alveolar and airway epithelium as well as on many leukocyte populations. TLRs 3, 7, and 8 can sense RNA viruses such as influenza virus. TLR3 is expressed in endosomal compartments and binds double-stranded RNA (dsRNA). It remains controversial whether TLR3 is important for type I IFN release, and/or activation of adaptive immunity in response to influenza infection in humans and mice. Mice deficient in TLR3 exhibit a normal cellular immune response; however, their antibody response is skewed in a Th2 direction.47 In these animals, virus titers in the lungs are increased when compared with wildtype mice, but morbidity and mortality are reduced.48 TLR7 activation by single-stranded RNA (ssRNA) leads to type I IFN production on pDCs in humans but not in mice. Influenza virus is an excellent inducer of pDCs and induction through this receptor may prove to be an important mechanism for interferon release in vivo during infection. The discovery of TLRs with specificity for ssRNA and dsRNA was heralded as the key to understanding the initiation of immunity to virus infection and undoubtedly TLRs do play an important role in the response to many viruses. However, with regard to the response to influenza virus, the data, while still controversial, suggest an important, if not paramount role for another family of receptors found in the cytoplasm of virtually all cells that may function in both innate and adaptive immunity. This family is called the retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs).

3.2 RIG-I-like Receptors The RIG-I-like receptors (RLRs) are DExD/H box RNA helicases that sense the presence of viral infection through their interaction with viral RNA. RIG-I is specifically activated by ssRNA with exposed 5′phosphates.49,50 Another RLR, the melanoma differentiation associated gene-5 (MDA-5), has specificity for dsRNA.51 A third RLR, LGP-2, lacks activation domains and may function as an inhibitor of downstream signaling.52 Influenza virus genomic RNA has been shown to trigger type I IFN production through RIG-I.53,54 However, for this triggering to occur, there

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are a number of obstacles that need to be bypassed during normal infection. First, the virus codes for an inhibitor, NS1 that specifically inhibits RIG-I recognition.55–57 Second, virus replication takes place in the nucleus where viral RNA is coated with nucleoprotein.

3.3 Type I IFN and Influenza NS1 Type I IFNs are multifunctional proteins that have considerable effects on both innate and adaptive immunity. They are secreted from virus-infected cells (or in the case of pDCs, “virus-exposed cells”) and bind to a ubiquitous receptor. This binding results in triggering of transcription of hundreds of genes, many having antiviral functions. The antiviral proteins produced are largely synthesized in an inactive form but can be activated by virus infection to interfere with its replication. Mx1, PKR, RNAseL, and 2′, 5′-oligoadenylate synthetase are the best described antiviral proteins, all of which have been shown to be effective against influenza virus.58–60 The NS1 protein of influenza virus prevents the production of type I IFNs from primarily infected cells by blocking RIG-I signaling in response to viral RNA. NS1 also blocks effector proteins such as PKR and 2′, 5′-oligoadenylate synthetase by preventing their activation through binding of dsRNA.59,60 The removal or inactivation of the NS1 protein which allows RIG-I to be activated renders the virus unable to establish infection.49,55–57 However, NS1 is unable to block type I IFN release in response to TLR signaling from pDCs. Thus, the type I IFN produced by pDCs during influenza virus infection does not seem to interfere with virus replication.

4. INNATE-ADAPTIVE INTERFACE 4.1 Virus-DC Interaction Conventional DCs (cDCs) are the primary cells involved in launching the adaptive immune response (Figure 2). Upon encountering microbes, cDCs are activated and undergo a maturational change from sentinel to antigen presenting cells. They then migrate to peripheral lymphoid tissue where they present microbial peptides to virus specific T cells. When triggered by

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Figure 2. Anti-influenza virus adaptive immune response. I: Lung epithelia infected with influenza virus provides inflammatory signals for the recruitment of proinflammatory cells including DCs. II: Activated DCs capture the antigen and transport it to the draining lymph node. III: DCs present antigen to specific T and B cells leading to their differentiation into effector and memory cells. IV: Effector T and B cells are recruited to the lung for clearance of the infection.

viruses, DC maturation is best characterized by the synthesis and release of type I IFNs, inflammatory cytokines, and changes to surface molecules that improve DC-T cell interactions. The activation of cDCs by many viruses is dependent upon autocrine or paracrine type I IFN signaling, while for others such as Sendai, no additional stimulus is needed.61–63 cDC maturation by influenza virus is triggered through the RIG-I pathway and is most likely independent of TLR signaling.64 Influenza virus abortively infects DCs and is generally a very poor activator of DC maturation65

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Table 1. Receptors That Activate the Type I Interferon Pathway in Human Cells Name

TLR3

TLR7

TLR8

TLR9

RIG-I

MDA5

Cell Respiratory distribution epithelium, cDC

pDCs, B cells

pDCs

pDCs, B cells

All cells

All cells

Ligand

dsRNA

ssRNA

ssRNA

Adaptor protein

TRIF

MyD88 MyD88

Unmethylated ssRNA ssDNA with with free CpGrich 5′phosphates, motifs dsRNA MyD88

MAVS/IPS1

dsRNA

MAVS/ IPS1

because of the NS1 protein. NS1 inhibits the transcription of both type I IFNs and many of the other genes associated with DC maturation.55,61,66–68 Thus, the stimulation of immunity to influenza may depend on a cross priming mechanism which may be explained as follows: virus-infected apoptotic cells or partial virions are engulfed by DCs and are shuttled into the MHC class I pathway where they associate with nascent MHC class I molecules, move to the cell surface, and interact with receptors on specific T cells.69 In contrast to cDCs, influenza virus does cause a rapid activation of pDCs through a TLR7-mediated event. However, strong evidence suggests that, in the absence of TLR signaling, cellular immunity is efficiently generated. Thus, activation of pDCs is either not essential for the generation of cellular immunity, or is compensated for by redundant mechanisms in TLR deficient animals.47,48

4.2 DC-T Cell Interface DCs carrying influenza virus proteins are directed to the draining mediastinal lymph nodes by CCR7-mediated migration, where they present viral peptides derived from many influenza virus proteins to T cells.70 It has been widely reported that influenza virus infection in the mouse model triggers rapid activation and migration of DCs to the draining nodes. The DCs arrive at the lymph nodes within the first 24 hours of infection and their migration ceases by 36 hours postinfection.71 This finding

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suggests that a rapid inflammatory response must occur shortly after the establishment of infection. However, a response to non-immunogenic aerosolized ovalbumin in mice can only be observed if the ovalbumin is administered two days after initiation of an influenza virus infection. This response presumably occurs because DCs carrying the OVA and activated by the virus are only beginning to migrate to the draining lymph node at that time point.72 Moreover, evidence suggests that T cell proliferation begins 72 hours after infection. The kinetics of these events is difficult to reconcile with the very early DC migration from the lung to the lymph node after infection. More studies are needed to clarify this issue. Resident CD8+ DCs in the lymph nodes have been reported to play an important role in the activation of T cells. They accumulate viral antigens from other lung derived CD8- DCs that apparently function to only transport antigen.73–75 However this finding has been recently amended by the observation that the CD8+ DCs may be particularly suited to activate memory cells, while lung derived DCs may activate naive T cells.76

4.3 Anatomical Location of Response There is little doubt that DCs carrying viral antigens migrate to draining lymph nodes in order to activate the T cells involved in adaptive immunity. However, it has been demonstrated that, in the absence of conventional peripheral lymphoid tissue, immunity can still be effectively generated in auxiliary sites, called the bronchus associated lymphoid tissue (BALT).77 The importance of these lung associated sites for immunity to influenza virus, in vivo, has yet to be elucidated.

5. ACUTE EFFECTOR RESPONSE TO INFLUENZA VIRUS INFECTION Despite a significant degree of control of influenza virus infection by innate immunity, elements from the adaptive immune response are necessary for the clearance of the infection. Adaptive immunity also provides life long immunological memory and confers protection against reinfection. Antigen presenting cells carrying viral antigens from the lungs and activated by the virus infection, initiate the differentiation and activation of

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specific T and B cells in the draining lymph nodes. In mice, both CD4+ and CD8+ T cells have been implicated in the clearance of primary influenza virus infection either through a direct effector function78–81 or as a result of their role in promoting B cell differentiation to antibody-secreting cells.82,83 T cells begin to appear in the lung at day 6 postinfection, which coincides with the onset of viral clearance. High titers of isotyped switched antibodies can be detected at day 10 postinfection. The titer of these antibodies peaks five days later.84 The contribution of each of these aspects of immunity to the clearance of a primary influenza virus infection will be discussed.

5.1 CD8+ T Cells The role for effector CD8+ T cells in the clearance of a primary influenza virus infection is well established. Mice receiving influenza specific CD8+ T cell lines or clones by adoptive transfer clear the virus more efficiently and demonstrate reduced mortality when compared with control groups.85,86 Conversely, mice deficient in T cells87,88 or MHC class I presentation (β 2microglubulin−/−)78 show a delayed viral clearance and increased mortality after challenge with a virulent strain of influenza virus when compared with wild-type mice. The importance of CD8+ T cells in the control of influenza virus in humans is demonstrated by the relatively high mutation rate observed in the NP gene affecting peptides recognized by human CD8+ T cells.89–92 In C57BL/6 mice, influenza virus-specific CD8+ T cells are restricted mainly to six epitopes distributed between H-2Db and H-2Kb.93 The most prevalent CD8+ T cells are those specific for epitopes derived from viral nucleoprotein (DbNP366-374) and the viral polymerase (DbPA224-233) although epitopes in other virus proteins have also been identified.94,95 The preponderance of diverse CD8 epitopes vary in different mouse strains.96 Effector CD8+ T cells specific for influenza virus are generated in the lymph nodes from naive T cells which are stimulated by activated antigen-presenting cells. After extensive proliferation and differentiation, specific CD8+ T cells migrate to the lung to eliminate the virus via the production of proinflammatory cytokines and the direct killing of virus-infected cells through perforin/granzyme- or Fas/FasL-mediated interactions.97 Influenza virus-responsive CD8+ T cells expressing different cytokine profiles are found in the lungs and airways one week after infection.

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Evidence suggests that the functional differentiation of influenza-specific CD8+ T cells is a consequence of different strengths of T cell receptor (TCR) signaling, which is determined by the avidity of TCR-MHC-peptide interactions, the load of antigen, and environmental factors.98 The majority of influenza-specific CD8+ T cells produce IFN-γ upon stimulation with peptides, a subset of these cells produce TNF-α, and only a small fraction of IFN-γ /TNF-α + cells also produce IL-2.98,99

5.2 CD4+ T Cells CD4+ T cells participate in the clearance of a primary influenza virus infection by improving CD8+ T cell and B cell responses. Mice depleted of CD8+ T cells by treatment with anti-CD8 antibodies, and mice lacking MHC class I because of a disruption in the β 2-microglobulin gene, clear the virus similar to wild-type mice and recover from infection.79 In contrast, mice lacking CD8+ T cells, or B cells, and depleted of CD4+ T cells show a significant delay in viral clearance and increased mortality.79,100,101 Work from our laboratory demonstrated that treatment of influenza virus-infected mice with the Th2-driving cytokine IL-4 during a primary infection suppresses the generation of specific CD8+ cytotoxic lymphocytes (CTLs) and causes a delay in virus clearance from the lungs.102 This evidence indicates that a Th1 type of CD4+ T-cell bias is required for the effective development of an anti-influenza virus immune response. In fact, effector CD4+ T cells of the Th1 type are found in the lungs of mice between six and eight days after infection. These cells secrete IFN-γ in the lungs when influenza antigen is present.103,104 IFN-γ has been shown to promote the expansion and recruitment of T cells to the lung and support class switching to the IgG2A and IgG3 isotypes. These factors contribute to the efficient clearance of virus infection.105,106

5.3 B Cells and Antibodies The role of B cells and influenza-specific antibodies in the clearance of a primary influenza virus infection remains controversial. Isotype switched, anti-influenza virus antibodies can be detected in infected mice from day 10 postinfection onwards; a time when the virus has already been

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eliminated.84 Moreover, B cell deficient mice ( µ MT) can recover from infection with a low pathogenicity influenza virus (strain X31) or sublethal doses of other virus strains at a rate similar to that of wild-type mice.80,101 However, immunodeficient SCID mice can clear influenza virus infection with the aid of anti-HA neutralizing antibodies83 and mice lacking both B cells and CD8+ T cells cannot recover from infection in contrast to mice depleted of CD8+ T cells only, which eventually clear the virus.100,101 A more definite role for B cells in the recovery from primary infection is seen upon infection of mice with highly pathogenic virus. B cell deficient mice are more susceptible to lethal doses of influenza virus than wild-type mice,80 and in the absence of CD4+ T cells, mice succumb to infection with the virulent influenza PR8 strain.107 Nevertheless, CD8+ T cells can autonomously clear the infection when injected to CD4+/B cell double deficient mice, clouding the role of B cells in the resolution of primary infection with influenza virus.80

6. MEMORY AND RECALL RESPONSE TO INFLUENZA VIRUS INFECTION During a primary virus infection, in addition to the development of effector cells that clear the virus, long term specific memory T and B cells are generated. Memory cells can control a secondary infection with the same or closely related virus more efficiently than naive cells because of the higher frequency of specific cells and their faster effector response.104,108 Memory T cells are generally classified as either effector or central according to their location within the organism, cell surface markers, and functional characteristics. Effector memory cells are found in peripheral organs and have effector functions that aid in controlling virus infection. Central memory T cells are localized in the lymph nodes and quickly proliferate in response to a secondary infection and give rise to effector memory cells.109

6.1 Antibodies: Role in Clearance and Protection During Reinfection Antibodies play a preponderant role in protection against reinfection with the same influenza virus or within viruses from the same subtype

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arising through antigenic drift (see below).110,111 Mucosal IgA and systemic IgG neutralize virus growth and help to control the infection.112 Neutralizing antibodies against the virus hemagglutinin (HA) protein play a critical role in limiting virus replication by interfering with infection. Anti-NA antibodies that prevent virus release from infected cells also reduce the rate of infection.113 Antibodies to proteins conserved among different influenza virus strains, such as the M2, NP and M1 are also induced in response to infection but only anti-M2 antibodies confer some protection.110,114 The influenza HA and neuraminidase (NA) epitopes “drift” in response to antibody selection because of the poor fidelity of the influenza polymerase. This ability supports the survival of the virus in human populations. As a consequence of this drift, a number of variants of the same virus circulate in the population at one given time.111,115 In addition, new influenza viruses arise from antigenic “shift” resulting from the reassortment of existing viruses into a new strain. This occurs when viruses circulating in different animal species coinfect the same cell. A progeny virus formed by a mixture of segments from the parent viruses is then formed. For example, a reassortment between a human H1N1 and an avian H2N2 virus is thought to be the origin of the human H2N2 influenza virus that caused the 1957 pandemic. Three different HAs and two NAs proteins are predominant among the human influenza viruses currently circulating within the population. However, 16 different HAs and nine NAs are circulating in nature, including the avian H5N1 and H7N7 viruses.116 It is feared that this diversity will soon result in reassortant viruses with devastating consequences for humans because of the lack of pre-existent neutralizing antibodies against these new viruses. Nevertheless, it has been extensively demonstrated in the mouse model that viruses resulting from antigenic “drift” or “shift” can be controlled significantly by heterosubtypic immunity.

6.2 Heterosubtypic Immunity Mice immunized with one virus subtype and challenged with a different subtype show a reduced disease state and a facilitated recovery when compared to wild-type mice, independent of preformed antibodies.117,118 This response has also been observed in humans in limited situations.110

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This phenomenon is known as heterosubtypic immunity and is mediated by virus-specific CD4+ and CD8+ T cell responses.118 Heterosubtypic immunity is directed against conserved viral proteins such as the internal NP protein and minimizes the transmission of variants of influenza virus resulting from antigenic “drift” or “shift”. It is important to note that some occasional drift of the more conserved NP genes has also been observed in humans,119 which compromises the ability of heterosubtypic immunity to control reinfection with a different strain of influenza virus. Circulating anti-influenza virus antibodies and both CD8+ and CD4+ T cells can independently contribute to protection against otherwise lethal secondary infection with influenza virus.108,109 The availability of mouse adapted strains of influenza virus, carrying different subtypes of HA and NA proteins, have allowed studies to determine the contribution of cellular immunity during secondary infection, by eliminating the impact of neutralizing antibodies.

6.3 Memory CD8+ T Cells Two decades ago it was demonstrated that adoptively transferred influenza virus-specific CD8+ T cells provided protection to counter the influenza virus in mice.81,86,120 It is now well established that, after clearance of a primary influenza virus infection, a number of CD8+ memory T cells including both NP and PA specificities persist for a long time in both lymphoid and non-lymphoid tissues. These cells contribute to the control of a second infection with the same or closely related virus by rapidly limiting its growth.121,122 Influenza-specific memory CD8+ T cells are generated in the lymph nodes early after the primary infection (days 3–4).123 Establishment of memory CD8+ T cells require CD4+ T cell help as mice deficient in MHC II (IAb−/−) have diminished memory and recall CD8+ T cell responses.124–126 Influenza-specific memory CD8+ T cells can be divided based on their cytokine profile (IFN-γ alone, IFN-γ /TNFα, and IFN-γ /TNFα /IL-2 secretors) similar to the profiles observed in effector CD8+ T cells.98 Memory T cells disseminate into various tissues including the lung. The expression of the integrin VLA-1 on the surface of CD8+ T cells have been shown to permit the retention of CD8+ T cells in the lung and other

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tissues.127 Large numbers of memory CD8+ T cells are found in the lung in the first few months after resolution of the infection. This number wanes with time until it stabilizes to a minimum level six months after infection. These cells persist for the life of the animals.108,109 Influenza virus-specific memory CD8+ T cells are greatly expanded after secondary exposure to the virus and substantial numbers of CD8+ T cells migrate to the lungs at this time. The magnitude of the CD8+ T cell response after the challenge is significantly greater than that of the primary response.122,128 At least three populations of memory CD8+ T cells participate in the recall response to influenza virus infection: resident lung memory T cells, the first to encounter the virus that function to reduce the viral load; non-proliferating effector memory T cells, recruited from the circulation to the lung, which react in response to non-specific inflammatory signals; and proliferating memory T cells that are recruited to the lung as fully mature effector cells.108 The participation of T cells from different compartments ensures a quick and sustained response to the virus after secondary challenge.

6.4 Memory CD4+ T Cells Studies in mice have shown that adoptively transferred CD4+ Th1 clones, generated in vitro, are able to protect against influenza infection in vivo, whereas Th2 clones do not provide protection.129 Similarly, adoptively transferred Th1-polarized transgenic CD4+ T cells abrogate weight loss and promote the survival of mice lethally challenged with the virulent influenza virus strain PR8.104 Additionally, mice depleted of CD4+ cells at the moment of challenge with influenza virus demonstrate a delay in virus clearance.130 These data, together with the observation that CD4+ T cells persist in the lung after influenza virus infection, in mice103 and humans,131 lend support to the role of memory CD4+ T cells in protection from secondary infection.

7. VACCINES AND PERSPECTIVES Recent infections of humans by avian influenza have heightened the fear of emerging influenza pandemics. This has served to stimulate research

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that may lead to the development of vaccines with broader cross reactivity across influenza species. Although the generation of antibodies following vaccination prevents reinfection by most pathogens, this is not true for influenza virus because of the selection for antigenic variants by population immunity. This variability makes it unlikely that any vaccine will be able to elicit an antibody response that will provide broader protection. The only way to achieve persistent protection is by using vaccine formulations that efficiently stimulate cellular immunity. Presently, the predominant anti-influenza virus vaccine contains killed virus of three different subtypes. The vaccine formulation is updated yearly on the basis of global virus surveillance for circulating or emerging strains. This vaccine has little impact on cellular immunity and probably fails to generate a virus-specific CD8+ response. Efforts are underway to develop live attenuated vaccines with a limited ability for dissemination. One such vaccine is the NS1 deleted or truncated vaccine. In the absence of NS1 protein virus spread is restricted by the type I IFN pathway yet both humoral and cellular responses are generated.132–134 Other efforts being undertaken are the insertion of influenza genes into those viruses which are unable to produce disseminated infection in humans such as Newcastle disease virus (NDV). NDV viruses that contain epitopes for both cellular and humoral immunity could trigger a response that would provide broad protection against many influenza virus subtypes.

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45. Piqueras B, Connolly J, Freitas H, Palucka AK, Banchereau J (2006). Blood 107, 2613-2618. 46. Barton GM, Medzhitov R (2002). Curr Top Microbiol Immunol 270, 81–92. 47. Heer AK, Shamshiev A, Donda A, Uematsu S, Akira S, Kopf M, Marsland BJ (2007). J Immunol 178, 2182–2191. 48. Le Goffic R, Balloy V, Lagranderie M, Alexopoulou L, Escriou N, Flavell R, Chignard M, Si-Tahar M (2006). PLoS Pathog 2, e53. 49. Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F, Reis e Sousa C (2006). Science 314, 997–1001. 50. Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, et al. (2006). Science 314, 994–997. 51. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, et al. (2006). Nature 441, 101–105. 52. Saito T, Hirai R, Loo YM, Owen D, Johnson CL, Sinha SC, Akira S, Fujita T, Gale M, Jr. (2007). Proc Natl Acad Sci USA 104, 582–587. 53. Loo YM, Fornek J, Crochet N, Bajwa G, Perwitasari O, Martinez-Sobrido L, Akira S, Gill MA, Garcia-Sastre A, Katze MG, et al. (2008). J Virol 82, 335–345. 54. Siren J, Imaizumi T, Sarkar D, Pietila T, Noah DL, Lin R, Hiscott J, Krug RM, Fisher PB, Julkunen I, et al. (2006). Microbes Infect 8, 2013–2020. 55. Mibayashi M, Martinez-Sobrido L, Loo YM, Cardenas WB, Gale M, Jr., Garcia-Sastre A (2007). J Virol 81, 514–524. 56. Opitz B, Rejaibi A, Dauber B, Eckhard J, Vinzing M, Schmeck B, Hippenstiel S, Suttorp N, Wolff T (2007). Cell Microbiol 9, 930–938. 57. Guo Z, Chen LM, Zeng H, Gomez JA, Plowden J, Fujita T, Katz JM, Donis RO, Sambhara S (2007). Am J Respir Cell Mol Biol 36, 263–269. 58. Staeheli P, Pravtcheva D, Lundin LG, Acklin M, Ruddle F, Lindenmann J, Haller O (1986). J Virol 58, 967–969. 59. Hatada E, Saito S, Fukuda R (1999). J Virol 73, 2425–2433. 60. Min JY, Krug RM (2006) Proc Natl Acad Sci USA 103, 7100–7105. 61. Lopez CB, Garcia-Sastre A, Williams BR, Moran TM (2003). J Infect Dis 187, 1126–1136. 62. Lopez CB, Yount JS, Hermesh T, Moran TM (2006). J Virol 80, 4538–4545. 63. Yount JS, Moran TM, Lopez CB (2007). J Virol 81, 7316–7319.

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64. Lopez CB, Moltedo B, Alexopoulou L, Bonifaz L, Flavell RA, Moran TM (2004). J Immunol 173, 6882–6889. 65. Lopez CB, Fernandez-Sesma A, Schulman JL, Moran TM (2001). J Interferon Cytokine Res 21, 763–773. 66. Fernandez-Sesma A, Marukian S, Ebersole BJ, Kaminski D, Park MS, Yuen T, Sealfon SC, Garcia-Sastre A, Moran TM (2006). J Virol 80, 6295–6304. 67. Lopez CB, Moran TM, Schulman JL, Fernandez-Sesma A (2002). Int Rev Immunol 21, 339–353. 68. Lopez CB, Yount JS, Moran TM (2006). J Virol 80, 3128–3134. 69. Shen L, Sigal LJ, Boes M, Rock KL (2004). Immunity 21, 155–165. 70. Jakubzick C, Tacke F, Llodra J, van Rooijen N, Randolph GJ (2006). J Immunol 176, 3578–3584. 71. Legge KL, Braciale TJ (2003). Immunity 18, 265–277. 72. Brimnes MK, Bonifaz L, Steinman RM, Moran TM (2003). J Exp Med 198, 133–144. 73. Belz GT, Shortman K, Bevan MJ, Heath WR (2005). J Immunol 175, 196–200. 74. Belz GT, Smith CM, Eichner D, Shortman K, Karupiah G, Carbone FR, Heath WR (2004). J Immunol 172, 1996–2000. 75. Carbone FR, Belz GT, Heath WR (2004). Trends Immunol 25, 655–658. 76. Belz GT, Bedoui S, Kupresanin F, Carbone FR, Heath WR (2007). Nat Immunol 8, 1060–1066. 77. Moyron-Quiroz JE, Rangel-Moreno J, Kusser K, Hartson L, Sprague F, Goodrich S, Woodland DL, Lund FE, Randall TD (2004). Nat Med 10, 927–934. 78. Bender BS, Croghan T, Zhang L, Small PA, Jr. (1992). J Exp Med 175, 1143–1145. 79. Eichelberger M, Allan W, Zijlstra M, Jaenisch R, Doherty PC (1991). J Exp Med 174, 875–880. 80. Graham MB, Braciale TJ (1997). J Exp Med 186, 2063–2068. 81. Lukacher AE, Braciale VL, Braciale TJ (1984). J Exp Med 160, 814–826. 82. Mozdzanowska K, Furchner M, Zharikova D, Feng J, Gerhard W (2005). J Virol 79, 5943–5951. 83. Gerhard W, Mozdzanowska K, Furchner M, Washko G, Maiese K (1997). Immunol Rev 159, 95–103.

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Swain SL, Dutton RW, Woodland DL (2004). Viral Immunol 17, 197–209. Wells MA, Ennis FA, Albrecht P (1981). J Immunol 126, 1042–1046. Taylor PM, Askonas BA (1986). Immunology 58, 417–420. Schulman JL, Petigrow C, Woodruff J (1977). Dev Biol Stand 39, 385–390. Wells MA, Albrecht P, Ennis FA (1981). J Immunol 126, 1036–1041. Berkhoff EG, Geelhoed-Mieras MM, Verschuren EJ, van Baalen CA, Gruters RA, Fouchier RA, Osterhaus AD, Rimmelzwaan GF (2007). Clin Exp Immunol 148, 296–306. Gog JR, Rimmelzwaan GF, Osterhaus AD, Grenfell BT (2003). Proc Natl Acad Sci USA 100, 11143–11147. Boon AC, de Mutsert G, Graus YM, Fouchier RA, Sintnicolaas K, Osterhaus AD, Rimmelzwaan GF (2002). J Virol 76, 2567–2572. Rimmelzwaan GF, Boon AC, Voeten JT, Berkhoff EG, Fouchier RA, Osterhaus AD (2004). Virus Res 103, 97–100. Zhong W, Reche PA, Lai CC, Reinhold B, Reinherz EL (2003). J Biol Chem 278, 45135–45144. Townsend AR, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ (1986). Cell 44, 959–968. Turner SJ, Kedzierska K, La Gruta NL, Webby R, Doherty PC (2004). Semin Immunol 16, 179–184. Lawrence CW, Ream RM, Braciale TJ (2005). J Immunol 174, 5332–5340. Topham DJ, Tripp RA, Doherty PC (1997). J Immunol 159, 5197–5200. La Gruta NL, Turner SJ, Doherty PC (2004). J Immunol 172, 5553–5560. Belz GT, Xie W, Doherty PC (2001). J Immunol 166, 4627–4633. Mozdzanowska K, Furchner M, Maiese K, Gerhard W (1997). Virology 239, 217–225. Topham DJ, Doherty PC (1998). J Virol 72, 882–885. Moran TM, Isobe H, Fernandez-Sesma A, Schulman JL (1996). J Virol 70, 5230–5235. Roman E, Miller E, Harmsen A, Wiley J, Von Andrian UH, Huston G, Swain SL (2002). J Exp Med 196, 957–968. Swain SL, Agrewala JN, Brown DM, Jelley-Gibbs DM, Golech S, Huston G, Jones SC, Kamperschroer C, Lee WH, McKinstry KK, et al. (2006). Immunol Rev 211, 8–22. Baumgarth N, Kelso A (1996). J Virol 70, 4411–4418.

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106. Turner SJ, Olivas E, Gutierrez A, Diaz G, Doherty PC (2007). J Immunol 178, 7616–7622. 107. Mozdzanowska K, Maiese K, Gerhard W (2000). J Immunol 164, 2635–2643. 108. Hikono H, Kohlmeier JE, Ely KH, Scott I, Roberts AD, Blackman MA, Woodland DL (2006). Immunol Rev 211, 119–132. 109. Wherry EJ, Barber DL, Kaech SM, Blattman JN, Ahmed R (2004). Proc Natl Acad Sci USA 101, 16004–16009. 110. Tamura S, Tanimoto T, Kurata T (2005). Jpn J Infect Dis 58, 195–207. 111. Gerhard W (2001). Curr Top Microbiol Immunol 260, 171–190. 112. Sangster MY, Riberdy JM, Gonzalez M, Topham DJ, Baumgarth N, Doherty PC (2003). J Exp Med 198, 1011–1021. 113. Johansson BE, Bucher DJ, Kilbourne ED (1989). J Virol 63, 1239–1246. 114. Black RA, Rota PA, Gorodkova N, Klenk HD, Kendal AP (1993). J Gen Virol 74 (Pt 1), 143–146. 115. Holmes EC, Ghedin E, Miller N, Taylor J, Bao Y, St George K, Grenfell BT, Salzberg SL, Fraser CM, Lipman DJ, et al. (2005). PLoS Biol 3, e300. 116. Doherty PC, Turner SJ, Webby RG, Thomas PG (2006). Nat Immunol 7, 449–455. 117. Hogan RJ, Usherwood EJ, Zhong W, Roberts AA, Dutton RW, Harmsen AG, Woodland DL (2001). J Immunol 166, 1813–1822. 118. Liang S, Mozdzanowska K, Palladino G, Gerhard W (1994). J Immunol 152, 1653–1661. 119. Berkhoff EG, Boon AC, Nieuwkoop NJ, Fouchier RA, Sintnicolaas K, Osterhaus AD, Rimmelzwaan GF (2004). J Virol 78, 5216–5222. 120. Lightman S, Cobbold S, Waldmann H, Askonas BA (1987). Immunology 62, 139–144. 121. Turner SJ, Diaz G, Cross R, Doherty PC (2003). Immunity 18, 549–559. 122. Marshall DR, Turner SJ, Belz GT, Wingo S, Andreansky S, Sangster MY, Riberdy JM, Liu T, Tan M, Doherty PC (2001). Proc Natl Acad Sci USA 98, 6313–6318. 123. Kedzierska K, Stambas J, Jenkins MR, Keating R, Turner SJ, Doherty PC (2007). Proc Natl Acad Sci USA 104, 9782–9787. 124. Topham DJ, Tripp RA, Sarawar SR, Sangster MY, Doherty PC (1996). J Virol 70, 1288–1291.

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125. Riberdy JM, Christensen JP, Branum K, Doherty PC (2000). J Virol 74, 9762–9765. 126. Belz GT, Wodarz D, Diaz G, Nowak MA, Doherty PC (2002). J Virol 76, 12388–12393. 127. Ray SJ, Franki SN, Pierce RH, Dimitrova S, Koteliansky V, Sprague AG, Doherty PC, de Fougerolles AR, Topham DJ (2004). Immunity 20, 167–179. 128. Flynn KJ, Riberdy JM, Christensen JP, Altman JD, Doherty PC (1999). Proc Natl Acad Sci USA 96, 8597–8602. 129. Graham MB, Braciale VL, Braciale TJ (1994). J Exp Med 180, 1273–1282. 130. Epstein SL, Lo CY, Misplon JA, Bennink JR (1998). J Immunol 160, 322–327. 131. de Bree GJ, Daniels H, Schilfgaarde M, Jansen HM, Out TA, van Lier RA, Jonkers RE (2007). J Infect Dis 195, 1718–1725. 132. Quinlivan M, Zamarin D, Garcia-Sastre A, Cullinane A, Chambers T, Palese P (2005). J Virol 79, 8431–8439. 133. Richt JA, Lekcharoensuk P, Lager KM, Vincent AL, Loiacono CM, Janke BH, Wu WH, Yoon KJ, Webby RJ, Solorzano A, et al. (2006). J Virol 80, 11009–11018. 134. Vincent AL, Ma W, Lager KM, Janke BH, Webby RJ, Garcia-Sastre A, Richt JA (2007). Vaccine 25, 7999–8009.

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CHAPTER 7

Signaling Pathways in the Host Cell Response to RSV Infection Allan R. Brasier, Ping Liu, Bing Tian & Sanjeev Choudhary

ABSTRACT Respiratory Syncytial Virus (RSV) is a negative-sense RNA virus of the family Paramyxoviridae and is responsible for significant numbers of human diseases. In children, RSV infection is a leading worldwide cause of severe lower respiratory tract infection (LRTI), clinically manifesting as pneumonia and bronchiolitis. RSV produce severe diseases in children with bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis and immunosuppressed states. In natural infections, RSV primarily replicates in the airway mucosa, principally epithelial cells, where it activates an innate immune response via signaling pathways. Although many of the clinical manifestations of RSV are associated with an exaggerated host response to the virus, surprisingly RSV does not induce long term protective immunity even in normal hosts. For these reasons understanding RSV-induced cellular signaling pathways, the genetic response of epithelial cells to RSV, and how this virus modifies the cellular response will provide greater insights into how to therapeutically modify RSV-induced LRTI. This chapter reviews the impact of RSV disease, the processes it uses to replicate in airway epithelial cells, the signaling pathways responsive to this virus, and how nonstructural proteins modulate this process. Specifically we will focus on recent studies describing the role of the pattern recognition receptors (TLR), and the cytoplasmic RNA helicases (RIG-I) in modulating 157

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transcription factor activation including nuclear factor-κ B (NF-κ B) and the interferon response factor (IRF).

1. INTRODUCTION Respiratory Syncytial Virus (RSV) is a negative-sense RNA virus of the family Paramyxoviridae, first isolated in 1956 during an outbreak of coryza in a primate colony. Like many RNA viruses, hRSV is a species adapted to primate hosts and by virtue of its transmission in immunologically naïve infants and children, it occupies a niche difficult to control by vaccination. In susceptible populations, RSV infection produces severe lower respiratory tract infections (LRTI) that manifest clinically as pneumonia or bronchiolitis. After large particle inoculation, RSV initially replicates in nasal epithelial cells, but will spread to epithelial cells of the lower airways. Here, RSV activates and modulates signaling pathways controlling the innate immune response such as the nuclear factor-κ B (NF-κ B) and interferon response factor (IRF) pathways. These signaling pathways control expression of a variety of chemotactic cytokines (chemokines) that participate in leukocyte recruitment, activation of adaptive immune response for clearance of the virus, and also mediate some of the major clinical features of the disease. In this review, we will discuss the impact of RSV disease, relevant features of the RSV life cycle, and the cell signaling response best understood in airway epithelium.

2. RSV DISEASE 2.1 Impact and Magnitude of the Problem RSV is a significant human pathogen that produces a variety of diseases in immunologically competent individuals, including simple upper respiratory tract infection (URI), lower respiratory tract infection (LRTI), and has been implicated as a causative agent in otitis media (OM). RSV infects virtually all children by the age of three years old.1 In the US, RSV is the leading viral pathogen responsible for LRTI in children requiring hospitalization.2 LRTI caused by RSV was

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responsible for 1.7 million office visits and 86 000 hospitalizations, resulting in estimated annual direct medical costs of $394 million in the year 2000. In addition, RSV is the most important cause of hospitalization of infants for any reason,3 and analysis of infant hospitalizations showed that hospitalization rates for bronchiolitis are increasing.4 This latter finding indicated RSV LRTI are having an increasing impact on childhood morbidity.

2.2 Transmission and Disease Spectrum RSV is spread via large particle aerosols, a mode that requires close contact with infected persons or contact with objects contaminated by infected respiratory secretions. The most common means of infection is via mucosal self-inoculation,1,5 where RSV attaches to nasal epithelial cells and initiates intracellular replication. In individuals with normal immune systems, RSV infection is associated with a spectrum of diseases ranging from a simple URI to various other severities such as otitis media,6 tracheobronchitis, or LRTI (pneumonia or bronchitis). RSV infections are associated with exacerbations of asthma in individuals with preexisting atopy, while severe LRTI infection is associated with recurrent wheezing.

2.2.1 Lower respiratory tract infection (LRTI) In immunologically naïve infants, upon their first infection, RSV spreads to the lower respiratory tract one to three days after first developing URI symptoms. The mechanism for lower airway spread of virus is not completely known but may probably include spread via specialized intercellular bridges,5 ciliary entry of columnar epithelial cells,7 and/or cell surface attachment to glycosaminoglycans.8 In the non-ciliated epithelium of the small airways, RSV replication produces pronounced epithelial necrosis and sloughing with excess mucous production. This process results in mucous plugging and obstruction of the small airways,9,10 that accounts for the characteristic clinical features of hyperinflation, atelectasis, and wheezing. In fatal cases, recent studies have shown the presence of viral staining in the alveoli as well as on bronchiolar

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epithelium.11 Additionally, intense perivascular mononuclear infiltration is seen.9,10 Due to its high morbidity and mortality, the pathogenesis of RSV LRTI has been examined in human populations with naturally occurring infections,9–11 animal models,12,13 and in vitro models. Although it was originally thought that RSV LRTI was partially driven by immunopathogenic mechanism,14 recent work has forced a reexamination of this conclusion. In fatal cases of RSV compared to influenza LRTI, RSV infection was found to induce lesser amounts of inflammatory mediators and reduced amounts of activated CD8 T lymphocytes and Natural Killer cells.11 This study also found evidence of intense epithelial apoptosis as indicated by caspase staining, suggesting that LRTI is associated with an inadequate adaptive immune response, robust viral replication and apoptotic crisis.11 In animal models, by contrast, RSV induces production of inflammatory mediators, and surprisingly, much of the clinical manifestations of disease can be attenuated by reducing the host inflammatory response.13 Finally, from our in vitro studies of human airway cells, RSV induces release of inflammatory mediators including networks of cytokines and type I interferons.15–17 Here, high throughput genomics studies have shown that RSV induces expression of diverse classes of chemokines of the C, CC and CXC classes in multiple expression patterns.15 More work will be required to understand the interface between the host inflammatory response, viral proteins, and adaptive immunity in the manifestations of clinical disease.

2.3 Susceptible Populations The severity of RSV infections is influenced by environmental and genetic modifiers. In children, a number of factors for the development of severe LRTI have been identified. These include day care attendance, passive smoke exposure, the presence of school-aged siblings, and birth within six months of RSV season.18 In addition, RSV infection has been found to be more severe in children with cystic fibrosis, congenital heart disease, and bronchopulmonary dysplasia. Children with transplant immunosuppression are at particularly high risk for RSV-induced complications.

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2.3.1 Cystic fibrosis (CF) Children with CF produce thick, tenacious secretions that obstruct the distal airways, creating an environment that ultimately results in colonization with Pseudomonas aeroginosa, recurrent pneumonia and respiratory insufficiency.19 Interestingly, the CF mutation results in an impaired innate immune response that prevents inducible nitric oxide synthase and expression.20 For this reason, infants with CF are prone to RSV infection. Prospective observational studies have shown that this infection results in greater hospitalization rates21 and faster declines in lung function.22 In vitro studies using epithelial cells engineered to have the CF mutation have shown that CF apparently induces activation of the NF-κ B signaling axis,23 a pathway responsible for producing chronic inflammation via enhanced production of the IL-8 chemokine in airway secretions and circulating in serum.24 These early inflammatory events may significantly influence the course of disease.

2.3.2 Congenital heart disease (CHD) and bronchopulmonary dysplasia (BPD) An early observation of children with CHD is that RSV infection is associated with greater rates of intensive care utilization and mortality.25 Although improvements in intensive care therapy have sharply reduced RSV-induced mortality for this group of children, RSV infections still result in more complicated hospitalizations.26 Children with lung immaturity, predominantly in premature infants, are at increased risk for LRTI and hospitalization.1

3. THE LUNG EPITHELIUM IN INNATE IMMUNE RESPONSE TO PATHOGENS The human airway is highly specialized to protect from noxious agents, chemicals, and microorganisms. Specifically, the airway epithelial cell is armed with pathogen-sensing membrane and cell-surface pattern recognition receptors (PRR), such as the Toll Receptor family and cytosolic PRRs, such as the RNA helicases, retinoic acid inducible gene-I (RIG-I) and

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melanoma differentiation antigen-5 (Mda5) to initiate innate immune response upon sensing the presence of viral patterns. The activation of these pathways result in the secretion of mucosal type I interferons (IFNs), responsible for producing an antiviral state in neighboring cells (Figure 1). In addition, inducible expression of antimicrobial peptides, cytokines, chemokines and other metabolites coordinate the complex processes of vascular permeability, paracrine activation, and leukocyte recruitment which are important in the immune response to RSV infection. Working in concert, the alveolar macrophage is a sentinel cell that is also activated by viral products in fundamentally different mechanisms than those activating epithelial cells. Activated macrophages result in local secretion of TNF/IL-1, IFN, and other chemokines important in leukocyte recruitment. In vivo

Figure 1. Cellular signaling interactions in the innate immune response of the distal airways. Shown is a schematic diagram of an alveolus with a resident alveolar macrophage. Upon contact with respiratory viruses, airway epithelial cells initiate host defense through the elaboration of interferons (IFN), or expression of CC and CXC chemokines. In addition, alveolar macrophages synthesize and secrete IL-1 and TNF and other cytokines upon contact with bacteria or respiratory viruses. These cytokines have both autocrine and paracrine functions to initiate antiviral defense mechanisms.

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viral pathogens are involved in a number of direct and paracrine signaling pathways (Figure 1), including the nuclear factor-κ B (NF-κ B), interferon response factor (IRF) and signal transducer and activator of transcription (STAT) pathways discussed in detail below. How cells signal in response to viral infection, and the physiological effects of the signal transduction pathways are major questions actively under investigation.

3.1 Cellular Responses to Viral Infection Epithelial cells are known to have at least two distinct mechanisms for detecting the presence of viral replication. The family of Toll-like receptors (TLRs) were first described as an important sensor for detecting foreign pathogens. Since TLR3 was discovered to bind double-stranded RNA (dsRNA),27 it has been thought that TLR3 plays a critical role in activating innate immunity in response to virus. However, TLR3 deficient mice show no difference in their response to several types of viral infections, a finding that resulted in the discovery of a new family cytoplasmic RNA receptors. These PRRs include the RNA helicases, RIG-I and Mda-5, that play significant roles in response to single-stranded RNA viruses.28,29 The signaling pathways initiated by these two types of sensors have also referred to as “TLR-dependent” or “TLR-independent” pathways; activation of both TLR-dependent and -independent pathways have been reported in viral infection.30–32

3.2 TLR-Dependent Pathways The TLR-family members are PRRs that recognize different types of pathogens by binding LPS, carbohydrate, peptide, double-stranded RNA, single-stranded RNA and DNA. So far, 12 TLR family members have been identified (Figure 2). Interestingly, different TLRs are expressed in different compartments of cells, whose location is dependent on the cell type and state of activation. In immune cells, TLR1, -2, -4, -5 and -6 localize at the cell surface, whereas TLR3, -7, -8 and -9 are contained within the endosomal compartment. In epithelial cells, TLR9 is cell surfaceassociated.33 The localization of TLR3 is dynamic, translocating to the cell surface upon cellular infection.34,35

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Figure 2. The cytoplasmic and endosomal Toll-like receptors (TLRs). A subgroup of the 11 TLRs, notably TLR3, TLR4 and TLR7/9, appear to mediate most of the host cell signaling responses to RSV. Recognizing distinct ligands, TLRs also differentially utilize the primary signaling adapters, Myd88 and TRIF. For simplicity the downstream adapters of Myd88 and TRIF are not illustrated. TLR signaling is upstream of the canonical NF-κ B, and IRF pathways.

TLRs are composed of an ectodomain of ligand binding leucine-rich repeats (LRRs), and a cytoplasmic Toll/interleukin-1 (IL-1) receptor (TIR) domain that interacts with TIR-domain-containing adaptor molecules.36 Two types of downstream adaptors have been identified to associate with different TLRs. Myeloid differentiation primary-response gene 88 (Myd88) interacts with most of the TLRs, except TLR3. Myd88, in turn binds downstream molecules including the IL-1R-associated kinase1 (IRAK1), IL-1R-associated kinase2 (IRAK2) and TNFα receptor-associated factor 6 (TRAF6). The other TLR adaptor, TIR-domain-containing adaptor protein inducing IFNβ (TRIF), is only recruited by TLR3 and TLR4. TRIF interacts with downstream TRAF6 and RIP. Through these adapter pathways, TLR signaling ultimately converges on activation of the latent

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cytoplasmic NF-κ B transcription factor through the Iκ B kinases (Figure 2). In addition, the IRF pathway is activated by a process involving recruitment of TANK-binding kinase 1 (TBK 1) and IKKε to TRIF.34,37 NF-κ B and IRF are key transcription factors in activation of type I IFNs. In response to RSV infection, TLR3, -4, -7/8 and -9 have been reported to be RSV sensors in different cell types. In airway epithelial cells, TLR3 and -4 have been shown to interact with RSV, whereas TLR7/8 and TLR9 functions in plasmacytoid dendritic cells (pDCs).

3.2.1 The TLR4 pathway TLR4 has been reported to recognize bacteria endotoxin (lipopolysaccharide, LPS) and RSV F protein.38 Normally, TLR4 is expressed at very low levels on the cell surface and as a result, epithelial cells are normally not LPS responsive. However, RSV infection increases both expression and cell surface translocation of TLR4.20 Clinically TLR4 may play a role in protection against LRTI because non-synonmous TLR4 polymorphisms were significantly overrepresented in children with RSV LRTI.39 Studies of TLR4 function in RSV-infected animal models have been controversial and strain dependent.40 In a separate study investigating NF-κ B activation in the airways of RSV infected BALB/c mice, the role of alveolar macrophages (AMs) and TLR4 were described in mediating two different patterns of NF-κ B activation. The first NF-κ B response occurs early after RSV inoculation, is AM- and TLR4-dependent, and is viral replication-independent, whereas the second response involves epithelial cells and/or inflammatory cells, is TLR4-independent, and requires viral replication.12 Studies of TLR4 function in response to other paramyxoviruses have produced similar conclusions.41,42 Together, these results suggest that TLR4 may not play a major role in protection against paramyxovirus infection. However, the association of TLR4 polymorphisms are intriguing, and suggest more work in humans will be required to resolve this issue.

3.2.2 The TLR3 pathway In airway epithelial cells, RSV replication is necessary for inducing downstream signaling.43 Given that RSV may produce dsRNA during its

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replication, the role of TLR3 has been studied. Downregulation of TLR3 expression decreased synthesis of CXCL10 and CCL5 in response to RSV infection but did not significantly reduce levels of IL-8,31 suggesting TLR3 plays a role in host signaling. Our group found most of TLR3 in unstimulated epithelial cells is contained in the endosomal compartment, whereas after viral infection, TLR3 expression is upregulated and redistributed to the cell surface.30,44 We also found that TLR3 expression is a paracrine effect-dependent IFN-β signaling.30 This result not only indicates the essential role of IFN-β for TLR3 induction, but also suggests the existence of other intracellular viral sensors at the early time point for RSV infection. In animal studies of TLR3−/− mice, an accumulation of eosinophils was found after RSV exposure. TLR3−/− mice also produced significant increases in Th2-type cytokines, IL-5, and IL-13, as well as an increase of mucus production compared with wild-type mice after RSV infection. However, RSV clearance is apparently not reduced in TLR3−/− mice.32 Together, these studies suggest that the anti-RSV function of TLRs may not be as important as initially expected, and the existence of a new signaling pathway to detect RSV infection was indicated.

3.3 DExD/H Box RNA Helicases, (“TLR-Independent” Pathways) In 2004, the DExD/H box RNA helicase RIG-I was identified as an essential regulator for dsRNA-induced signaling.45 DExD/H box helicases have the potential to unwind dsRNA, and RIG-I in particular, contain two caspase recruitment domains (CARD) that interact with downstream molecules to activate the transcription factors NF-κ B and IRF-3. The RIG-I helicase domain contains ATPase activity and is responsible for dsRNA or ssRNA recognition.46 Melanoma differentiation-associated gene-5 (Mda5), is another DExD/H box RNA helicase containing tandem CARD domains.47 It is now known that RIG-I and Mda5 recognize different virus types, with RIG-I responding to most ssRNA viruses, whereas Mda5 responds to picornavirus.28 Recently, another IFN-inducible DExD/H box helicase, LGP2, has also been identified.48 LGP2 lacks the CARD region and may act as a feedback regulator of RIG-I and Mda5.

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Interestingly, all known DExD/H box RNA helicases converge on the same downstream adaptor (Figure 3) known as mitochondrial antiviral signaling (MAVS). MAVS contains an NH2-terminal CARD domain, proline-rich domain and a COOH-terminal transmembrane domain (TM).49 Functional analysis of this molecule has shown that the CARD domain is an essential motif to interact with CARD domains in RIG-I or Mda5 and the TM domain is critical for mitochondrial location. Deletion of either

Figure 3. Intracellular pattern recognition receptors (PRRs) and downstream signaling pathways in epithelial cells. Shown is a schematic diagram of a cell infected with RNA virus. Upon binding viral RNA (vRNA), the cytosolic PRRs associate on the surface of mitochondria with mitochondrial activator of viral signaling (MAVS) via caspase recruitment domains (shaded). This association results in the activation of two parallel signaling pathways, mediated by the Iκ B kinase (IKK) and the atypical IKKs (TBK1/ IKKι). As a result enhanced nuclear translocation of the nuclear factor-κ B (NF-κ B) and interferon response factor (IRF) occurs, resulting in activation of IFN and chemokine gene expression.

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the CARD or TM domains result in a molecule being unable to initiate downstream signaling. In 2006, our group first reported that RIG-I is the intracellular RSV sensor. In this study, siRNA-mediated knockdown of RIG-I significantly inhibited RSV-induced NF-κ B and IRF3 activation at early points of infection, but not at later times.30 Consistent with this finding, RSV-induced IFN-β , IP-10, CCL-5 and ISG15 expression levels were decreased in RIG-Isilenced cells during the early phase of infection. We concluded that RIG-I is the primary anti-RSV sensor in airway epithelial cells, and in the later time of infection, other antiviral signaling including TLR3 might be activated.30 Consistent with our study, Seya and colleagues proved that RSVinduced IFN-β production is initiated by RIG-I but not the TLR3 pathway.50 In summary, the TLR-independent pathway mediated by RIG-I as an initial intracellular sensor for detecting RSV in airway epithelial cells. During the later stage of RSV infection, other PRRs, including TLRs, may be involved.

3.4 The NF-κ B Activation Pathways NF-κ B is a family of cytoplasmic transcription factors that play a central role as a mediator of inflammation. The NF-κ B family includes the transactivating subunits, RelA, RelB, c-Rel, and the post-translationally processed DNA binding subunits, NF-κ B1 (p50) and NF-κ B2 (p52).51 The NF-κ B dimers are cytoplasmically sequestered by interacting with a group of inhibitory ankyrin repeat-containing proteins, collectively referred to as Iκ Bs (Iκ Bα, Iκ Bβ , Iκ Bε , p100 and p105), whose phosphorylationcoupled proteolytic degradation are required for NF-κ B release.52 Using a tightly regulated dominant-negative inhibitor of the canonical NF-κ B pathway, we have recently identified 144 NF-κ B dependent genes that were altered by RSV in epithelial cells.53 These genes encoded a wide range of functional proteins including chemokines, NF-κ B isoforms, as well as a spectrum of diverse intracellular function.53 In follow up studies in a mouse model of RSV infection, we have shown that intranasal administration of a specific cell permeable Iκ B kinase inhibitor, NF-κ B DNA binding activity, chemokine gene expression, and airway inflammation were markedly reduced in response to RSV infection,13 indicating that

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NF-κ B plays a central role in viral induced airway inflammation. Recently it has been appreciated that NF-κ B activation can be controlled by at least three major pathways: the RIG-I-MAVS pathway (discussed above), the “canonical” and the “non-canonical” pathways.

3.4.1 Canonical/classical pathway of NF-κ B activation The canonical (or so-called “classical”) pathway is rapidly and transiently activated by various stimuli, such as inflammatory cytokines (TNFα and IL1), mitogens and DNA damage (Figure 4, Ref. 54). In airway

Figure 4. The canonical NF-κ B activation pathway. The canonical NF-κ B activation pathway is coupled to the cytokine receptors, TNF and IL-1. These receptors initiate the formation of a submembranous complex containing TNF receptor-associated factors (TRAF) encoding ubiquitin ligase activity. This results in the recruitment of the MAP3K members, notably TGFβ activated kinase (TAK). The activated TAK complex activates the IKK multiprotein complex, representing the effector kinase controlling of Iκ Bα phosphorylation and degradation. Once liberated NF-κ B enters the nucleus to activate target genes, including IL-8, IL-6, Gro-β and others.103

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epithelial cells, this pathway is likely to be activated in a paracrine response to macrophage-mediated TNF/IL-1 release (Figure 1). In the example of TNF stimulation shown in Figure 4, the activated TNF receptor-recruits TNF receptor-associated adapter proteins. This process leads to the phosphorylation of IKK, a multiprotein complex containing at least two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ .55 Another protein rich in glutamine, lysine and serine (ELKS) functions by recruiting Iκ B to the IKK complex for phosphorylation.56 Activation of IKK complex leads to Iκ Bα phosphorylation at specific N-terminal serine residues targeting them for ubiquitination by ubiquitin ligase complex and subsequent degradation by the 26S proteosome.57 This process releases sequestered Rel A•NF-κ B1 to undergo modification and translocation to the nucleus where it can bind NF-κ B specific promoter sequence and regulate expression of NF-κ B dependent genes. We have previously reviewed the genetic network under NF-κ B control in canonical and RSV infection.58,59

3.4.2 Non-canonical pathway of NF-κB activation In contrast, the non-canonical pathway liberates Rel B•NF-κ B2 complexes into the nucleus (Figure 5). Recently, it has been observed that the noncanonical pathway can be activated in response to specific stimuli, including lymphotoxin β ,60,61 CD40 ligand,62 DNA virus infection,63 and B-cell activating factor [BAFF, Ref. 64]. Interestingly, neither IKKα or IKKβ, key regulators of the canonical pathway, are required for activation of the non-canonical pathway.61,65 Rather, a NIK and IKKα kinase activates post-translational processing of the NF-κ B2 precursor, p100, into the 52 kDa active DNA binding isoform. Newly formed 52 kDa NF-κ B2 then dimerizes with cytoplasmic Rel B and translocates into the nucleus. In this pathway, NIK serves to activate IKKα as well as provide a docking site to recruit both p100 NF-κ B2 and IKKα into a complex.63 NIK therefore is an essential component of the non-canonical NF-κ B activation pathway. Interestingly, in our genomic analysis of RSV infection, one of the genes found to be highly inducible in response to RSV infection was NIK itself. On further investigating the role of NIK and the activation of noncanonical NF-κ B activation pathway in RSV-induced inflammation lead to the conclusion that RSV infection rapidly activates the non-canonical

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Figure 5. The non-canonical NF-κ B activation pathway. The non-canonical NF-κ B activation pathway is coupled to a distinct set of receptors related to the TNFR; shown is the response to B-cell activating factor (BAFF). Downstream, the NF-κ B inducing kinase (NIK) and IKKα are activated to phosphorylate the 100 kDa precursor of NF-κ B2. As a result, the ankyrin repeat-containing COOH-terminus of the NF-κ B2 precursor is cleaved, liberating the 50 kDa DNA binding forms of NF-κ B2 and RelB. The NF-κ B2•RelB transcription complex activates a distinct set of target genes, including stromal derived factor (SDF), EB11-ligand chemokine (ELC) and BAFF.

NF-κ B activation pathway prior to the more potent canonical pathway activation. This appears to be through a novel mechanism involving the induction of NIK kinase activity, expression, and nuclear translocation of a ternary complex with IKKα and processed NF-κ B2/p52.66 The role of the nuclear NIK-IKKα complex will require further investigation.

3.5 The Interferon Response Factor (IRF) Pathway In addition to the NF-κ B pathway, virus infection also activates the IRFs, a family of transcription factors controlling type I IFN expression, a group

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of important cytokines inhibiting viral replication in airway epithelial cells.67 So far, nine human IRFs have been reported (IRF1-9); of these, IRF-3, -1 and -7 are the key regulators of type 1 IFN gene expression in response to viral infection.68,69 IRF3 is constitutively expressed in the cytoplasm. In response to viral infection, IRF3 is phosphorylated, inducing dimerization and nuclear translocation.70–72 By contrast, IRF7 is expressed at a low level in most of cell lines and is strongly induced in response to viral infection. The virusinduced phosphorylation, dimerization and nuclear translocation of IRF7 are similar to the activation of IRF3.68,73 As IRF7 is strongly induced by type I IFN through an ISRE binding site in its promoter,74 a two-phase model of secretion of type I IFN in response to viral infection has been proposed. The first phase involves the detection of virus by cytoplasmic PRRs, activation of IRF3 and initial production of IFNβ /IFNα 4. Activated IRF3 or the secreted type I IFN increases IRF-1 and -7 transcription. The second phase includes the activation of IRF-1 and -7 and the induction of other type I IFNs. The IRF-controlled second phase of IFN secretion ensures a maximum antiviral response from host cells.75 RSV-induced IRF activation was described by Casola and colleagues, showing IRF-1 and -7 induction in response to RSV infection. They showed that signal transducers and activators of transcription (STAT) was a necessary factor for IRF induction. In addition, they also demonstrated that RSV-induced reactive oxygen species (ROS) is required for the activation of STAT. Using NADPH oxidase inhibitors, BHA and DPI, they successfully blocked the production of ROS, inhibited the phosphorylation of STAT, and consequently downregulated the induction of IRF-1 and -7.76

3.6 The Janus Activated Kinase (Jak)-Signal Transducer and Activator of Transcription (STAT) Pathway In vitro, RSV replication in epithelial cells is a potent inducer of Type I interferon (IFN) production,16,17,77 an antiviral cytokine that plays a central role in mucosal immunity.78 The Type I IFN primarily produced by epithelial cells is IFN-β, a highly inducible cytokine that works in a paracrine manner to limit viral replication. Upon binding target cells, IFN-β induces

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gene expression programs to produce an antiviral state through several mechanisms. One involves enhancing peptide production from intracellular pathogens by inducing 26S proteasome catalytic activity by expression of LMP2, and inducing cytosolic to ER transport by expression of TAP1 and TAP2. Together, coordinate expression of the LMP2/TAP genetic element results in the extracellular display of pathogen-derived peptides within the context of the MHC I. As a result, cytotoxic CD8-expressing T lymphocytes can then recognize and clear the infected cells. The second mechanism is to induce expression of various IFN stimulated genes, including MxA, oligoadenylate synthetase, Protein Kinase R, speckled protein-100, and others that produce an antiviral state by inhibition of viral translation and replication.79 Because of its potent antiviral effects, RSV has adapted specific proteins that serve to antagonize IFN production. For example, using recombinant virus, several groups have shown that RSV non-structural proteins antagonize the activation of IRF3, one of the major activators of IFN production.80,81 The role and elements of IFN-induced signaling pathways are intensively being investigated.82 Type I IFNs signal cells by activating receptor (IFNAR)-associated tyrosine kinases, known as the janus kinases (Jak)-1 and Tyk2 kinases, followed by recruitment of the cytoplasmic STAT-1 and -2 isoforms, and their phosphorylation on critical Tyrosine residues 701 and 689, respectively (Figure 6). This modification produces intermolecular SH2-SH3 domain interactions, which results in STAT homoand hetero-typic association, forming distinct types of complexes. Newly created STAT1 homodimers, STAT1•2 heterodimers and a complex of STAT1•2 heterodimers and IRF9, termed ISGF3, are transported into the nucleus, where they bind high affinity sequences in target genes, recruiting p300/CBP coactivators, producing chromatin and factor-induced acetylation, and inducing gene expression.83 Subsequently, STATs are dephosphorylated and exported from the nucleus.82 Although the classic view of STAT activation involves kinase activation, the mechanisms by which RSV induces STAT activation appear more complex. In earlier studies, we have shown that RSV replication is a rapid inducer of ROS species, an event associated with accumulation of tyrosine phosphorylated STAT, without detectable changes in Jak/Tyk kinase activity.84

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Figure 6. The IFN activated Jak-STAT pathway. The cell surface receptor type I IFN receptor is composed of two chains, α and β. Upon activation, the IFN receptor signals through the janus kinase (jak) and tyrosine kinase (tyk)-2. These proteins induce tyrosine phosphorylation of the STAT1 and 2, binding IRF9, resulting in formation of the ISGF3 complex.

4. MECHANISMS FOR RSV INDUCED MODULATION OF THE INNATE IMMUNE RESPONSE Because of the potent inhibitory roles of type I IFNs, RSV has adapted to modulate these signaling pathways in ways not fully understood yet (Figure 7). Using reverse genetics, the Collins group has shown that the NS1/NS2 proteins are potent inhibitors of IRF3 activation and type I IFN gene expression.17,81 In related studies, Conzelmann and colleagues also demonstrated that the NS1 and NS2 protein from bovine respiratory syncytial virus (bRSV) inhibited type I interferon production. They observed a significant increase of phosphorylation of IRF3 as well as its transcriptional activity in the cells infected by mutant recombinant bRSV lacking

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Figure 7. Sites of signaling inhibition by RSV proteins. Schematic diagram of the known points of inhibition of signaling by RSV proteins expressed late in infection.

both NS1/NS2 genes, but not by wild-type bRSV. This indicated that the NS1 and NS2 protein of RSV are negative regulators for IRF pathway by inhibiting IRF3 phosphorylation.80,85 These studies also discovered that the IRF3 antagonism was most pronounced when both NS1 and NS2 are expressed, and that there is evidence for species adaptation for the IRF antagonism. Finally, NS2 plays additional roles, it induces proteasomal STAT2 degradation as well as antagonizes IFN signaling responses.86,87 RSV-infected cells release a soluble form of the G glycoprotein (sG), a protein that has immunomodulatory properties. This may be of importance because the RSV G protein binds to the fractalkine (CX3CL1) chemokine receptor as a form of molecular mimicry.88 More work will be needed to understand the influence of RSV on non-epithelial immune cells, and its effect on disease pathogenesis.

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5. RSV SIGNALING IN IMMUNE CELLS 5.1 Alveolar Macrophages/Monocytes Alveolar macrophages(AMs), along with respiratory epithelial cells, are the first cells to encounter RSV in the airways.89 Here, AM play an important part in controlling the immune response to viral infection through multiple mechanisms — pathogen phagocytosis, cytokine production, direct interaction with helper and cytotoxic T-cells, and antigen presentation.90 Studies in vitro have indicated that AM and epithelial cells respond to RSV infection in fundamentally distinct ways, in kinetics, magnitude and TLR utilization. As discussed earlier, in vivo, AM mediate a rapid viral replication independent activation of airway NF-κ B, whereas epithelial cells and/or inflammatory cells, contribute a second, more potent contribution to NF-κ B activation that requires viral replication.12 Mechanistically, Kurt-Jones et al. discovered that the RSV fusion (F) protein induces proinflammatory cytokines in AM/monocytes via the TLR4 PRR.38 Because TLR4 is not strongly expressed in epithelial cells, this pathway is not a major activator of epithelial cell signaling, thereby accounting for the lack of viral replication independent signaling in this cell type. As a result RSV exposed AM secrete IL-1ß, IL-6, IL-8, IL-10, IL-12 and TNF,91 whereas RSV-infected respiratory epithelial cells secrete 17 distinct groups of CC, CXC and CX3C cytokines.15 In addition, it has been recently reported that RSV induces the expression of the proinflammatory cytokine IL-15 in the human monocytic cells92 in a mechanism involving PKC-α /β pathway converging on NF-κ B/RelA. These investigators also found IL-15 gene upregulation occurs as a result of cell surface interaction with virus particles from infectious and inactivated preparations (i.e., UV- and heatinactivated) comparatively with the mock-treated cells. These data indicate that RSV replication is not necessary for the induction of IL-15 as well. Comparative studies on mononuclear cells from neonatal cord blood and adults have shown that production of IL-6 and TNF-α in response to RSV infection is less efficient in neonates compared to adults.93 A separate study suggested that a low monocyte IL-12 response during

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initial RSV infection might adversely affect the clinical outcome of patients with severe RSV bronchiolitis.94 These observations may help to explain the mechanism behind the more severe disease seen in young infants.

5.2 Eosinophils Eosinophils have been of interest in RSV LRTI for several reasons. Pulmonary eosinophilia is a characteristic finding in children who developed exaggerated disease after exposure to formalin inactivated vaccine,95 and eosinophil degranulation products have been found in children with naturally occurring RSV infection.96 This phenomenon has been observed also in mouse model of RSV infection as well, although there is some controversy on this point. Eosinophils express MHC-I and MHC-II, antiviral ribonucleases, cytokines, chemokines, and can engage T cells, supporting the concept that they may contribute to the regulation of both innate and adaptive immunity. Transcripts encoding TLR1, -4, -7, -9, and -10 are also expressed constitutively by eosinophils.97 Eosinophils are able to recognize RSV-associated molecular patterns in a TLR-MyD88–dependent manner and to orchestrate an innate antiviral host response to RSV. Furthermore, by infecting the airways of hypereosinophilic (IL-5 Tg) mice with RSV or by the adoptive transfer of MyD88-sufficient, but not MyD88-deficient eosinophils to wild-type (WT) mice, it has shown that eosinophils mediate accelerated viral clearance via MyD88-dependent pathways, leading to the suppression of RSV-induced pathology, including AHR.98 These results suggest that eosinophils contribute to antiviral immunity and play a beneficial role in limiting RSV-induced lung dysfunction. Airway eosinophils function as effector cells in airway allergic inflammation by releasing basic granule proteins, including major basic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, and eosinphil cationic protein (ECP). In RSV-infected children, the levels of ECP are highly increased in bronchoalveolar lavage fluid.96 Eosinophil chemotaxis is mediated in part via elaboration of eosinophilic chemokines, such as M1P-1α and RANTES secreted primarily by lower respiratory epithelial cells.15,99 Moreover, RSV infected epithelial cells produce CD18,

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a factor important in stimulating eosinophil degranulation of ECP.100 These interrelationships may be highly relevant to the pathogenesis of RSV LRTI.

5.3 Dendritic Cells Dendritic cells (DCs) are antigen-presenting cells that determine T-cell differentiation and play an important role in both allergy and viral infection. In vivo, RSV increases the numbers of plasmacytoid DCs (pDCs) in the lung and draining lymph nodes, where their action may downregulate Th1 and augment Th2 responses101 as well as induce the expression of inflammatory and immunomodulatory cytokines, including TNF-α, IL-6, IL-1β, IL-10, and IL-12p70.102 Other studies have found that RSV can productively replicate in monocyte-derived DC, which severely impairs their capacity to stimulate CD4+ T-cell proliferation.102 This is an actively expanding field, and the signaling mechanisms through which these phenomena occur are only beginning to be understood.

6. SUMMARY AND FUTURE DIRECTIONS RSV is a major human pathogen that activates and modulates intracellular signaling pathways in host sentinel cells of the airways. Investigated most systematically in airway epithelial cells, RSV infections induces myriad effects on signaling, affecting innate immune pathways including the NF-κ B, IRF, and Jak-STAT pathways. These signaling interactions are mediated by PRR including DExD/H box helicases and TLRs. Our findings indicate that the antiviral response is closely coordinated between the helicase and TLR pathways in epithelial cells and suggest that innate signaling between cytoplasmic RNA helicase signaling and TLR are interdependent. Further work needs to be done to define the intermediate regulators of the NF-κ B and IRF pathways and determine their relationship to antiviral immunity, inflammation and disease in vivo. RSV has evolved a number of strategies to modulate the inflammatory pathways, whose precise molecular mechanisms are still not understood. Finally, the role of TLR signaling in mediating human disease still requires elucidation.

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This work holds promise for the modulation of the clinical manifestations of RSV LRTI and could significantly impact the morbidity of this infectious agent.

ACKNOWLEDGMENTS Work in this lab was supported by NIH grants RO1 AI40218, PO1 AI062885 (to A.R.B.), NHLBI contract BAA-HL-02-04 (A. Kurosky), and AHA (S.C). Core Laboratory support was from NIEHS P30 ES06676 (to J. Halpert, UTMB).

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54. Pahl H (1999). Oncogene 18, 6853–6866. 55. Schmidt C, Peng B, Li Z, Sclabas GM, Fujioka S, Niu J, Schmidt–Supprian M, Evans DB, Abbruzzese JL, Chiao PJ (2003). Molecular Cell 12, 1287–1300. 56. Sigala JLD, Bottero V, Young DB, Shevchenko A, Mercurio F, Verma IM (2004). Science 304, 1963–1967. 57. Ghosh S, Baltimore D (1990). Nature 344, 678–682. 58. Tian B, Brasier AR (2003). Recent Prog Horm Res 58, 95–130. 59. Tian, B, Brasier, AR. (2005). Microarrays and Transcription Factor Networks. Landes Bioscience. 60. Dejardin E, Droin NM, Delhase M, Haas E, Cao Y, Makris C, Li ZW, Karin M, Ware CF, Green DR (2002). Immunity 17, 525–535. 61. Xiao G, Harhaj EW, Sun SC (2001). Molecular Cell 7, 401–409. 62. Coope HJ, Atkinson PG, Huhse B, Belich M, Janzen J, Holman MJ, Ley SC (2002). EMBO J 21, 5375–5385. 63. Xiao G, Fong A, Sun SC (2004). J Biol Chem 279, 30099–30105. 64. Claudio E, Brown K, Park S, Wang H, Siebenlist U (2002). Nat Immunol 3, 958–965. 65. Senftleben U, Cao Y, Xiao G, Greten FR, Krahn G, Bonizzi G, Chen Y, Hu Y, Fong A, Sun SC, et al. (2001). Science 293, 1495–1499. 66. Choudhary S, Boldogh S, Garofalo RP, Jamaluddin M, Brasier AR (2005). J Virol 79, 8948–8959. 67. Kim TK, Maniatis T (1997). Molecular Cell 1, 119–129. 68. Marie I, Durbin JE, Levy D (1998). EMBO J 17, 6660–6669. 69. Matsuyama T, Kimura T, Kitagawa M, Pfeffer K, Kawakami T, Watanabe N, Kundig TM, Amakawa R, Kishihara K, Wakeham A, et al. (1993). Cell 75, 83–97. 70. Lin R, Heylbroeck C, Pitha PM, Hiscott J (1998). Mol Cell Biol 18, 2986–2996. 71. Sato M, Tanaka N, Hata N, Oda E, Taniguchi T (1998). FEBS Lett 425, 112–116. 72. Yoneyama M, Suhara W, Fukuhara Y, Fukuda M, Nishida E, Fujita T (1998). EMBO J 17, 1087–1095. 73. Sato M, Hata N, Asagiri M, Nakaya T, Taniguchi T, Tanaka N (1998). FEBS Lett 441, 106–110. 74. Lu R, Au WC, Yeow WS, Hageman N, Pitha PM (2000). J Biol Chem 275, 31805–31812.

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75. Sharma S, Tenoever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J (2003). Science 300, 1148–1151. 76. Liu T, Castro S, Brasier AR, Jamaluddin M, Garofalo RP, Casola A (2004). J Biol Chem 279, 2461–2469. 77. Garofalo R, Mei F, Espejo R, Ye G, Haeberle H, Baron S, Ogra PL, Reyes VE (1997). J Immunol 157, 2506–2513. 78. Pestka S, Krause CD, Walter M (2004). Immunol Rev 202, 8–32. 79. Der SD, Zhou A, Williams BRG, Silverman RH (1998). Proc Natl Acad Sci USA 95, 15623–15628. 80. Bossert B, Marozin S, Conzelmann KK (2003). J Virol 77, 8661–8668. 81. Spann KM, Tran KC, Collins PL (2005). J Virol 79, 5353–5362. 82. Schindler C, Levy DE, Decker T (2007). J Biol Chem 282, 20059–20063. 83. Horvai A, Xu L, Korzus e (1997). Proc Natl Acad Sci USA 94, 1074–1079. 84. Liu T, Castro S, Brasier AR, Jamaluddin M, Garofalo RP, Casola A (2003). J Biol Chem 279, 2461–2469. 85. Bossert B, Conzelmann KK (2002). J Virol 76, 4287–4293. 86. Ramaswamy M, Shi L, Monick MM, Hunninghake GW, Look DC (2004). Am J Respir Cell Mol Biol 30, 893–900. 87. Lo MS, Brazas RM, Holtzman MJ (2005). J Virol 79, 9315–9319. 88. Tripp RA, Jones LP, Haynes LM, Zheng H, Murphy PM, Anderson LJ (2001). Nat Immunol 2, 732–738. 89. Kimpen JL (2001). Am J Respir Crit Care Med 163, S7–S9. 90. Gordon SB, Read RC (2002). Br Med Bull 61, 45–61. 91. Becker S, Quay J, Soukup J (1991). J Immunol 147, 4307–4312. 92. Ennaciri J, Ahmad R, Menezes J (2007). J Leukoc Biol 81, 625–631. 93. Matsuda K, Tsutsumi H, Sone S, Yoto Y, Oya K, Okamoto Y, Ogra PL, Chiba S (1996). J Med Virol 48, 199–203. 94. Bont L, Kavelaars A, Heijnen CJ, van Vught AJ, Kimpen JL (2000). J Infect Dis 181, 1772–1775. 95. Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, Parroty RH (1969). Am J Epidemiol 89, 422–434. 96. Garofalo R, Kimpen JLL, Welliver RC, Ogra PL (1992). J Pediatrics 120, 28–32. 97. Nagase H, Okugawa S, Ota Y, Yamaguchi M, Tomizawa H, Matsushima K, Ohta K, Yamamoto K, Hirai K (2003). J Immunol 171, 3971–3982.

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98. Phipps S, Lam CE, Mahalingam S, Newhouse M, Ramirez R, Rosenberg HF, Foster PS, Matthaei KI (2007). Blood 110, 1578–1586. 99. Olzewska B, Casola A, Saito T, Alam R, Crowe S, Mei F, Ogra PL, Garofalo R (1998). J Virol 72, 4756–4764. 100. Olszewska-Pazdrak B, Pazdrak K, Ogra PL, Garofalo R (1998). J Immunol 160, 4889–4895. 101. Qureshi MH, Durre K, Yang W (2007). J Immunol 178, S25–S2a. 102. Guerrero-Plata A, Casola A, Suarez G, Yu X, Spetch L, Peeples ME, Garofalo RP (2006). Am J Respir Cell Mol Biol 34, 320–329. 103. Tian B, Nowak D, Brasier AR (2005). BMC Genomics 6, 137.

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CHAPTER 8

Protective and Pathologic Host Responses to Pulmonary Respiratory Syncytial Virus Infection Dennis M. Lindell & Nicholas W. Lukacs

ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infection in infants, with nearly all infants infected by the end of their second year. Infections with RSV do not confer completely protective immunity, and reinfections throughout life are common. Severe RSV infection in infancy may predispose individuals to later development of asthma. Additionally, RSV infection can serve to exacerbate existing asthmatic diseases. RSV-induced bronchiolitis is characterized by intense peribronchial and perivascular mononuclear inflammation, often including eosinophilia. In the airways, mucus hyper secretion and epithelial damage result in mucus plugging and edema. Susceptibility to disease may be related to altered innate immune function. Viral recognition by epithelial cells, antigen presenting cells, and other innate immune cells via pattern recognition receptors results in chemokine and cytokine production. This in turn, shapes the development of adaptive immunity. The development of adaptive immunity contributes to viral clearance, but also enhances immunopathology associated with RSV-induced disease.

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1. INTRODUCTION Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infection in infants, with nearly all infants infected by the age of three.1 Premature infants are at higher risk of contracting severe disease. Prophylactic passive antibody therapy is beneficial in these children, but carries with it a high cost of therapy.2 Infections with RSV do not confer completely protective immunity, and reinfections throughout life are common. These vary, but can have severe consequences for individuals with compromised immune function, such as immunosuppressed transplant recipients, and the elderly.3 Additionally, RSV is becoming increasingly recognized as an important pathogen for individuals with chronic lung disease such as chronic obstructive pulmonary disease (COPD) and asthma.3 Evidence suggests that severe infection in infancy may predispose individuals to the later development of asthma.4 Furthermore, RSV infection can serve to exacerbate existing asthmatic disease. Cumulatively, RSV accounts for at least 17 000 deaths per year in the US, although RSV infection in adults in not routinely documented.5 RSV-induced bronchiolitis is characterized by intense peribronchial and perivascular mononuclear inflammation, often including eosinophilia. In the airways, mucus hypersecretion and epithelial damage result in mucus plugging and edema. Susceptibility to disease may be related to altered innate immune function. Toll-like receptors are a family of pattern recognition receptors that recognize components common to microbes. In particular, TLR4 has been implicated in the recognition and early response to RSV infection, both in mice and in humans. Chemotactic cytokines (chemokines) promote differential leukocyte recruitment, and evidence suggests that chemokine production by structural and innate immune cells of the lungs controls the type of adaptive immune response generated. The development of adaptive immunity to RSV contributes to viral clearance, but also enhances immunopathology associated with disease. Evidence from humans and animal models support the concept that Th2 cytokines, especially IL-13, play a role in promoting disease. Both CD4+ and CD8+ T cell responses are generated in response to RSV infection. The preponderance of the evidence suggests that CD8+ T cells are largely protective, whereas, CD4+ T cells contribute predominantly to pathology. Considerable

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efforts have been made to understand the basis for the unfortunate history of RSV vaccination. Recipients of alum-precipitated formalin-inactivated RSV vaccines became severely ill (and some even died) upon subsequent exposure to the virus, thus nullifying any benefits from enhanced clearance of the virus. A better understanding of the host response to RSV is essential for the development of RSV vaccines or other therapeutics. This chapter will summarize and discuss the literature regarding host response to RSV infection, including viral recognition, innate immunity, and the establishment of adaptive immunity.

2. CLINICAL IMPACT Respiratory syncytial virus (RSV) is an important respiratory pathogen of infants and young children worldwide. In the United States, RSV infects nearly 70% of infants in their first year of life and virtually all children by the age of three.1 RSV-induced disease is a leading cause of mechanical ventilation and respiratory failure in infants in the United States, with approximately one to two percent of RSV infected children requiring hospitalization. In temperate climates, RSV-induced disease is a seasonal epidemic, typically beginning in late fall and lasting through winter. In the tropics, RSV-induced disease can be more variable, but often coincides with seasonal heavy rains. Immunity to RSV is incomplete, with reinfections common throughout life, especially in young children. Immune responses in healthy adults tend to be self-limiting, but are comparable to, or exceed the duration and severity of influenza.6 Some epidemiologic evidence has suggested that severe RSV infection may predispose individuals to the subsequent development of asthma. Furthermore, RSV is increasingly recognized as an important cause of asthma exacerbation, in elderly and patients with chronic obstructive pulmonary disease (COPD), as well as in immunosuppressed transplant recipients.3 In the US, although morbidity in infants in the US has a significant impact on childhood hospitalization, RSV-associated death is highest among the elderly.5 Thus, an unrealized impact of RSV on health in the adult population is becoming apparent. No vaccine for RSV is currently available. The standard therapy for RSV infection is largely supportive. Severe cases may involve admission

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to the intensive care unit and mechanical ventilation. Treatment with corticosteroids or the broad-spectrum antiviral Ribavirin do not shorten the duration of acute illness, and their efficacy in reducing severe disease is questionable. A recent therapeutic option is the use of prophylactic passive monoclonal antibody therapy (palivizumab/Synagis). Although some studies of palivizumab have reported decreased hospitalization in susceptible populations, the cost of therapy is prohibitively expensive.2

3. INNATE IMMUNE SYSTEM 3.1 Anatomical Issues RSV is acquired via inhalation into the upper airways. The virus infects epithelial cells by entering via the direct fusion with cell membranes. The virus uncoats in the cytosol and replicates, generating infectious virus via budding and release from host epithelial cells. Additionally, RSV may spread between neighboring cells via the formation of syncytia. Damage to host epithelial cells results in epithelial sloughing leading to the release of damaged epithelial cells into the airways. One of the pathologic manifestations of RSV infection is often mucus overproduction. Normally, mucus provides a non-specific barrier to viral infection, with removal aided via the mucocilliary elevator. However, mucus can also provide a source of moisture that protects infectious virus from the environment, making transmission more likely. Other non-specific immune mechanisms such as defensins, and (in the lower airways) surfactant can limit viral entry or spread. If the virus colonizes the upper airways effectively, it can spread to the lower airway resulting in more severe bronchiolitis. Some risk factors for severe RSV infection are anatomic and physiologic, rather than immunologic. Very young infants, especially premature children, have incompletely developed smaller airways, as lungs are among the last organs to become fully functional during gestation. Smaller airways are thought to be a primary contributing factor, as small changes in airway diameter have dramatic consequences on the ability to move air. This is complicated by the apparent ability of RSV to induce airway constriction and obstruction with mucus overproduction. Infant lungs are less effective at producing surfactant, which facilitates gas

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exchange in the alveoli and serves as a non-specific barrier to infection. Several studies suggest that surfactant plays an important protective function against RSV. Polymorphisms of surfactant protein A, B, and C genes are associated with severe RSV infection.7 SP-A and D can function as opsonins for RSV clearance by phagocytic cells including alveolar macrophages, as well as having neutralization function. RSV infection may lead to dysregulated surfactant production, as altered surfactant is present during severe RSV infection.8 In vitro, surfactant proteins A and D bind RSV, and neutralization is likely to be protective for the host. One study, however, suggested that SP-A may enhance the infectivity of RSV.9 In animal studies, SP-A deficient mice are more susceptible to RSV-induced disease.10 Cumulatively, these studies support the conclusion that surfactant plays a protective role in RSV infection, and that impaired surfactant production may contribute to RSV-induced disease. In addition to surfactant, clara cell secretory protein (CCSP) has also been shown to have anti-inflammatory and immunomodulatory functions in the lung.11 In addition, the production of CCSP correlates with gestational age, so preterm infants with lower levels of CCSP are also at increased risk of severe RSV infection.11 RSV infection of CCSP−/− mice results in exacerbated disease as assessed by AHR, mucus production, and viral persistence.12 This appears to be accompanied by a Th2 shift in the cytokine profile that may provide the mechanism of activation for the altered pathogenesis.12 Thus, deficiency in CCSP represents an additional mechanism that may contribute to RSV disease susceptibility in preterm infants.

3.2 Viral Recognition The existence of pathogen associated molecular patterns (PAMPs) and host pattern recognition receptors (PRRs) were originally hypothesized by Charles Janeway. PAMPs are structures common to microbes, usually essential structural features of the pathogens. Viruses can be potentially recognized on the surface by PRRs located on the host cell surface. However, once fusion and entry have taken place, cell surface recognition is unlikely to affect viral replication or disease pathogenesis. Mammalian cells also have intracellular mechanisms to detect viral infection. Toll-like

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receptors 3, 7, 8 and 9 are located on the lumenal surface of endosomes, and are localized to detect intracellular pathogens, bacterial and viral infection. RSV is a single-stranded RNA virus, but like other ssRNA viruses, replication of RSV proceeds via a dsRNA intermediate. Thus, RSV nucleic acids could be potentially recognized by TLR7 (ssRNA) or TLR3 (dsRNA). In addition to TLRs, recent evidence has demonstrated that intracellular helicases (including RIG-I) play an important role in the recognition of viral nucleic acids (Figure 1). Recognition of viral infection is essential for the production of IFNα and IFNβ, and the generation of the type I interferon-dependent antiviral state. A number of studies have demonstrated roles for Toll-like receptors (TLRs) in the recognition of RSV. TLR3 is involved in the recognition of double-stranded RNA. RSV infection of human epithelial cells and fibroblasts induces the expression of TLR3, and the induction of RSVinduced CXCL10 and CCL5 is TLR3-dependent.13 This effect is not dependent upon signaling through the type I interferon receptor IFAR, and does not affect viral load.13 Increased expression of TLR3 also results in increased responsiveness to subsequent stimulation with synthetic TLR3 agonist dsRNA. Thus, in addition to inflammatory chemokine production mediated directly by RSV recognition, the upregulation of TLR3 on respiratory epithelium may sensitize the pulmonary barrier surface for subsequent activation to other viruses. TLR7, which recognizes singlestranded RNA, may also be involved in recognition of RSV, although no studies demonstrating a role for TLR7 have been published to date. TLR7 is expressed by B cells and plasmacytoid dendritic cells. Two studies have demonstrated that plasmacytoid dendritic cells are essential for the type I IFN response generated during RSV infection in mice14,15 and may provide the initial protective mechanism involved in the inhibition of viral replication. One controversial aspect of RSV recognition by TLRs has been the role of TLR4. In studies using recombinant protein, RSV fusion (F) protein was shown to elicit innate immune responses from human PBMC (as assayed by monocyte production of IL6 and CXCL8) in a TLR4and CD14-dependent manner.16 Additionally, genetically TLR4-deficient C57BL10/ScCr mice displayed delayed clearance of the virus.16 However, C57BL10/ScCr mice are also defective in the expression of IL-12Rα,

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mDC TLR 4 RIG-I ssRNA dsRNA

MyD88

TRIF TLR 3 NFkB

Cytokines (IL-12) Chemokines:(CCL2, CCL5, CXCL10, etc.)

pDC ssRNA

Type I IFN

TLR 7

TLR 4

NFkB RIG-I

dsRNA

MyD88

Figure 1. Recognition and Th1 promoting immune signal generation by myeloid (mDC) and plasmacytoid dendritic cells (pDC) during RSV infection. TLR4 is expressed by both subsets, and recognizes RSV F protein on the viral surface. RSV is a single-stranded virus (ssRNA). Entry of the virus through the cell membrane into the cytosol is followed by encoating and conversion to a double-stranded RNA intermediate (dsRNA). Single-stranded RNS is recognized by TLR3, predominantly expressed in the endosomes of mDC. TLR7 is also localized to endosomes, but expressed predominantly by pDC. Binding and activation of TLR3 results in the recruitment of adaptor protein TRIF, leading to NF-κ B, and ultimately to cytokine (IL-12) and chemokine production (CCL5, CXCL10, etc.). In pDCs TLR7 may recognize single-stranded RSV. TLR4 and TLR7 both signal through the adapter protein MyD88, leading to NF-κ B and other downstream signaling. Cytosolic pattern recognition receptors, including RIG-I, recognize RSV nucleic acids and promote Type I IFN production by pDC. Together, signals from mDC and pDC cooperate to promote Th1 responses to RSV.

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which could account for delayed viral clearance as well. This potential problem was addressed in a subsequent study using a number of mouse strains deficient in IL-12 signaling or TLR4.17 TLR4-deficient mice of various backgrounds had no defect in viral clearance and had indistinguishable inflammatory responses, compared to controls, suggesting that IL-12 plays a more dominant role in viral clearance in mice in vivo.17 In humans, there are a number of known polymorphisms in TLR4. Two polymorphisms (299)Gly and (399)Ile in TLR4 correlate with severe RSV-induced disease.18 Furthermore, cloning and expression of these polymorphic TLR4s into human bronchial epithelial cells results in differential proinflammatory cytokine production in response to RSV or LPS. Using PBMCs from individuals carrying the polymorphisms, surface TLR4 expression and NF-κ B signaling in heterozygotes are impaired following in vitro challenge with LPS.19 Other studies have suggested that TLR4 ligation by RSV increases expression of TLR4, thereby sensitizing airway epithelium for subsequent responses to endotoxin. Taken together, there is substantial evidence from humans to suggest that TLR4 plays a role in recognition of RSV, and may contribute to severity of disease. The reason for the minimal effect of TLR4 deletion on RSV pathogenesis in mice is as yet unclear, but may represent species-specific differences in TLR4 dependence by the host. MyD88 is an adapter molecule involved in TLR signaling. MyD88 is required for the all known TLR signaling except TLR3. Additionally, TLR4 has both MyD88-dependent and MyD88-independent pathways. TLR3 signaling, and the MyD88-independent pathway of TLR4 are both mediated via another adapter molecule, TRIF. Studies in our laboratory have demonstrated that siRNA-mediated blockade of MyD88 signaling in human epithelial cells blocks the induction of CXCL8, but not CCL5 or CXCL10, which are TLR3-dependent.13 RSV infection in MyD88−/− mice results in exacerbated disease, which is associated with an enhanced Th2 cytokine profile.20 These studies were performed using mice on a C57/Bl6 background, which are relatively resistant to RSV-induced disease. RSV infection in B6 mice is associated with the early and dramatic induction of IL-12 and a Th1 cytokine profile. Bone marrow-derived dendritic cells isolated from MyD88−/− mice, however, could not produce IL12 or the Notch ligand delta-4 in response to RSV infection.20 Together,

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these results demonstrate that both MyD88-dependent and MyD88independent responses are generated in response to RSV. Furthermore, MyD88 signaling plays an instructive role in promoting Th1 responses to RSV. In addition to TLRs, mammalian cells can also recognize viruses via soluble intracellular sensors, including retinoic acid inducible (RIG-I), and MDA-5, as well as the RNA-dependent protein kinase R (PKR). Of these, the roles of PKR and RIG-I have been investigated in RSV recognition.21,22 The induction of IFNα in human pDCs is independent of PKR. Additionally, IFNα induction by RSV is not chloroquine sensitive (i.e., not dependent upon endosomal maturation). This is in contrast to the TLR9 stimulus, CpG, and TLR7 signaling which are chloroquinesensitive.22 Using an siRNA approach, knockdown of RIG-I in human epithelial cells results in decreased RSV-induced IFNβ , CXCL10, CCL5, and TLR3. Knockdown of TLR3 results in decreased IFNβ and chemokine production, but at a later time point.21 These results would suggest that RIG-I-mediated recognition of RSV precedes TLR3mediated recognition, at least in epithelial cells which do not express TLR3 at baseline.21 RIG-I and MDA-5 also contain caspase activation and recruitment (CARD) domains, which provide a link to cell apoptotic machinery. Cumulatively, these data demonstrate that RSV infection induces TLR-dependent and TLR-independent signaling. TLR-independent signaling appears to contribute to the type I IFN response, whereas TLRdependent signaling shapes the resulting response via IL-12 and chemokine induction.

3.3 Chemokines Chemokines (chemotactic cytokines) are a family of small molecular weight proteins named for their ability to promote the migration of leukocytes. Chemokine ligand production and receptor expression are differentially regulated under a variety of disease, resulting in the differential recruitment of specific leukocyte subsets. Additionally, it is becoming increasingly clear that chemokines play a greater role in immunity than simply chemotaxis, including affecting angiogenesis, cytokine regulation, T cell phenotype, etc.

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RSV infection in epithelial cells in vitro induces the production of a variety of chemokines including CCL2, CCL3, CCL5, and CXCL8. Evidence from clinical studies suggests that increased chemokine production is associated with more severe disease. In one study comparing RSVinfected infants with varying degrees of bronchiolitis, elevated levels of CCL3 and CCL5 were associated with severe bronchiolitis.23 Additionally, CCL3 and CCL5 both signal via CCR5, and CCR5 polymorphisms are associated with severe RSV bronchiolitis.24 In the mouse model of RSV infection, many of the same chemokine mediators are induced as in humans. Studies in our laboratory have shown that viral replication is necessary for optimal chemokine production.25 In addition to epithelial cell production of CCL2 and CCL5, CXCL10 and CXCL1 are induced during RSV infection.25 In mice, CCL5 is induced in the lungs throughout RSV infection. Treatment of RSV-infected mice with anti-CCL5 antibodies results in attenuated RSV-induced AHR.26 Assessment of Th1/Th2 cytokine profiles demonstrates that CCL5 induction is regulated by IL-13. Conversely, anti-CCL5 treatment increases the production of IL-12, favoring a Th1 environment. Other studies have demonstrated a role for CCL3 in RSV-induced inflammation. RSVinfected CCL3−/− mice have attenuated inflammation, compared to controls, without any increase in viral load.27 Antibody-mediated neutralization of CXCR2 (the CXCL8 receptor) attenuates RSV-induced disease as assessed by mucus hypersecretion and AHR.28 These findings are recapitulated in CXCR2−/− mice.28 Although a contributing factor to inflammation and immunopathology, it is as yet unclear whether chemokine production observed during RSV infection actually confers susceptibility. The question remains — is the induction of CXCL8, CCL5, etc. an “inappropriate” response to infection? At least one study in humans suggests that chemokine induction may be a common response to RSV infection, not particular to those that will develop severe disease. In this study, human volunteers were infected intranasally with RSV, and chemokine production was assessed in nasal lavages.29 Subjects with established infection had increases in early CXCL8, and sustained increases in CCL2, CCL3, and CCL5 during virus shedding.29 Thus, similar chemokine profiles were observed in otherwise healthy adults in response to RSV infection. Taken together, these data demonstrate that RSV

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infection induces the production of chemokines CCL2, CCL5, CXCL8, and CCL3. The expression of these chemokines contributes to the development of immunopathology during RSV infection. The magnitude and localization of chemokine production may contribute as much to pathology, as the production of any particular chemokine itself.

3.4 Eicosanoids Prostaglandins and leukotrienes are eicosanoid lipid mediators derived from arachidonic acid. Leukotrienes are synthesized by the enzyme 5-lipoxygenase, and cysteinyl leukotrienes in particular, play an important role in allergic asthma, promoting airway hyperreactivity (the “slow reacting substance of anaphylaxis”). Cysteinyl leukotrienes signal via CysLT1 and CysLT2 on target cells to promote smooth muscle contraction, vascular permeability, mucus production, leukocyte recruitment, and activation. Mast cells in particular, are targets of cysteinyl leukotrienes. In vitro, RSV infection in epithelial cells results in the induction of 5-lipoxygenase (5-LO), necessary for the generation of leukotrienes from arachidonic acid.30 Clinically, cysteinyl leukotrienes are elevated in the nasal lavages of infants during and up to one month after RSV bronchiolitis,31 independent of exposure to cigarette smoke or family history of allergy/atopy.31 Similar results are found in bronchoalveolar lavages, and higher levels of CysLTs correlate with the presence of eosinophils. In the mouse model, cysteinyl leukotrienes in the BAL and lungs correlate temporally with RSV-induced disease.32 Treatment (daily, starting one day prior to infection) using the leukotriene inhibitor zileuton reduced airway constriction, inflammation, and weight loss.32 Attenuated airway hyperresponsiveness (but notably not inflammation) is observed in RSV-infected mice treated with the leukotriene receptor antagonist MK-571.33 Thus, substantial evidence suggests that leukotrienes play a role in promoting RSV-induced disease.

4. DENDRITIC CELLS — BRIDGING INNATE AND ADAPTIVE IMMUNITY The role of dendritic cells (DC) in the development, maintenance and exacerbation of RSV-induced lung disease continues to be a challenge to

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resolve. Most studies of dendritic cells have used splenic or bone-marrow derived cells (BMDC) to study DC biology. Due to the local microenvironment of the lung and the nature of the mucosal immune response, it is likely that bone marrow or splenic derived DC subsets do not fully reflect the DC that migrate into the lung during disease or are sentinel. Some studies have suggested that a subset of myeloid lineage dendritic cells (mDC) can preferentially skew the immune response toward a Th2 phenotype. Conversely, plasmacytoid DC (pDC) have been suggested to exert a predominantly regulatory effect within the pulmonary immune environment. The function of myeloid dendritic cells may be best defined based upon co-stimulatory molecule expression and cytokine profile. Subsets of mDC that express high levels of CD40, CD80 and produce high levels of IL-12 are more efficient at promoting a Th1 type response, whereas DC that express OX40L and lower levels of IL-12 will promote a predominantly Th2 response. The determinants of these profiles appear to depend upon the type of antigenic signal the DC encounters. The most clearly defined set of signals that dictate mDC function are those provided by TLRs. Ligation of TLRs promotes the expression of important instructive signals to be expressed, including co-stimulatory molecules (CD40, CD80), IL-12, as well as notch ligands. Together, these signals promote a Th1 type response. While the signals that prompt mDC to become Th2 cell induction are less well known, one airway epithelial cell-derived molecule, TSLP, has piqued the interest of researchers. TSLP directly activates DC and promotes Th2 responses through the expression of OX40. Thus, the determination of whether an mDC will promote a Th1- or Th2mediated response is dependent upon the nature of the signal that it receives from the pulmonary environment. The role of pDCs in shaping the pulmonary immune response has been more elusive. Depletion of pDC in allergic responses results in greatly enhanced allergic responses. Consequently, pDC were initially suggested to have a predominantly suppressive effect on T cell activation. Subsequently, experiments examining the role of pDC on viral responses have demonstrated a key role for these cells in promoting viral clearance. This aspect of pDC function may relate to their ability to produce large amounts of type I IFN. Other studies have suggested that pDCs are

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required for optimal mDC function.34 In a more recent set of studies, the depletion of pDC during RSV infection led to a decrease in viral clearance and increased Th2 responses, further supporting the requirement of pDC during viral responses.14,15 Another factor that distinguishes mDC from pDC is the differential expression of TLRs. While some TLRs are expressed by both (such as TLR4), TLR3 is preferentially expressed on mDCs, whereas TLR7 and TLR9 are expressed at high levels by pDCs (Figure 1). Type 1 IFN from pDCs and IL-12 from mDCs likely serve to cooperatively promote Th1 immunity against RSV. Thus, a complex relationship is beginning to develop between the different DC subsets and the regulation of immune responses within the lung. A better understanding of these activation events may allow additional avenues of therapeutic control during complex disease phenotypes within the lung immune environment as well as to allow more efficient vaccine development.

5. “RECRUITED INNATE” IMMUNITY Natural killer cells are lymphocytes that possess inherent antigennonspecific cytotoxic activity. NK cells respond to signals, such as the absence of MHC-I expression by lysing target cells via perforin and granzymes. Additionally, NK cells are potential early source of IFNγ that can activate macrophages, and influence T cell differentiation. In mice, RSV infection results in enhanced early NK cell-mediated cytotoxicity, and the frequency of IFNγ producing NK cells at day 4 is greater than any other lymphocyte subset, including CD8 T cells.35 This early IFNγ limits eosinophilia and dissemination of RSV.36 NK cell recruitment to the lungs and NK-mediated cytotoxicity are impaired in IL-12−/− mice or IL-12R−/− RSV-infected mice.17 Together, these studies suggest that IL-12-dependent recruitment and activation of NK cells contributes to early control of viral replication, while promoting the establishment of Th1 cell-mediated immunity. Natural Killer T cells (or NKTs) are a set of distinct subtypes of lymphocytes, bearing both NK cell markers and CD3. The majority of NKT cells are invariant Vα 4 expressing, CD1d restricted. CD1d is a nonclassical MHCI-like molecule, which binds glycoproteins. One study,

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which sought to determine whether CD1-restricted NKT cells play a role in RSV host response, found decreased CD8 responses in CD1−/− mice. Transient activation of NKT using the agonist α GalCer led to reduced illness, but delayed viral clearance. Gamma delta T cells (γδ T cells) are a subset of T lymphocytes that express γδ T cell receptors instead of the classical αβ TCR receptors. Although the mechanisms by which γδ T cells are stimulated are not completely understood, they can respond to antigens in an MHC-independent manner. γδ T cells represent a minority of T cells overall, but are enriched in the peripheral blood during some bacterial or viral infections; however, γδ T cells do not constitute a significant proportion of the T cells recruited to the airways during RSV infection in mice or humans. In the bovine RSV model, depletion of γδ T cells alters antibody responses, but does not affect inflammation or viral clearance.37 Thus, NKT cells and γδ T cells likely play minor roles in RSV infection.

6. ADAPTIVE IMMUNITY 6.1 Humoral Immunity Infection with RSV induces antibody responses in humans and mice. The fact that reinfections with RSV are common throughout life suggests that humoral immunity is not lasting, or is insufficient to protect against secondary infection. There is evidence to support both of these concepts. Primary RSV infection results in the generation of serum and localized antibody responses. These do not appear to be maintained from one RSV season to the next, however, as RSV-specific antibody titers in infants are at or near detectable levels one year postinfection. RSV infections tend to become less severe with each occurrence, and secondary infection is associated with a rapid, high titer antibody response.38 In a study of virusassociated hospitalization, patients with non-RSV related hospitalizations had higher RSV-neutralizing antibody titers than those hospitalized for RSV-associated disease.39 While RSV-specific humoral immunity may not prevent reinfection, these studies suggest that antibody responses to RSV provide protection against severe disease. Evidence from mice also supports a role for B cells and antibodies in RSV infection. B cell-depleted mice have more severe illness, increased

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pathology, and impaired secondary immunity. Conversely, when mice allowed to recover from primary RSV infection are treated with T celldepleting antibodies prior to secondary challenge, they still exhibit enhanced viral clearance, suggesting that antibodies afford a level of protection against reinfection.40 More recent studies have suggested that repeated intranasal infection with RSV results in dampening of local RSV-specific plasma cell responses, while those in bone marrow are maintained.41 In premature infants, or very young infants with increased susceptibility to RSV infection, prophylactic passive antibody therapy has been used to prevent infection. Purified RSV immune globulin (RSV-IGIV), a concentrated preparation of RSV-neutralizing from adult human serum was used with some success. Palivizumab has largely replaced the use of RSV-IGIV. Palivizumab is a humanized murine monoclonal IgG antibody directed toward RSV F (attachment) protein. Palivizumab blocks entry of RSV via blocking the RSV F protein-dependent fusion. This monoclonal antibody has advantages in that it can be produced in standardized fashion in large quantities, without the risk of transmitting infection (risks of pooled immunoglobulin). Although therapy has been successful at decreasing the rates of RSV-associated hospitalization, and improving a number of outcomes, it is costly.2 Detailed prospective cost-benefit studies have not been done, the savings afforded by treatment may be outweighed by the cost of therapy in all but very premature infants, and those with chronic lung disease of prematurity (CLD). Therefore, although safe and effective, passive antibody therapy for RSV remains limited in its usefulness.

6.2 Cell-Mediated Immunity — CD4 vs. CD8 T Cells T cells play an important role in the host response to RSV, contributing to both viral clearance and virus-induced immunopathology. In the murine model of primary RSV infection, depletion of both CD4 and CD8 T cells leads to a dramatic delay in the clearance of RSV, while depletion of either subset only modestly extends viral clearance.40 However, mice depleted of both CD4 and CD8 T cells are protected from RSV-induced disease as assessed by weight loss and pulmonary inflammation.40

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A number of studies support the model that CD4 T cells are responsible for immunopathology in primary infection, as well as vaccine-induced immunopathology. The role of CD4 T cells has been studied extensively in vaccine — induced models of RSV disease. In these models, enhanced pulmonary disease is observed in mice primed with either formalininactivated RSV (FI-RSV) or vaccinia virus-expressing RSV proteins (VV-RSV). In the FI-RSV model, enhanced disease is abrogated by depletion of CD4 T cells.42 Passive transfer of RSV-specific CD4 or CD8 T cells into RSV-infected mice augments both clearance and immunopathology.43 In the VV-RSV model, pre-vaccination of mice with VV expressing different RSV genes results in differential T-cell responses upon subsequent challenge with live RSV. RSV-G (glycoprotein), nucleoprotein (N), and phosphoprotein (P) yield a predominantly CD4 T-cell response, second matrix (22K) yields a predominantly CD8, and RSV-F (fusion) yields a mixed response.44 F-protein priming results in a primarily Th1 response, whereas G-protein priming is predominantly Th2 driving and results in eosinophilia.44 CD8 or IFNg depletion in F-protein primed mice results in the development of eosinophilia.45 Thus, most studies using the VV-RSV or FI-RSV models have suggested that CD8 cells mediate clearance of RSV, and antagonize CD4 T cell-induced immunopathology.45–48 Effector CD4 T cell responses can be characterized as Th1 (IFNγ ) or Th2 (IL-4, -5, and -13) based on the predominating cytokine patterns. These responses in T cells are regulated by several instructive signals, such as IL-12 that is induced during RSV infection. Treatment of RSVinfected mice with anti-interleukin 12 resulted in increased AHR, mucus production, and airway eosinophilia. Similar results were obtained using STAT4 deficient mice, which is required for IL-12 responsiveness.49 Together, these results demonstrate that IL-12, which promotes Th1 responses and antagonizes Th2 responses promotes clearance and limits immunopathology during RSV infection. One cytokine that has received considerable attention in animal models of RSV infection is IL-13. IL-13 promotes mucus production and airway hyperreactivity in a variety of lung disease models. Among Th2 cytokines, IL-13 is highly induced during RSV infection in susceptible mice, which temporally correlates with AHR.50 Treatment of mice with anti-IL-13, but notably, not anti-IL-4 antibodies inhibited AHR. Anti-IL-13

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also resulted in an early increase in IL-12. RSV-induced AHR in stat6 deficient mice was similar to IL-13, and further decreased mucus production, relative to control RSV-infected mice.50 One study suggested that IL-13 might have beneficial effects as well. Overexpression of IL-13 in RSV-infected mice decreased viral titers compared to non-transgenic littermates. Conversely, RSV infection of the IL-13 deficient mice resulted in increased viral titers at day 4 postinfection, as did neutralization of IL-13 in STAT1 deficient mice.51 The induction of IL-13 likely depends upon factors in both the host and the virus. Recent data suggest that different RSV isolates produce disparate cytokine responses in genetically identical hosts.52 Thus, IL-13 production during RSV infection may have both protective and pathogenic consequences. Several recent discoveries have dramatically advanced the understanding of the kinetics of RSV-specific T cell responses in mice. In the Balb/c mouse model of RSV infection, several immunodominant epitopes have been identified. The CD8 T cell response to one of these, RSV M82-90, accounts for up to 50% of CD8 T cells in the lungs of mice during primary infection. Primary effector functions of cytolytic T cells include the production of IFNγ and perforin-dependent lytic activity. At the peak of the CD8 T cell response in RSV infected mice, only half of the tetramer CD8 T cells in the lungs of RSV-infected mice produced IFNγ upon restimulation. As a comparison, influenza infection resulted in nearly all of the tetramerspecific cells elaborating interferon upon stimulation ex vivo. This defect in effector function was only detected in CD8 T cells in infected lungs, not CD8 T cells in lymphoid organs. The discrepancy in CD8 effector function between influenza and RSV-infected mice suggested that active RSV infection may impair the effector function in CD8 T cells. Overall, a better understanding of the role of CD4 and CD8 T cells and their epitopes that promote specific responses will be useful for establishing potential vaccine candidates as well as assessing pathogenic potential of different RSV infections.

7. RSV AND CHRONIC LUNG DISEASE: ASTHMA AND COPD Several studies have suggested that early severe RSV infection predisposes individuals to subsequent development of lung disease, including

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asthma.4 Children hospitalized in infancy for severe RSV infection were more likely (at age 13) to have asthma/recurrent wheezing, allergic sensitization, and allergic rhinoconjunctivitis.4 Other studies have failed to support such a link. In one study, wheezing following RSV lower respiratory tract infection was followed prospectively for six years. Late wheezing (at six years of age) was associated with polymorphisms of the IL-13 gene, but not related to early postbronchiolytic wheezing following RSV infection.53 It is possible that the same underlying factors (genetic, physiological, or other) contribute to both early severe RSV infection and the subsequent development of asthma. Thus, although early severe RSV infection correlates with the subsequent development of reactive airway disease, it is yet unclear whether the two are causally related or perhaps RSV merely uncovers an underlying phenotype. In addition to primary infections, an important clinical scenario involving RSV infection is virus-induced exacerbation of asthma, COPD, or other chronic lung diseases. In the past decade, viral infection has been increasingly recognized as a contributor to exacerbations of asthma. RSV is the most common respiratory virus associated with wheezing in children under the age of two.54 RSV exacerbation of asthmatic disease was more likely to be severe, and more likely to be accompanied by eosinophilia, compared with influenza, the second most common pathogen associated with asthma exacerbation in the study.55 Although the causal basis for viral exacerbation of asthma is as yet unclear, one possibility is that asthmatic disease alters the immune tendency of the host, resulting in a Th2 or atopic environment in the lungs. Since the protective (“appropriate”) response to RSV is thought to be a well-controlled Th1 type response, this is antagonized by the existing Th2 environment, resulting in a non-protective Th2 response to the virus. One study suggested that secondary RSV responses in the context of allergic lung disease are more Th2 prone and more detrimental.56 In addition to exacerbation of allergic disease, concomitant viral infection and allergic asthma can result in delayed clearance of RSV.57 Another model places less emphasis on the Th1/Th2 paradigm, and posits more generally that asthma and “inappropriate” RSV infection activate common inflammatory pathways, such that the pathogenesis of one predisposes the lung environment for more robust response to the other.

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A number of commonalities between RSV-induced disease and asthma have been discussed, including: (1) inflammation — lymphocytic and eosinophilic, (2) mucus hypersecretion, (3) production of chemokines, (4) induction of Th2 cytokines, including IL-13, (5) induction of CysLTs and other eicosanoids.58 Recent data has indicated that in the most severe asthmatics, there is an alteration in the ratio of CD4:CD8 T cells that can be found in the airway, such that the ratio is significantly reduced compared to either non-asthmatics or mild asthmatics with an increase in CD8+ T cells.59 In addition, a number of studies have identified that CD8+ T cells play a role in allergic and virus responses in animal models.60,61 Thus, the role of CD8 T cells for asthma exacerbation has become an area of greater interest due to the realization that these cells can influence Th2 type cytokines during asthmatic responses that may be related to viral responses. Some insights into the mechanistic basis for virus-induced exacerbation of asthma have come from studies using murine models. In a mouse model of RSV exacerbation of dust-mite asthmatic disease, treatment with either corticosteroids or a leukotriene receptor antagonist attenuated virus-induced inflammation.62 In a model of RSV exacerbation of cockroach antigen-induced asthma, studies in our laboratory have pointed to the importance of CCL5 and CCR1 in viral exacerbations of allergic disease.63,64 The activation and recruitment of T cells that amplify and skew the immune response toward more intense pathology, including mucus production and remodeling of the airways, are likely scenarios that lead to more severe disease and clinical crisis in asthmatic patients. Exacerbations of chronic obstructive pulmonary disease are often associated with viral infection. Viruses such as rhinovirus (RV) and RSV, which typically cause upper respiratory infection, are a significant cause of lower respiratory infection in patients with COPD.65 Although less common than rhinovirus-mediated exacerbation, RSV is associated with more severe disease in stable COPD.66 RSV was also associated with higher inflammatory marker levels, suggesting a relationship with disease.66 RSV is increasingly recognized as a complicating factor in COPD and other chronic lung disease.

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8. RSV VACCINOLOGY The history of RSV vaccination is an unpleasant one. In the 1960s, formalininactivated RSV (FI-RSV) vaccines were given to children as young as two months old. In children that had not been previously exposed to RSV, the vaccine generated virus-specific antibodies, suggesting efficacy of the vaccine. However, upon natural infection with live virus, it became clear that, not only was the vaccine not protective, it was actually detrimental. Vaccine-enhanced disease resulted in hospitalizations and even deaths in vaccinated children.67 Based on decades of studies into the mechanisms of vaccine-enhanced RSV disease, it is now generally believed that FI-RSV induces a population of Th2 skewed RSV-specific memory CD4+ T cells, which are reactivated and expanded upon subsequent infection.68 These Th2 cells promote the recruitment and activation of large numbers of eosinophils to the lungs. Additionally, recent studies of FI-RSV have suggested that formalin-inactivated vaccines are inherently Th2 biasing via a mechanism involving enhanced uptake by scavenger receptors binding to reactive carbonyl groups.69 The immunobiology of RSV makes it a particularly difficult pathogen for vaccine development. Ideally, a vaccine would be capable of inducing protective immunity in neonates, elderly, asthmatics, and other at-risk populations. Secondly, in individuals with preexisting immunity to RSV, the vaccine should at the very least, not cause disease. Finally, the history of the adverse effects of previous vaccines makes the burden of demonstrating safety even higher than that typically required for vaccines. A variety of vaccine strategies have been investigated for RSV, including live attenuation, inactivated RSV (often with the addition of other PAMPs), subunit vaccines, recombinant carrier microbes such as salmonella or sendai virus expressing RSV antigens, DNA-based vaccines and others. Substantial effort has been recently invested in the generation of a safe and effective RSV vaccine. The widespread use of any potential vaccine must overcome issues of efficacy and safety, both actual and perceived. This is indeed a high hurdle.

REFERENCES 1. Glezen WP, Greenberg SB, Atmar RL, Piedra PA, Couch RB (2000). Jama 283, 499–505.

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2. Harkensee C, Brodlie M, Embleton ND, McKean M (2006). J Infect 52, 2–8. 3. Falsey AR (2007). Semin Respir Crit Care Med 28, 171–181. 4. Sigurs N, Gustafsson PM, Bjarnason R, Lundberg F, Schmidt S, Sigurbergsson F, Kjellman B (2005). Am J Respir Crit Care Med 171, 137–141. 5. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K (2003). Jama 289, 179–186. 6. Hall CB, Long CE, Schnabel KC (2001). Clin Infect Dis 33, 792–796. 7. Hallman M, Haataja R (2006). Semin Perinatol 30, 350–361. 8. Kerr MH, Paton JY (1999). Am J Respir Crit Care Med 159, 1115–1118. 9. Hickling TP, Malhotra R, Bright H, McDowell W, Blair ED, Sim RB (2000). Viral Immunol 13, 125–135. 10. LeVine AM, Gwozdz J, Stark J, Bruno M, Whitsett J, Korfhagen T (1999). J Clin Invest 103, 1015–1021. 11. Lassus P, Nevalainen TJ, Eskola JU, Andersson S (2000). Pediatr Pulmonol 30, 466–469. 12. Wang SZ, Rosenberger CL, Bao YX, Stark JM, Harrod KS (2003). J Immunol 171, 1051–1060. 13. Rudd BD, Burstein E, Duckett CS, Li X, Lukacs NW (2005). J Virol 79, 3350–3357. 14. Smit JJ, Rudd BD, Lukacs NW (2006). J Exp Med 203, 1153–1159. 15. Wang H, Peters N, Schwarze J (2006). J Immunol 177, 6263–6270. 16. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, et al. (2000). Nat Immunol 1, 398–401. 17. Ehl S, Bischoff R, Ostler T, Vallbracht S, Schulte-Monting J, Poltorak A, Freudenberg M (2004). Eur J Immunol 34, 1146–1153. 18. Puthothu B, Forster J, Heinzmann A, Krueger M (2006). Dis Markers 22, 303–308. 19. Tulic MK, Hurrelbrink RJ, Prele CM, Laing IA, Upham JW, Le Souef P, Sly PD, Holt PG (2007). J Immunol 179, 132–140. 20. Rudd BD, Schaller MA, Smit JJ, Kunkel SL, Neupane R, Kelley L, Berlin AA, Lukacs NW (2007). J Immunol 178, 5820–5827. 21. Liu P, Jamaluddin M, Li K, Garofalo RP, Casola A, Brasier AR (2007). J Virol 81, 1401–1411. 22. Hornung V, Schlender J, Guenthner-Biller M, Rothenfusser S, Endres S, Conzelmann KK, Hartmann G (2004). J Immunol 173, 5935–5943.

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23. Garofalo RP, Patti J, Hintz KA, Hill V, Ogra PL, Welliver RC (2001). J Infect Dis 184, 393–399. 24. Hull J, Rowlands K, Lockhart E, Moore C, Sharland M, Kwiatkowski D (2003). J Infect Dis 188, 904–907. 25. Miller AL, Bowlin TL, Lukacs NW (2004). J Infect Dis 189, 1419–1430. 26. Tekkanat KK, Maassab H, Miller A, Berlin AA, Kunkel SL, Lukacs NW (2002). Eur J Immunol 32, 3276–3284. 27. Haeberle HA, Kuziel WA, Dieterich HJ, Casola A, Gatalica Z, Garofalo RP (2001). J Virol 75, 878–890. 28. Miller AL, Strieter RM, Gruber AD, Ho SB, Lukacs NW (2003). J Immunol 170, 3348–3356. 29. Noah TL, Becker S (2000). Clin Immunol 97, 43–49. 30. Behera AK, Kumar M, Matsuse H, Lockey RF, Mohapatra SS (1998). Biochem Biophys Res Commun 251, 704–709. 31. Da Dalt L, Callegaro S, Carraro S, Andreola B, Corradi M, Baraldi E (2007). Pediatr Allergy Immunol 18, 100–104. 32. Welliver RC, 2nd, Hintz KH, Glori M, Welliver RC, Sr. (2003). J Infect Dis 187, 1773–1779. 33. Fullmer JJ, Khan AM, Elidemir O, Chiappetta C, Stark JM, Colasurdo GN (2005). Pediatr Allergy Immunol 16, 593–601. 34. Yoneyama H, Matsuno K, Toda E, Nishiwaki T, Matsuo N, Nakano A, Narumi S, Lu B, Gerard C, Ishikawa S, et al. (2005). J Exp Med 202, 425–435. 35. Anderson JJ, Norden J, Saunders D, Toms GL, Scott R (1990). J Gen Virol 71 (Pt 7), 1561–1570. 36. Hussell T, Openshaw PJ (2000). J Immunol 165, 7109–7115. 37. Taylor G, Thomas LH, Wyld SG, Furze J, Sopp P, Howard CJ (1995). J Virol 69, 6658–6664. 38. Welliver RC, Kaul TN, Putnam TI, Sun M, Riddlesberger K, Ogra PL (1980). J Pediatr 96, 808–813. 39. Piedra PA, Jewell AM, Cron SG, Atmar RL, Glezen WP (2003). Vaccine 21, 3479–3482. 40. Graham BS, Bunton LA, Wright PF, Karzon DT (1991). J Clin Invest 88, 1026–1033. 41. Singleton R, Etchart N, Hou S, Hyland L (2003). J Virol 77, 11303–11311. 42. Connors M, Kulkarni AB, Firestone CY, Holmes KL, Morse HC, 3rd, Sotnikov AV, Murphy BR (1992). J Virol 66, 7444–7451.

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43. Alwan WH, Record FM, Openshaw PJ (1992). Clin Exp Immunol 88, 527–536. 44. Alwan WH, Record FM, Openshaw PJ (1993). J Immunol 150, 5211–5218. 45. Hussell T, Baldwin CJ, O’Garra A, Openshaw PJ (1997). Eur J Immunol 27, 3341–3349. 46. Sharma AK, Woldehiwet Z (1995). Clin Exp Immunol 101, 288–294. 47. Hussell T, Georgiou A, Sparer TE, Matthews S, Pala P, Openshaw PJ (1998). J Immunol 161, 6215–6222. 48. Olson MR, Varga SM (2007). J Immunol 179, 5415–5424. 49. Tekkanat KK, Maassab H, Berlin AA, Lincoln PM, Evanoff HL, Kaplan MH, Lukacs NW (2001). Am J Pathol 159, 631–638. 50. Tekkanat KK, Maassab HF, Cho DS, Lai JJ, John A, Berlin A, Kaplan MH, Lukacs NW (2001). J Immunol 166, 3542–3548. 51. Zhou W, Hashimoto K, Moore ML, Elias JA, Zhu Z, Durbin J, Colasurdo G, Rutigliano JA, Chiappetta CL, Goleniewska K, et al. (2006). Microbes Infect 8, 2880–2889. 52. Lukacs NW, Moore ML, Rudd BD, Berlin AA, Collins RD, Olson SJ, Ho SB, Peebles RS, Jr. (2006). Am J Pathol 169, 977–986. 53. Ermers MJ, Hoebee B, Hodemaekers HM, Kimman TG, Kimpen JL, Bont L (2007). J Allergy Clin Immunol 119, 1086–1091. 54. Rakes GP, Arruda E, Ingram JM, Hoover GE, Zambrano JC, Hayden FG, PlattsMills TA, Heymann PW (1999). Am J Respir Crit Care Med 159, 785–790. 55. Zhao J, Takamura M, Yamaoka A, Odajima Y, Iikura Y (2002). Pediatr Allergy Immunol 13, 47–50. 56. Kondo Y, Matsuse H, Machida I, Kawano T, Saeki S, Tomari S, Obase Y, Fukushima C, Kohno S (2004). Clin Exp Allergy 34, 1307–1313. 57. Hassantoufighi A, Oglesbee M, Richter BW, Prince GA, Hemming V, Niewiesk S, Eichelberger MC (2007). Clin Exp Immunol 148, 218–229. 58. Lukacs NW, Tekkanat KK, Berlin A, Hogaboam CM, Miller A, Evanoff H, Lincoln P, Maassab H (2001). J Immunol 167, 1060–1065. 59. O’Sullivan S, Cormican L, Faul JL, Ichinohe S, Johnston SL, Burke CM, Poulter LW (2001). Am J Respir Crit Care Med 164, 560–564. 60. Schaller MA, Lundy SK, Huffnagle GB, Lukacs NW (2005). Eur J Immunol 35, 2061–2070. 61. Schwarze J, Makela M, Cieslewicz G, Dakhama A, Lahn M, Ikemura T, Joetham A, Gelfand EW (1999). J Immunol 163, 5729–5734.

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62. Matsuse H, Kondo Y, Machida I, Kawano T, Saeki S, Tomari S, Obase Y, Fukushima C, Mizuta Y, Kohno S (2006). Ann Allergy Asthma Immunol 97, 55–60. 63. John AE, Berlin AA, Lukacs NW (2003). Eur J Immunol 33, 1677–1685. 64. John AE, Gerard CJ, Schaller M, Miller AL, Berlin AA, Humbles AA, Lukacs NW (2005). Eur J Immunol 35, 108–116. 65. Seemungal TA, Wedzicha JA (2003). Curr Opin Pulm Med 9, 111–116. 66. Seemungal T, Harper-Owen R, Bhowmik A, Moric I, Sanderson G, Message S, Maccallum P, Meade TW, Jeffries DJ, Johnston SL, et al. (2001). Am J Respir Crit Care Med 164, 1618–1623. 67. Fulginiti VA, Eller JJ, Sieber OF, Joyner JW, Minamitani M, Meiklejohn G (1969). Am J Epidemiol 89, 435–448. 68. Connors M, Giese NA, Kulkarni AB, Firestone CY, Morse HC, 3rd, Murphy BR (1994). J Virol 68, 5321–5325. 69. Moghaddam A, Olszewska W, Wang B, Tregoning JS, Helson R, Sattentau QJ, Openshaw PJ (2006). Nat Med 12, 905–907.

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CHAPTER 9

Innate Recognition of Viral Infection and the Involvement of Autophagy Balaji Ramanathan & Akiko Iwasaki

ABSTRACT Immune defense against virus infection is initiated through the process of virus recognition and signaling by the host cells. Such recognition activates a set of antiviral genes that ultimately limit virus replication. Specific motifs associated with virus and its replication intermediates are recognized by the host cell as pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Viruses enter cells by fusion at the plasma membrane or within endosomes, followed by entry into the cytoplasm, where it initiates replication. Recognition of viral PAMPs leads to the initiation of antiviral responses such as production of type I interferons. PRRs such as Toll-like receptors (TLRs) and RNA helicases are responsible for the recognition of viral pathogens in different compartments of the cell. In plasmacytoid dendritic cells (pDCs), primary innate sensing of endocytosed viruses is thought to occur in the endosomes by TLRs without direct infection. Recently, viral replication intermediates have been identified as an additional innate recognition targets of endosomal TLR for certain RNA viruses such as the vesicular stomatitis virus (VSV), which requires autophagy. Originally identified as a process involved in nutrient acquisition during starvation and maintenance of quality control of long-lived proteins, autophagy is now being appreciated as a pathway used by the innate immune system to recognize and destroy viruses. In this chapter, we 209

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provide a review of host gene responses to RNA viral infection with emphasis on VSV and the latest studies characterizing novel mechanisms of innate viral recognition and immunity.

1. INTRODUCTION The immune system has evolved to provide protection from microorganisms. Innate immunity, a highly effective set of conserved mechanisms used by multicellular organisms, represents the first line of defense against invading pathogens, including viruses, before adaptive immune response is launched against such pathogens.1,2 Defense responses by mammals often rely on intimate communication between the innate and adaptive arms of the immune system. Adaptive immunity depends on lymphocyte cell-surface receptors that recognize an infinite number of antigens, due to the ability of these cells to generate diverse receptors based on gene rearrangement. Instead, the innate immune system relies on a definite set of receptors, called pattern recognition receptors (PRRs).3,4 In initiating the immune response against the invading pathogens, PRRs recognize pathogen-associated molecular patterns (PAMPs) mostly via direct pathogen-PRR interaction.2 PAMPs are defined as molecular structures that are conserved within a group of pathogens. For instance, lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) are PAMPs associated with gram-negative bacteria and viruses, respectively. Recognition of pathogens can occur at one of these sites — extracellular, plasma membrane or intracellular (cytoplasmic space or in the lumen of intracellular vesicles). The proteins of PRR families such as complement, pentraxin, and collectin present in extracellular space play a main role in pathogen opsonization for phagocytic clearance and in activation of complement pathways. PRRs on the cell membrane have two major functions: promotion of phagocytosis and initiation of intracellular signaling pathways. Cytoplasmic PRRs can be grouped into at least three families: interferon (IFN)-inducible proteins, caspase-recruiting domain (CARD) helicases, and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). IFN-inducible proteins such as dsRNA-activated protein kinase (PKR), and CARD helicases such as retinoic acid-inducible protein

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I (RIG-I), mediate antiviral defense, whereas NLRs primarily mediate antibacterial defense. The main functions carried out by PRRs include enhancement of phagocytosis, production of inflammatory cytokines, activation of complement cascades, and maturation of various immune cells such as dendritic cells (DCs). Cytokine production and DC maturation primarily rely on the ability of PRRs to trigger signal transduction upon receptor engagement by PAMPs.5–7 In this chapter, we will summarize recent progress in the field of innate immune viral recognition and the role of autophagy in this process.

2. VIRAL RECOGNITION: SELF AND NON-SELF DISCRIMINATION In order for a cell to sense a pathogen, it has to distinguish “self ” from infectious “non-self ”.2,8 To understand the cellular responses raised by different viruses, it is important to know which PRRs become activated during viral infections and which viral molecules serve as ligands for different PRRs. TLRs play a major role in viral recognition by plasmacytoid dendritic cells (pDCs), which are specialized sensors of viral infections.9 Viruses with their intracellular lifecycles, are capable of being recognized by intracellular TLRs. Viral nucleic acids of certain DNA viruses10 are marked by higher frequency of CpG motifs, and DNA from several DNA viruses have been demonstrated to stimulate TLR 9. The RNA isolated from reovirus (dsRNA virus) stimulates TLR 3, which is also activated artificially by the dsRNA poly I:C.11 The complexity of viral glycoproteins makes it possible for them to interact with different surface TLRs. TLR 2 and 4 are expressed on the cell surface and are thus susceptible to being bound by glycoproteins on the virion. For instance, TLR 2 interacts with a component of the human cytomegalovirus (CMV) particle12 and is also activated upon binding to hemagglutinin of measles virus.13 As for TLR 4, the fusion protein of respiratory syncytial virus (RSV) is a potent trigger of proinflammatory cytokine expression, and this occurs via ligation to TLR 4.14 The envelope protein of mouse mammary tumor virus and murine leukemia virus have been reported to activate DCs and stimulate B cell proliferation through TLR 4.15 It is noteworthy that the viral ligands for TLR 2 and TLR 4 characterized so far are glycoproteins used by viruses

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for fusion and entry into host cells. Thus, these viruses may exploit engagement of TLRs to mediate environment suitable for infection in host cells.

2.1 TLR-Mediated Virus Recognition TLRs are the best described class of PRRs, expressed at the plasma membrane or within intracellular vesicles depending on the TLR member. TLRs contain characteristic domains defined by an extracellular luminal ligand-binding leucine-rich region (LRR) domain, and a cytoplasmic signaling Toll/Interleukin (IL)-1 receptor (TIR) domain. TLRs 1, 2, 4, 5, 6, and 10 are expressed on the cell-surface, while TLRs 7, 8, and 9 are localized in endosomes.16–18 The cellular distribution of TLR3 appears to be cell type dependent, since this receptor is expressed on the surface of human fibroblasts, whereas it is expressed in endocytic compartments in monocyte-derived immature DCs and CD11c cells.19 The specific nature of the signal transduction activated by TLRs is at least in part determined by the adaptor proteins employed by different TLRs. Four different TIR domain containing adaptor proteins have been described to mediate signal transduction by TLRs. These are myeloid differentiation factor (MyD) 88, MyD88-adapter-like (Mal), TIR-domain-containing adaptor inducing IFN-β (TRIF), and TRIF-related adaptor molecule (TRAM). MyD88 is used by all TLRs, except TLR 3, whereas the other adaptor proteins are much more restricted in their TLR interactions. For example, Mal is involved in signaling from TLR 2 and TLR 4, TRIF is a component of the signaling machinery from TLR 3 and TLR 4, while TRAM is involved in signal transduction from TLR 4. Downstream of the adaptor proteins, all the TLRs activate nuclear factor (NF)-κ B and mitogen-activated protein (MAP) kinases, thereby inducing expression of proinflammatory cytokines and antimicrobial peptides. This proceeds through members of the IL-1 receptor-associated kinase (IRAK) family and tumor necrosis factor (TNF) receptor-associated (TRAF) 6, with the exception of TRIF, which initiates signaling to NF-κ B via direct interaction with TRAF6. In addition to this, TRIF is able to activate signal transduction through TRAF family member-associated NF-κ B activator binding kinase (TBK) 1 and

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inhibitory protein κ B kinase (IKK) ε, which subsequently phosphorylate and activate IFN regulatory factor (IRF) 3. Since induction of IFN-α /β requires IRF 3 and IRF 7, TRIF appears to play a central role as the adaptor molecule that enables TLR 3 and TLR 4 to trigger IFN-α /β expression. TLRs 7 and 9, which are closely related TLRs, signal through mechanisms independent of TRIF, yet are capable of triggering IFN expression. This is due to their ability to recruit IRF7 to the signaling complex, whereby MyD88 forms a complex with IRF7, IRAK4 and TRAF6 and induce transcription of type I IFN genes.20

2.1.1 Double-stranded DNA virus recognition CpG motif-rich DNA sequences are known to activate TLR 9.21 It was first identified that HSV-1 and HSV-2 (dsDNA viruses) stimulate cytokine production through a TLR 9-dependent pathway in pDCs.22 Since TLR 9 is activated in the lysosome, this requires that the viral genetic material be delivered to this compartment. Indeed, purified DNA of HSV-2 does induce cytokine expression in pDCs through TLR 9, and HSV-1 and murine CMV also activate signaling through this receptor,23 strongly suggesting that viral DNA genomes serve as TLR 9 ligands for these herpesviruses. Thus, the endocytic TLRs appear to serve as receptors for viral nucleic acids present within the cellular endocytic compartments during a viral infection. Interestingly, certain isolates of HSV-1 can also stimulate TLR 2 expressed on the surface of macrophages and dendritic cells.24 Since TLR 2 is expressed in brain cells as well, induction of cytokines through TLR 2 leads to encephalitis. Animal experiments indicate that TLR 2 knockout mice are much less susceptible to challenge with HSV-1, and the lack of susceptibility correlates with decreased cytokine production in the brain. In DCs, which express TLR 2 on the cell surface and TLR9 in the lysosome, these HSV-1 isolates are recognized in a serial manner.25 In other words, TLR 2-mediated recognition of HSV-1 at the plasma membrane enables full activation of TLR 9 in the endosome by the same DC.25 VZV can also induce the production of cytokines through a TLR 2-mediated pathway.26 In this case, however, the ability of the virus to elicit cytokine induction, same as the

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ability of the virus to cause disease, is species-restricted, with human cells being capable of producing cytokines in response to VZV infection while mouse cells, even those expressing human TLR 2, do not respond. Human CMV has been shown to stimulate the production of cytokines via a TLR 2-dependent pathway in macrophages.12 In addition, a role for both TLR 3 and TLR 9 in response to MCMV has been reported in knockout mice.27 In a recent study, TLR 3 deficiency in humans was found to be associated with herpes encephalitis.28

2.1.2 Single-stranded RNA virus recognition Among TLR family members, the highest degree of similarity exists between TLR 7 and 8, and they are both involved in recognition of singlestranded RNA viruses.29–31 TLR 7 responds to synthetic imidazoquinoline components (resiquimod and imiquimod) as well as guanosine nucleotide analogues (loxoribine).32 On the other hand, TLR 8 is only functional in humans, but not in mice and it responds to resiquimod but not imiquimod or loxoribine.33,34 Delivery of viral or self-RNA to endosomes is sufficient to activate TLR 7, arguing against the recognition of unique viral nucleic acid motifs as the sole basis for activation. Oligonucleotides containing simple mixtures of free guanosine and uridine nucleosides appear sufficient to activate human PBMC, presumably through TLR 7 or 8.35 In addition, polyuridine was shown to be sufficient to activate both mouse and human TLR 7, suggesting that uridine and ribose, the two defining features of RNA, are necessary for TLR 7 stimulation.36 Based on these findings, it seems unlikely that specific ssRNA motifs serve as the signature of viral infection. Coxsackie B virus has been shown to interact with TLR 8.37 West Nile virus is a mosquito-borne cause of encephalitis. It has been demonstrated that this virus appears to use its interaction with TLR 3 to cause disruption of the blood-brain barrier that leads to viral entry into the brain.38 Without such interaction, TLR 3 knockout mice do not succumb to encephalitis. This effect is presumably mediated by virtue of the ability of this positive strand RNA virus to produce dsRNA. Both influenza and vesicular stomatitis virus (VSV) have been shown to stimulate induction of IFN through a TLR 7mediated pathway.29 Sendai virus, a paramyxovirus, interacts with TLR 7

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and 8 or RIG-I in a cell-dependent manner.39,40 TLR 3 does not seem to play a major role in vivo against infection by VSV.41

2.1.3 Double-stranded RNA virus recognition TLR 3 was the first nucleic acid-specific TLR implicated in viral recognition.11 The dsRNA mimic poly I:C has long been appreciated for its immunostimulatory ability. Although recent work has implicated the cytosolic receptors RIG-I and Mda-5 in detection of Poly I:C, the analysis of knockout mice demonstrated an important role for Mda-5 in responses to intracellular poly I:C.42,43 TLR 3 can mediate activation of immunologically relevant cell types including dendritic cells, macrophages, and B cells. RNA isolated from reovirus has been shown to activate TLR 3 in vitro.11 TLR 3 has also been shown to recognize dsRNA produced during viral infection in the context of phagocytosed dying cells. Based on its dsRNA recognition, somewhat surprising little phenotype has been found to a specific viral challenge using TLR 3-deficient mice. In fact, the lack of a phenotype in TLR 3-deficient mice challenged with a diverse set of viruses has led to speculation that TLR 3 may not be generally involved in antiviral immunity.41 It is still unknown precisely what feature of dsRNA TLR 3 recognizes during a viral infection, especially given its endosomal location. The stimulatory capacity of the synthetic ligand poly I:C argues against recognition of specific sequence motifs. It remains possible that specific secondary structures adopted by certain dsRNAs are necessary for TLR3 stimulation, although a more likely scenario seems to be that the dsRNA backbone itself may be recognized by TLR 3. A recent study indicated that RNaseL generates ligands for RIG-I and Mda-5 by cleaving self RNA.44 Crystal structures of TLR 3 demonstrated that it is largely masked by carbohydrates, with one face glycosylation-free, suggesting its potential role in ligand binding and oligomerization.45 There were two patches of positively charged residues on TLR 3, which might provide an appropriate binding site for double-stranded RNA. In another study, it was found that mutation of H539E and N541A resulted in the loss of TLR 3 activation and ligand-binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR 3 toward the C-terminus of the ectodomain and could represent a dsRNA binding site.46

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2.1.4 Non-nucleic acid-based TLR activation by viruses It is important to consider several notable examples of viral recognition by TLRs that do not involve nucleic acids.47 In particular, a number of viruses appear to have the ability to stimulate TLR 2 or 4. Both HSV-1 and HSV-2 can activate TLR 2, although the exact viral proteins binding TLR 2 have not been identified.24 TLR 2 has also been implicated in the recognition of HCMV and MCMV.12,48 In addition, the hemagglutinin protein of measles virus is able to stimulate TLR 2.13 Finally, respiratory syncytial virus (RSV) and mouse mammary tumor virus (MMTV) have been shown to activate TLR 4 in a CD14-dependent fashion.14 CD14, a glycosyl phosphatidyl inositol-anchored cell surface protein has been known for its interaction with LPS.49 In the absence of CD14, mice do not develop septic shock when exposed to LPS.50 A logical explanation for this phenomenon was the hypothesis that CD14 could bind to LPS and function as a transporter to TLR 4 which then mediated signaling events. Since CD14 also exists as a soluble protein, and is present in serum, cell surface expression was not necessary for this phenomenon to occur. With respect to viruses, RSV has been shown to signal through TLR 4. At the same time, it was noted that the signal transduction events associated with triggering via RSV were, like those triggered by LPS, amplified by CD14. A number of other viruses such as LCMV51 and CMV12 have also been demonstrated to interact with CD14. The exact nature of this interaction and how it is related to TLR signaling is still not well defined.

2.2 Cytosolic Viral Recognition 2.2.1 RIG-I/Mda-5-mediated recognition of ssRNA viruses RNA helicases such as RIG-I and Mda-5 with two amino-terminal signaling domains and a carboxy-terminal helicase domain become induced in response to IFN stimulation.52,53 These helicases are kept in an inactive state in the absence of ligand. Viral infection results in the introduction of RNA replication intermediates, which are recognized by the helicase domain and activate through a mechanism dependent on the ATPase activity

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of the helicase domain. It has been reported that NF-κ B and IRF-3 become activated in response to activation of RIG-I.53 The ability to activate IRF3 and induce type I IFN expression revealed a critical role for these RNA helicases in antiviral defense. RIG-I recognizes a specific set of RNA viruses (Paramyxoviridae, Flaviviridae, and Rhabdoviridae), whereas Mda-5 is responsible for the antiviral defense against RNA viruses of the Picornaviridae family.1,43 Recently, it was demonstrated that intracellular poly(I:C) is a ligand for Mda-5 but not RIG-I, whereas long cytosolic dsRNA was found to activate RIG-I but not Mda-5.42,43 It has been shown that uncapped 5′-triphosphate RNA (3pRNA) generated by viral polymerases is detected by RIG-I.54 The adaptor molecule that connects RIGI sensing of incoming viral RNA to downstream signaling and gene activation has recently been elucidated by four independent groups,55–58 and has been ascribed four different names: mitochondrial antiviral signaling (MAVS), IFN-β promoter stimulator 1 (IPS-1), virus-induced signaling adaptor (VISA) and Cardif. Overexpression of IPS-1 activates the IFN-α, IFN-β and NF-κ B promoters, and TBK-1 is required for the activation of these promoters.55 Similar to RIG-I, IPS-1 consists of an N-terminal CARD domain and a C-terminal effector domain that recruits the adaptor Fas-associated death domain protein (FADD) and the kinase receptor interacting protein 1 (RIP1). The same RIG-I adaptor molecule, MAVS, was shown to utilize C-terminal transmembrane domain, in addition to its essential role in RIG-I-dependent signaling, to localize MAVS to the mitochondrial membrane, thus suggesting a novel role for mitochondrial signaling in the cellular innate response. Later, another group demonstrated that VISA is a crucial component of IFN-β signaling.58 VISA interacts with TRIF (also known as TICAM-1), TRAF 2 and 6 through a proline-rich domain, suggesting that VISA might mediate the bifurcation of the NF-κ B and IRF-3 activation pathways and have an essential role in the antiviral response through both TLR 3 and RIG-I virus-triggered pathways. Finally, Cardif, the same RIG-I adaptor molecule, was shown to interact with RIG-I and recruit IKKα, IKKβ and IKKε kinases through its C-terminal region.56 Overexpression of Cardif results in IFN-β- and NF-κ B-promoter activation, and knockdown of Cardif by short-interfering RNA (siRNA) inhibited RIG-I-dependent antiviral responses.

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2.2.2 DNA virus recognition Stetson et al., demonstrated that the presence of DNA from Listeria monocytogenes bacterium activates a receptor that was shown to be independent of TLR 9 and RIG-I/Mda-5 or NF-κ B. In addition, B-form dsDNA, when introduced into cytosol, can trigger a similar pathway.59 A recent study revealed that DAI (DLM-1/ZBP1), a cytoplasmic recognition receptor activated by DNA from a variety of sources (viral, bacterial and mammalian), leading to type I IFN gene induction through the activation of IRF3 and probably, IRF7.60 Notably, unlike the RNA sensor RIG-I, which becomes constitutively active in the absence of its RNA interaction domain, DAI (DLM-1/ZBP1) may require both its amino-terminal DNA-binding region and carboxy-terminal TBK1/IRF binding region for its activity. Therefore, DNA is not only critical to initiate but also to sustain the active signaling complex. Gene knockout mice studies will help identify the contribution of the cytosolic DNA-mediated and/or virus-mediated activation of innate immune responses.

3. AUTOPHAGY IN INNATE ANTIVIRAL IMMUNITY Autophagy, a process involved in nutrient acquisition during starvation and maintenance of quality control of long-lived proteins,61 is now being recognized to restrict viral infections and replication of intracellular bacteria and parasites.62–65 Additionally, this pathway delivers cytoplasmic antigens for MHC class II presentation to the adaptive immune system.66 Autophagy is composed of at least three distinct pathways: microautophagy, chaperone-mediated autophagy, and macroautophagy.61 Microautophagy involves budding of small cytosol-containing vesicles directly in lysosomal lumen, while in chaperone-mediated autophagy, proteins are directly imported into lysosomes via the LAMP-2a transported assisted by cytosolic and lysosomal chaperones. On the other hand, in macroautophagy, a cup-shaped isolation membrane encloses cytosolic constituents called autophagosome, which then fuses with lysosomes for degradation of the engulfed cargo. Multiple proteins such as members of the autophagyrelated genes (Atg) and enzymes such as class III phosphatidylinositol

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3-kinase (PI3K) are actively involved in the macroautophagy process. Autophagy can be divided into three stages: initiation, execution and maturation. The initiation of autophagy can be triggered by a variety of extracellular signals such as nutrient starvation and treatment with hormones that target TOR (target of rapamycin), a kinase that inhibits the autophagic pathway. Upon inactivation by dephosphorylation of TOR, execution stage of autophagy is mediated by two covalent-conjugation pathways: the covalent linkage of Atg5 and Atg12, and the covalent lipidation of Atg8 by phosphatidylethanolamine.67 The second conjugation pathway results in the covalent addition of the lipid phosphatidylethanolamine to the newly generated carboxyl-terminus of microtubule-associated-protein light-chain 3 (LC3). LC3 remains associated with autophagosomes until destruction in the autolysosome. Replication of many viruses is associated with specific intracellular compartments called virus factories or virioplasm.68 The nidovirales and picornaviridae are positive-stranded RNA viruses that assemble a replication complex containing the RNA polymerase, as well as proteins with helicase and nucleotide triphosphase activity, on the cytoplasmic face of cellular membranes. Viruses may also induce autophagy to generate a scaffold for the replication complex. This hypothesis is demonstrated in a couple of studies, wherein loss of Atg proteins resulted in a drop in yields of coronavirus69 and poliovirus.70 However, in the case of coronavirus infection, it was recently shown that Atg5 deficient primary cells support normal viral replication,71 and that autophagy is dispensable for coronavirus replication in vivo.

3.1 Autophagy in Innate Effector Responses Macroautophagy also serves as an innate effector mechanism against viral infections. Restriction of HSV-1 infection by macroautophagy in vitro and in vivo was found to be dependent on the type I IFN-inducible dsRNAdependent PKR.72,73 In order to develop neurovirulence, HSV-1 carries the ICP34.5 protein, which inhibits PKR-dependent macroautophagy induction by binding to Beclin-1.74 Recently, it was shown that HSV-1 lacking ICP34.5 gene are attenuated in growth and pathogenesis in animal models and in primary cultured cells.75 This growth defect has been attributed

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to the inability of an ICP34.5-deficient virus to counteract the induction of translational arrest through the PKR antiviral pathway. Macroautophagy induction via TNF family members has been established in several celldeath research studies. TNF-α was found to upregulate macrophagy in cells lacking NF-κ B activation. TNF-related apoptosis-inducing ligand (TRAIL) has also been shown to induce autophagy in human epithelial cells.76 Consistent with this, inactivation of Fas-associated death domain (FADD), the signaling adaptor protein of the TRAIL receptor, decreases autophagy induction by TRAIL.77 It is conceivable that the cells of the innate and adaptive immune systems could potentially use this macroautophagy induction mediated by TNF family (including TNF-α secreting dendritic cells, TRAIL-expressing NK and T cells) to mediate antiviral defense.

3.2 Autophagy and Viral Recognition A recent study demonstrated the role of autophagy in innate recognition of viral replication products. Plasmacytoid cells, a predominant IFN producing cells responds to DNA viruses such as HSV and CMV through TLR 9 while single-stranded RNA viruses such as influenza and VSV respond through TLR 7.9 Many such viruses are detected in the absence of viral replication. However, VSV and Sendai virus appear to require live viral infection.78 It was shown that recognition of VSV by pDCs occurred in the acidified endosomal/lysosomal compartment via TLR 7.28 How could the cytosolic replication event be required for triggering a receptor in the lysosome? We demonstrated that response to VSV live infection was diminished in pDCs deficient in autophagy, revealing the necessity for viral recognition through autophagy.78 Thus, TLR 7 recognizes, in addition to viral genome nucleic acid that has been taken up through the endocytic pathway (such as in influenza virus), signatures of viral replication in the cytosol by way of autophagy. An additional piece of information we obtained from this study was that not only is autophagy required for viral recognition of VSV, it was also required to induce IFN production in pDCs. This was revealed by the fact that in response to HSV or CpG (TLR9 agonists), Atg5-deficient pDCs failed to secrete IFN-α despite normal levels of IL-12 production.

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Table 1. Molecular Signatures of Viral Recognition. PRRs TLR2

Viral Components Viral proteins

TLR3

dsRNA

TLR4

Viral proteins

TLR7/8

ssRNA

TLR8

CpG DNA

RIG-I

5′ triphosphate RNA/dsRNA

Mda-5

dsRNA

Viruses

References

Herpes simplex viruses (HSV) Mouse and Human cytomegalovirus (MCMV/HCMV) Lymphocytic choriom eningitis virus (LCMV) VZV Reovirus West Nile Respiratory syncytial virus (RSV) Mouse mammary tumor virus (MMTV) Coxsackie B Influenza VSV Sendai HSV MCMV Newcastle disease virus Sendai Influenza Japanese encephalitis virus VSV Encephalomyocarditis Theiler’s virus Mengo virus

22, 23 12, 48 51 26 11 38 14 15 37 29, 30 29 29, 39 22, 89 23 79 79 43 43 79 42, 43 43 43

The implication of our finding is that TLR 7 must be able to somehow distinguish self RNA from non-self RNA that is delivered via autophagy into the lysosome. The nature of the ligand of viral replication intermediates that is detected by TLR 7 is not known. Furthermore, how Atg5 is required for the induction of IFN-α is unclear. Studies to address these and other questions are expected to reveal the mechanisms by which autophagy and Atg5 is involved in viral recognition in pDCs. In non-pDCs, RIG-I mediates recognition of VSV.79 To this end, a recent report has indicated that Atg5-Atg12 complex is involved in the

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regulation of RIG-I IPS-1 signaling.80 Atg5-Atg12 conjugate directly interacts with both IPS-1 and RIG-I and inhibits transmission of CARDmediated signaling, ultimately resulting in a suppression of immunostimulatory RNA-mediated type I IFN production and subsequent innate antiviral immune responses. Thus, in contrast to the requirement of autophagy in ssRNA recognition by TLR 7 in pDCs, RIG-I mediated recognition of VSV is negatively regulated by the machinery used in autophagy. It will be important to determine the etiology by which molecules involved in autophagy are utilized to keep IPS-1-dependent signaling at bay.

3.3 Antigen Presentation In addition to limiting pathogen replication in host cells, autophagy also delivers bacterial, viral and parasitic antigens to late endosomal compartments. Amphisomes, a fusion between autophagosomes and lysosomes, structurally resemble the major histocompatibility complex (MHC) class II loading compartments (MIICs), which are now considered to be conventional late endosomes that are equipped within the molecular machinery to load antigenic fragments onto MHC class II molecules for presentation to CD4+ T cells.81 It has been shown that autophagosomes fuse with MIICs, as identified by the presence of MHC class II, and the lysosomal membrane protein LAMP-2.82 This cellbiological evidence suggests that autophagy frequently delivers autophagosomal content, including pathogen-derived proteins, to MIICs. Biochemical studies in isolation of natural MHC class II ligands revealed characteristics of autophagy substrates. Some proteins, which appear to be preferentially degraded by autophagy have been frequently found to give rise to MHC class II peptide cargo. For example, nuclear antigen 1 of Epstein-Barr virus (EBV) is presented on MHC class II after macroautophagy.83 Similarly, influenza matrix protein 1 (MP1) is intracellularly processed onto MHC class II.84 Studies using pharmacological inhibition of autophagosome formation revealed decreased MHC II presentation of complement C5 protein in mouse macrophages and B cells.85 It has also been shown that viral antigen EBNA1 is processed intracellularly for MHC class II presentation to CD4+ T cells via

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macroautophagy in EBV-transformed B cells and EBNA-1 transfected EBV-negative Hodgkin’s lymphoma cells.66,86 Collectively, these data suggest that several antigens can enter the MIIC via macroautophagy and that this pathway leads to efficient MHC class II presentation to CD4+ cells. In addition to macroautophagy, chaperone-mediated autophagy has also been implicated in delivering cytosolic antigens for MHC class II presentation.87,88

4. CONCLUSION The discovery of PRRs as essential components of the innate immune system has greatly advanced our knowledge and understanding of immune responses to infection and how these are regulated. Innate immunity in general and TLRs in particular not only play a crucial role in the front line of host defenses against microbes, but also provide a key link to the adaptive immune system at large in vertebrate animals. It has become clear that autophagy is a fundamental and general process which plays a role in viral infections. Their role in innate and adaptive immunity will benefit in understanding the role of defense against pathogens. Moreover, viruses have evolved strategies to counteract autophagy mechanism to ensure survival, while some use autophagosomes for replication. Unraveling the specific role of autophagy in different viral infections will undoubtedly reveal the mechanism by which this ancient pathway is involved in antiviral immunity.

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10. Cornelie S, Hoebeke J, Schacht AM, Bertin B, Vicogne J, Capron M, Riveau G (2004). J Biol Chem 279, 15124–15129. 11. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA (2001). Nature 413, 732–738. 12. Compton T, Kurt-Jones EA, Boehme KW, Belko J, Latz E, Golenbock DT, Finberg RW (2003). J Virol 77, 4588–4596. 13. Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, Duprex WP, Wagner H, Kirschning CJ, Ter Meulen V, Schneider-Schaulies S (2002). J Virol 76, 8729–8736. 14. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, Finberg RW (2000). Nat Immunol 1, 398–401. 15. Jude BA, Pobezinskaya Y, Bishop J, Parke S, Medzhitov RM, Chervonsky AV, Golovkina TV (2003). Nat Immunol 4, 573–578. 16. Kawai T, Akira S (2007). TLR Semin Immunol 19, 24–32. 17. Lee MS, Kim YJ (2007). Mol Cells 23, 1–10. 18. Uematsu S, Akira S (2007). J Biol Chem 282, 15319–15323. 19. Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, Yamamoto A, Seya T (2003). J Immunol 171, 3154–3162. 20. Honda K, Yanai H, Mizutani T, Negishi H, Shimada N, Suzuki N, Ohba Y, Takaoka A, Yeh WC, Taniguchi T (2004). Proc Natl Acad Sci USA 101, 15416–15421. 21. Krieg AM (2002). Annu Rev Immunol 20, 709–760. 22. Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A (2003). J Exp Med 198, 513–520. 23. Krug A, French AR, Barchet W, Fischer JA, Dzionek A, Pingel JT, Orihuela MM, Akira S, Yokoyama WM, Colonna M (2004). Immunity 21, 107–119. 24. Kurt-Jones EA, Chan M, Zhou S, Wang J, Reed G, Bronson R, Arnold MM, Knipe DM, Finberg RW (2004). Proc Natl Acad Sci USA 101, 1315–1320. 25. Sato A, Linehan MM, Iwasaki A (2006). Proc Natl Acad Sci USA 103, 17343–17348. 26. Wang JP, Kurt-Jones EA, Shin OS, Manchak MD, Levin MJ, Finberg RW (2005). J Virol 79, 12658–12666. 27. Delale T, Paquin A, Asselin-Paturel C, Dalod M, Brizard G, Bates EE, Kastner P, Chan S, Akira S, Vicari A, Biron CA, Trinchieri G, Briere F (2005). J Immunol 175, 6723–6732.

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28. Zhang SY, Jouanguy E, Ugolini S, Smahi A, Elain G, Romero P, Segal D, Sancho-Shimizu V, Lorenzo L, Puel A, Picard C, Chapgier A, Plancoulaine S, Titeux M, Cognet C, Von Bernuth H, Ku CL, Casrouge A, Zhang XX (2007). Science 317, 1522–1527. 29. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA (2004). Proc Natl Acad Sci USA 101, 5598–5603. 30. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis eSoC (2004). Science 303, 1529–1531. 31. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S (2004). Science 303, 1526–1529. 32. Sato S, Takeuchi O, Fujita T, Tomizawa H, Takeda K, Akira S (2002). Int Immunol 14, 783–791. 33. Heil F, Ahmad-Nejad P, Hemmi H, Hochrein H, Ampenberger F, Gellert T, Dietrich H, Lipford G, Takeda K, Akira S, Wagner H, Bauer S (2003). Eur J Immunol 33, 2987–2997. 34. Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, Qiu X, Tomai MA, Alkan SS, Vasilakos JP (2005). J Immunol 174, 1259–1268. 35. Jurk M, Kritzler A, Schulte B, Tluk S, Schetter C, Krieg AM, Vollmer J (2006). Eur J Immunol 36, 1815–1826. 36. Diebold SS, Massacrier C, Akira S, Paturel C, Morel Y, Reis eSoC (2006). Eur J Immunol 36, 3256–3267. 37. Triantafilou K, Orthopoulos G, Vakakis E, Ahmed MA, Golenbock DT, Lepper PM, Triantafilou M (2005). Cell Microbiol 7, 1117–1126. 38. Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA (2004). Nat Med 10, 1366–1373. 39. Melchjorsen J, Jensen SB, Malmgaard L, Rasmussen SB, Weber F, Bowie AG, Matikainen S, Paludan SR (2005). J Virol 79, 12944–12951. 40. Rothenfusser S, Goutagny N, DiPerna G, Gong M, Monks BG, Schoenemeyer A, Yamamoto M, Akira S, Fitzgerald KA (2005). J Immunol 175, 5260–5268. 41. Edelmann KH, Richardson-Burns S, Alexopoulou L, Tyler KL, Flavell RA, Oldstone MB (2004). Virology 322, 231–238. 42. Gitlin L, Barchet W, Gilfillan S, Cella M, Beutler B, Flavell RA, Diamond MS, Colonna M (2006). Proc Natl Acad Sci USA 103, 8459–8464. 43. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T,

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65. Levine B (2005). Cell 120, 159–162. 66. Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, Tuschl T, Munz C (2005). Science 307, 593–596. 67. Yorimitsu T, Klionsky DJ (2005). Cell Death Differ 12 (Suppl 2), 1542–1552. 68. Wileman T (2006). Aggresomes. Science 312, 875–878. 69. Prentice E, Jerome WG, Yoshimori T, Mizushima N, Denison MR (2004). J Biol Chem 279, 10136–10141. 70. Jackson WT, Giddings TH, Jr., Taylor MP, Mulinyawe S, Rabinovitch M, Kopito RR, Kirkegaard K (2005). PLoS Biol 3, 861–871. 71. Zhao Z, Thackray LB, Miller BC, Lynn TM, Becker MM, Ward E, Mizushima NN, Denison MR, Virgin HW 4 t (2007). Autophagy 3 (Epub). 72. Talloczy Z, Virgin HW 4 t, Levine B (2006). Autophagy 2, 24–29. 73. Talloczy Z, Jiang W, Virgin HW, Leib DA, Scheuner D, Kaufman RJ, Eskelinen E-L, Levine B (2002). Proc Natl Acad Sci USA 99, 190–195. 74. Orvedahl A, Alexander D, Talloczy Z, Sun Q, Wei Y, Dennis ZB, Leib DA, Levine B (2007). Cell Host Microbe 1, 23–35. 75. Alexander DE, Ward SL, Mizushima N, Levine B, Leib DA (2007). J Virol 81, 12128–12134. 76. Mills KR, Reginato M, Debnath J, Queenan B, Brugge JS (2004). Proc Natl Acad Sci USA 101, 3438–3443. 77. Thorburn J, Moore F, Rao A, Barclay WW, Thomas LR, Grant KW, Cramer SD, Thorburn A (2005). Mol Biol Cell 16, 1189–1199. 78. Lee HK, Lund JM, Ramanathan B, Mizushima N, Iwasaki A (2007). Science 315, 1398–1401. 79. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, Matsui K, Tsujimura T, Takeda K, Fujita T, Takeuchi O, Akira S (2005). Immunity 23, 19–28. 80. Jounai N, Takeshita F, Kobiyama K, Sawano A, Miyawaki A, Xin KQ, Ishii KJ, Kawai T, Akira S, Suzuki K, Okuda K (2007). Proc Natl Acad Sci USA 104, 14050–14055. 81. Zwart W, Griekspoor A, Kuijl C, Marsman M, Van Rheenen J, Janssen H, Calafat J, Van Ham M, Janssen L, Van Lith M, Jalink K, Neefjes J (2005). Immunity 22, 221–233. 82. Menendez-Benito V, Neefjes J (2007). Immunity 26, 1–3. 83. Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Muller M, Kreymborg K, Altenberend F, Brandenburg J, Kalbacher H, Brock R, Driessen C,

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CHAPTER 10

VSV Infection Elicits Distinct Host Responses in the Periphery and the Brain Carol Shoshkes Reiss

ABSTRACT Vesicular stomatitis virus (VSV), a natural epizootic among farm animals which is spread by sand-flies, has been used for experimental acute infections of mice since the 1930s when Sabin and Olitzky did pioneering investigations. This chapter will summarize the contributions of many laboratories to our understanding of host innate and adaptive immune responses, and viral evasion of innate responses. In addition, the potential power of this virus for vaccine platforms and oncolysis will be discussed. The virus has an evasive strategy which inhibits host cell gene expression. VSV readily elicits Type I Interferon (IFN) responses in the periphery, but fails to trigger this critical antiviral response in the CNS. VSV is a deceptively simple virus whose study has led to unexpected insights into the complexities of cell biology and host responses to infection.

1. INTRODUCTION 1.1 Description of Virus, Agricultural Importance, and Transmission VSV is a bullet-shaped, single-stranded, non-segmented, enveloped negativesense RNA virus of the Rhabdovirus family. Replication is exclusively cytoplasmic.1 229

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VSV has just five translated gene products: Nucleoprotein (N) which binds to the RNA genome; Matrix Protein (M) with many biological functions; Phosphoprotein (P); a Glycoprotein (G) which is expressed as a Type 1 glycoprotein in homotrimers; and Large protein (L). The RNAdependent RNA polymerase is comprised of the L and P proteins.1 The M protein is post-translationally modified by a single ubiquitin moiety, while the G is both modified by oligosaccharide side-chains and palmitoylated, and both M and P are phosphorylated.2–5 In nature, VSV is an arbovirus, transmitted among domestic livestock by sand flies. Transmission is associated with acute, self-limited infections, which have the characteristic eponymous oral rash.6 This symptom is also found in other infections, including the picornavirus Hand, Foot and Mouth Disease, thus distinguishing among the causative agents critical in the handling of infected herds. There are two serotypes, Indiana and New Jersey, which are distinguished by epitopes on the glycoprotein. VSV is not a human pathogen. Humans readily seroconvert when bitten by infected sand flies, but are generally asymptomatic.7–10 It is rare to find people who are immune to the virus. This characteristic may be exploited in vaccines and in novel treatments for cancer. Closely related to VSV are Lyssaviruses, including Rabies. There are Rhabdoviruses of many species, including fish.1 Many of these viruses are neurotropic.

1.2 Relevance and Cell Biology Studies VSV has been studied extensively in cell biology, basic immunology, pathogenesis, vaccine vector, and oncolysis studies for more than 70 years. Its agricultural/veterinary medical importance, the relative safety of studying it as a model for the more pathogenic Rabies virus, the ease of reverse genetics, and its wide host range have contributed to its value in basic and translational research. The cloning of the viral genome11 has led to extensive studies in reverse genetics, pseudotyping, and vaccine generation (to be discussed in 4.1). The virus is exquisitely sensitive to the antiviral activities of interferons (IFNs; discussed below in 2.4) and thus, tumor cells which have impaired IFNdependent responses, are susceptible to VSV replication (see 4.2).

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It is easy to propagate in vitro and replicates in cells ranging from established lines to primary cultures, from insects to mammalian hosts. The laboratory safety of the model is enhanced by the fragile nature of the enveloped virus as it is susceptible to inactivation by exposure to soap and water, alcohol, by drying or UV irradiation. At high multiplicities of infection, defective interfering particles are generated;12 these modulate viral virulence but still elicit host responses.13,14 In contrast to the apical replication of many other viruses (e.g., influenza), the traffic of the G protein in polarized epithelial cells to the basolateral face results in basolateral budding and invasiveness.15–17 This characteristic is important in the murine model of encephalitis.

1.3 Historic Murine Studies Pioneering studies on VSV pathogenesis in mice were conducted by Sabin and Olitsky in the 1930s.18–21 They observed an age-dependent susceptibility, where young hosts were more likely to develop disease and succumb than are older hosts. These investigations were followed by many others, which were aided by pure virus and the development of inbred strains of mice. As the study of viral pathogenesis and host immunology became more nuanced and sophisticated, VSV infections have been used extensively to expand our understanding of these mechanisms. More recently, the contributions of distinct pathways have been elucidated with immunocompromised, knockout and transgenic mice as hosts. I will distill many of these observations in the balance of this review. Essentially, administration of virus parenterally (routes include intraperitoneal, intramuscular, subcutaneous, intravenous) to immunocompetent mice results in induction of both innate and adoptive immune responses in the absence of disease; in fact, it is extremely difficult to isolate infectious virus two days after administration of quantities as large as 108 p.f.u. However, injection of only a few p.f.u. intraperitoneally to scid/scid, nu/nu, or other immunocompromised hosts is rapidly fatal.22–25 Intranasal (i.n.) administration of infectious virus leads to infection of the olfactory sensory neurons and retrograde infection of the CNS via the olfactory nerve.26–29 In the olfactory neuroepithelium, as a result of the acute infection, there is degeneration and regeneration of the tissue from

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local stem cells (Eng, Palian, Stolberg, Talamo & Reiss, in preparation). Functionally, this is associated with transient anosmia. Additionally, i.n. administration of small volumes containing 200 p.f.u. to immunocompetent mice leads to transient dissemination of infectious virus to lung, spleen and lymph nodes,30–32 and also viral encephalitis. Death ensues in approximately half of the infected hosts, due to breakdown of the blood-brain barrier (BBB), infection of motor neurons of the lumbar-sacral spinal chord, or infection of the locus ceruleus.27–29,33 This distinction has been the focus of my research for the last decade.

2. INNATE IMMUNITY Innate immune responses are immediate, invariant and enormously diverse and complex. These include pre-existing and newly produced proteins, cellular responses (cell autonomous pathways, secreted products, activation), changes in vascular permeability, and recruitment of circulating cells by a variety of inflammatory mediators. Some responses are systemic, others local. The exclusive properties of the BBB regulate the availability of some systemic responses in the CNS during VSV encephalitis. While many components of the innate immune response are beneficial and promote containment and recovery, others may contribute to pathology or are antagonistic to inhibitory pathways. The use of VSV has helped elucidate many important cellular pathways, most recently is the contribution of host microRNAs as antiviral effectors, as dicer-deficient cells and mice are hypersensitive to VSV infection.34 The importance and effectiveness of these diverse innate pathways to regulate the early stages of infection have led to the development of evasive pathways by many viruses, with VSV being one of them.

2.1 Cells Both cells in organs and circulating cells of the reticuloendothelial system play essential roles in the innate immune responses to viral infections. I will describe the contributions of some of these in VSV encephalitis. The kinetics of the entry of cells to the CNS are described in the following.

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2.1.1 Plasmacytoid dendritic cells PDC in the lymph nodes and spleen are sentinels, the first cells to respond during VSV infections, irrespective of the route of administration. PDC are triggered by Pathogen-Associated Molecular Patterns via Toll-Like receptors (TLR) 3, 7, and 8 signaling through MyD88 in endocytic vesicles. In addition, PDC may be stimulated by the RNA helicases MDA5 and RIG-I which use the adapter IPS-1 in cytoplasm. The result of PDC activation is the production of Type I IFNs within the first 24 hours of exposure.35–40 Additional evidence suggests that a TLR4-dependent pathway may also trigger PDC.41,42 The level of IFN-α /β is sufficiently high in circulation to be detected by ELISA or bioassay.30 Dendritic cells not only produce IFNs, they also present viral Ags to CD4 T cells and produce cytokines.43 The maturation of myeloid dendritic cells is stimulated by VSV M protein.44 PDC are not present in the uninfected brain45 but are recruited and/or differentiated from precursors, late in the course of VSV infection.30 This Type I IFN may regulate both expansion of system innate responses and the development of the acquired immune response to viral infection; however, IFN α /β produced in the periphery cannot cross the BBB.46 The delay in the IFN response in the brain plays a major factor in the ability of VSV to invade the CNS.

2.1.2 Neutrophils PMN are rapidly recruited from circulation to the site of VSV infection.47–49 Among the chemoattractants for PMN are the anaphylatoxin complement fragments (especially C5a), chemokines, and LTB4.48–51 These cells produce many inflammatory mediators and promote clearance and containment. Antibody depletion of PMN or the blockage of the enzyme responsible for LT synthesis is detrimental to host responses to VSV encephalitis.49

2.1.3 Natural killer cells, macrophages/microglia, astrocytes The second circulating cell type to be recruited to the CNS in response to VSV infection is the NK cell.52 Like the neutrophil, it is present

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transiently and is an important inflammatory participant. Both chemokines and IL-12 are responsible for NK attraction. Tissue macrophages in the CNS parenchyma are called microglia. They are readily activated during VSV encephalitis.53 Among their products are eicosinoids, cytokines, chemokines, complement components, reactive oxygen and nitrogen intermediates. Circulating monocytes enter the CNS at about day 7 p.i. and are found in a perivascular distribution.54 This cell type has been more extensively studied in experimental allergic encephalomyelitis and bacterial infections than in VSV encephalitis. Astrocytes play many supporting roles in the CNS. While they are immediately reactive to VSV infection, as indicated by induction of NOS-3 and GFAP, the mechanism(s) of communication between infected neurons and adjacent astrocytes are unknown.55 They secrete cytokines, chemokines, eicosinoids, reactive oxygen and nitrogen products. In addition, they are intimately involved in the regulation of the BBB, via the apposition of astrocyte end-feet on the brain microvascular endothelial cells.

2.1.4 Neurons Although other cell types in the CNS parenchyma can be infected with VSV, disease is manifested by initial infection of olfactory receptor neurons and retrograde transport, probably along synapses. Unlike other cell types, neurons (with some few exceptions) are incapable of expressing major histocompatibility antigens56 and are thus precluded as potential cytolytic T cell targets. Note that in the periphery, CTLs are very effective at eliminating viral infections by killing infected cells. Neurons (with the exception of the olfactory neuroepithelium and rare stem cells) are nonrenewing and their loss would potentially result in loss of their cognitive or motor function. Therefore cell autonomous antiviral pathways are critical in neurons to eliminate VSV replication and clear infection. One of the most important pathways is Nitric Oxide Synthase-1 (NOS-1)-dependent production of NO. The enzyme is constitutively expressed because NO is an important neurotransmitter. NOS-1 is positively regulated by neuronal activity-driven Ca2+ release. The activity of this pathway is enhanced by inflammatory cytokines (IFN-γ , TNF-α, and

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IL-12).57–59 In fact, for IFN-γ , this is the only effector pathway, since 7-nitroindazole-inhibition of NOS-1 reverses the antiviral activity both in vitro and in vivo.57,60,61

2.2 Cytokines, Chemokines The roles of chemokines and cytokines in host responses in vitro and in vivo to VSV challenges have been studied in several labs. In both C57Bl/6 mice and BALB/c, which are characteristic of Th1 and Th2 responses to other viruses, respectively, VSV elicits a Th1 inflammatory cytokine response.49,52,62–65 Administration of IL-12 to mice, if within the first 48 hours after infection, promotes clearance of virus, survival and recovery.60,66,67 IL-12 and IFN-γ are effective as they activate the accumulation of NOS-1 which exerts antiviral activity in neurons.54,57,58,68–72 However, IFN-γ , IL-12, and IL-23 are not required for host recovery.71,73,74 IL-18 is expressed in the CNS constitutively, but does not have an effect when administered to mice.75 RNAse protection has been used to study the expression of cytokine and chemokine genes in the CNS following VSV infection, and largely confirms the expectations of responses associated with recruitment of infiltrating cells.49

2.3 Lipid Mediators Eicosinoids are often overlooked by investigators focused on gene expression or protein blots. But these effector molecules are potent in immunomodulation and inflammation. Among the critical mediators are prostaglandins (e.g., PGE2, PGJ2), leukotrienes (e.g., LTB4), and cannabinoids (endocannabinoids include arachidonic ethanolamide). The contributions of these small molecules have been determined by pharmaceutical treatments (inhibitors of Cyclooxygenase or Lipoxygenase) and/or by examining pathogenesis in knockout hosts. PGE2 is a potent molecule which is not only associated with pain and fever, opening the BBB via activation of VEGF production, it also antagonizes the production of NO, thus suppressing the ability of the neuron to suppress viral replication.51,76,77 In the periphery, PGE2 can inhibit T cell responses. PGJ2 which is an activating ligand for the anti-inflammatory

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transcription factor PPAR-γ , limits pathology associated with the inflammatory response to infection.38 Cannabinoids are immunoregulatory. Thus, in the CNS it may regulate appetite, euphoria, and pain perception. In neurons, because Ca2+ mobilization is inhibited following activation of CB1 receptors by agonists, production of NO is inhibited, and thus higher yields of virus were observed (Herrera, Oved & Reiss, submitted). LTB4 which is one of the chemoattractant molecules for neutrophils enhances the host’s systemic response to the local CNS infection.48,49,77

2.4 IFNs IFN receptor knockout studies by the Zinkernagel laboratory showed a profound requirement for Type I IFN responses, but not for IFN-γ R.78,79 When STAT1 or downstream effectors such as PKR, RNAse L have been knocked out, mice showed substantial increase of susceptibility to lethal infection, even from intraperitoneal administration of virus.23,24,80–83 IFN-γ responses may contribute to the increased number of females surviving from infection84 (Chen, Hodges & Reiss, unpublished), which may also be attributable to the presence of an estrogen-response element in the IFN-γ promoter.85 Type I IFN is rapidly made by plasmacytoid dendritic cells in the lymph nodes and spleen following intranasal infection (see Section 2.1.1, above),35–40 and the PDC appear to require either lymphatic or vascular exposure of infectious virus to trigger this response.30 IFN-α /β is ∼100-fold more potent as an antiviral effector than Type II IFN.30 It does not use the NOS-1-dependent pathway (or PKR, RNAaseL),86 but appears to alter posttranslational modification of viral proteins, interfere with viral particle assembly and release from infected neurons (D’Agostino, Amenta, Botwinick & Reiss, submitted). Type I IFN cannot cross the BBB46 and is not synthesized in the CNS early in the course of disease,49 when it might have an antiviral effect.

2.5 Evasion by M protein M protein has many biological functions including a principle role in viral assembly. M protein associates with the nuclear pore complex and inhibits

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nuclear export of cellular mRNAs, effectively shutting off host responses, including synthesis of IFNs.87–90 This is associated with suppression of alert to neighboring cells and is a pathogenic factor for host infection.30 Another cell autonomous effect is the targeting of mitochondria91 and induction of apoptosis.92–97 Some engineered mutations in M, including M51R, prevent the nuclear pore blockade and are being exploited in oncolytic therapies (see Section 4.2).

3. ACQUIRED IMMUNITY In contrast to innate immunity, which is invariant, reflexive, and in many cases preformed, adaptive immunity is highly specific to host recognition of pathogen peptides by T cell receptors (TCR) and antibody (Ab). VSV proteins are processed by antigen presenting cells including dendritic cells and macrophages, and presented to CD8+ or CD4+ T cells, which recognize peptide fragments associated with major histocompatibility complex (MHC) I or II molecules, respectively.98

3.1 T cells 3.1.1 Ag processing and presentation; MHC crystalline structure Even before the structure of MHC molecules were known,99 studies have indicated the requirements of MHC α 1 and α 2 domains on H-2Ld, the exclusive restriction molecule for VSV N protein in the BALB/c mouse, which were essential for presentation of VSV to CD8+ T cells.100–106 Other studies mapped the peptide sequences,107,108 MHC II-restriction, and cellular pathways necessary to present VSV G protein to CD4+ T cells.109–111

3.1.2 CD4 and CD8 CTL Among the earliest in vitro studies were the assessment of cytolytic T cell effector function of CD8+ cells derived from parenterally immunized mice.101,102,105,106,112 These were followed by many studies using

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knockout hosts, which elucidated critical pathways for induction or effector phases of CTL function, largely performed by the Zinkernagel laboratory.63,65,113,114 We also made use of the natural H-2Ld-deficient mouse, the dm2 strain, and showed that Class II was necessary and sufficient, eliciting CD4-restricted CTLs against VSV G.13,115,116 CD4+ Th1 cells are CTLs62,117 and are effective in vivo in adoptive transfer during VSV encephalitis.116 Many basic studies on T cell memory have been carried out in murine studies of VSV responses.43,118–120

3.2 B Cells 3.2.1 Natural Antibody B-1 B cells produce “natural antibodies”, that are germline-derived molecules which do not undergo antigen-driven affinity maturation. These molecules pre-exists and is a form of innate immunity. The Zinkernagel laboratory showed the important contribution of this to host susceptibility and response to VSV.121,122

3.2.2 T cell-dependent Antibody Adaptive T cell-dependent B cell responses to VSV are rapid and include effective neutralizing antibodies. IgM is very rapidly produced followed by IgGs. The viral G is the target of neutralizing antibodies, and distinguishes between the Indiana and NJ serotypes.108,123 Passive transfer of IgG3, especially, or exposure to G vectored by another virus are able to protect mice from experimental encephalitis.25,98,111

3.2.3 Complement Complement components are involved in many aspects of immune responses to VSV, including lysis of membrane-bound cells and viruses,124 activation of B cells,125,126 as well as in promoting ADCC and recruitment of neutrophils to sites of inflammation.50

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4. NEW APPLICATIONS OF VSV 4.1 Vectored Vaccines The availability of cloned VSV, its ability to pseudotype other viruses, incorporate genes via reverse genetics, induction of strong serological responses in naïve hosts, and finally the virtual absence of anti-VSV antibody in the general population has led to the development of VSV-vectored vaccines for HIV, HCV, Ebola, and other pathogens.31,32,127–129 However, although very effective in many ways, the neurovirulence of VSV remains a potential problem in vaccine development.130

4.2 Tumor Oncolysis Many tumors are deficient in function of the Type I IFN pathway, and thus viruses may replicate much more readily in those cells than in normal tissue. This “flaw” is being exploited by a new class of treatments for cancer, viral oncolysis. Because VSV is exquisitely sensitive to IFNs and normal cells readily produce IFN in response to VSV infection, it is potentially a good candidate virus and is in development by several laboratories for multiple tumor targets.93,93,131–134 Additionally, the rare prior exposure to VSV, and therefore absence of pre-existing immunity, makes this a far more attractive virus to use as compared to Adenovirus, HSV-1, or other human pathogens which are also under study. However, we have not yet reached the stage of development where the kinks have been worked out. In many published reports, an unacceptable negative outcome of viral encephalitis has been observed in experimental animals,93,131–133 so that, although VSV can replicate in many tumors, some modification(s) of the virus will be necessary before it can be used clinically.

5. PATHOGENESIS: THE CNS VS. THE PERIPHERY Rapid, robust, and effective antiviral responses to VSV rapidly take place in the periphery, ranging from immediate production of IFNs to CTLs days later, which suppress viral replication and disease in immunocompetent hosts. Deaths are rarely seen except in immunocompromised hosts.

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In contrast, the brain is susceptible to VSV replication and a failure to produce detectable mRNA specific for IFN even in immunocompetent adult hosts,30,49 will lead to encephalitis. A high proportion (approximately 50%) of infected immunocompetent mice has died from encephalitis. Intranasal infection is associated with local replication of virus in the olfactory neuroepithelium and retrograde transport of virus to the olfactory bulb and then more rostral areas, and may result in breakdown of the BBB and host death.22,25,28,29,33,52,59,135–137 The CNS can be protected from disease with attenuated viruses,30 DI particles,27 or with vaccination which elicits neutralization of antibodies.25,98,111 B cells do not travel to the CNS in response to VSV infection, and there is no evidence of antiviral antibody production in the CNS.53 While in the periphery, host CTLs eliminate all virally-infected cells, the infected neurons in the CNS lack the target MHC molecules and cannot be recognized or killed by CTLs; however, CD4+ and CD8+ T cells differentiate and travel to the CNS far too late in the course of disease to promote recovery. Their contribution has been shown in late stages of infection,25,98 but the critical responses which restrict viral replication and disease are innate.

ACKNOWLEDGMENTS This work has been supported by the U.S. National Institutes of Health grants DC003536 and NS039746. This review was written during the last month of a Sabbatical leave at New York University.

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54. Reiss CS, Komatsu T, Barna M, Bi Z (1996). Ann NY Acad Sci 795, 257–265. 55. Barna M, Komatsu T, Reiss CS (1996). Virology 223, 331–343. 56. Joly E, Mucke L, Oldstone MB (1991). Science 253, 1283–1285. 57. Komatsu T, Ireland DD, Chen N, Reiss CS (1999). Virology 258, 389–395. 58. Komatsu T, Bi Z, Reiss CS (1996). J Neuroimmunol 68, 101–108. 59. Komatsu T, Ireland DD, Chung N, Dore A, Yoder M, Reiss CS (1999). Nitric Oxide 3, 327–339. 60. Bi Z, Quandt P, Komatsu T, Barna M, Reiss CS (1995). J Immunol 155, 5684–5689. 61. Bi, Z, Reiss CS (1995). J Virol 69, 2208–2213. 62. Cao BN, Huneycutt BS, Gapud CP, Arceci RJ, Reiss CS (1993). Cell Immunol 146, 147–156. 63. Burkhart C, Freer G, Castro R, Adorini L, Wiesmuller KH, Zinkernagel RM, Hengartner H (1994). J Virol 68, 1573–1580. 64. Kundig TM, Hengartner H, Zinkernagel RM (1993). J Immunol 150, 2316–2321. 65. Zinkernagel RM (1996). Science 271, pp. 173–178. 66. Ireland DD, Bang T, Komatsu T, Reiss CS (1999). Viral Immunol 12, 35–40. 67. Komastu T, Ireland DD, Reiss CS (1998). Cytokine Growth Factor Rev 9, 277–285. 68. Komatsu T, Reiss CS (1997). J Immunol 159, 3444–3452. 69. Komatsu T, Barna M, Reiss CS (1997). Viral Immunol 10, 35–47. 70. Chesler DA, McCutcheon JA, Reiss CS (2004). J Interferon Cytokine Res 24, 141–149. 71. Chesler DA, Reiss CS (2002). J Neuroimmunol 131, 92–97. 72. Chesler DA, Reiss CS (2002). Cytokine Growth Factor Rev 13, 441–454. 73. Oxenius A, Karrer U, Zinkernagel RM, Hengartner H (1999). J Immunol 162, 965–973. 74. Ireland DD, Palian BM, Reiss CS (2005). Viral Immunol 18, 397–402. 75. Hodges JL, Ireland DD, Reiss CS (2001). Viral Immunol 14, 181–191. 76. Chen N, Restivo A, Reiss CS (2002). Prostaglandins Other Lipid Mediat 67, 143–155. 77. Chen N, Reiss CS (2002). Viral Immunol 15, 133–146.

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78. Muller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M (1994). Science 264, 1918–1921. 79. van den Broek MF, Muller U, Huang S, Aguet M, Zinkernagel RM (1995). J Virol 69, 4792–4796. 80. Stojdl DF, Abraham N, Knowles S, Marius R, Brasey A, Lichty BD, Brown EG, Sonenberg N, Bell JC (2000). J Virol 74, 9580–9585. 81. Khabar KS, Dhalla M, Siddiqui Y, Zhou A, Al-Ahdal MN, Der SD, Silverman RH, Williams BR (2000). J Interferon Cytokine Res 20, 653–659. 82. Chesler DA, Dodard C, Lee GY, Levy DE, Reiss CS (2004). J Neurovirol 10, 57–63. 83. Chesler DA, Munoz-Jordan JL, Donelan N, Garcia-Sastre A, Reiss CS (2003). Viral Immunol 16, 87–96. 84. Barna M, Komatsu T, Bi Z, Reiss CS (1996). J Neuroimmunol 67, 31–39. 85. Fox HS, Bond BL, Parslow TG (1991). J Immunol 146, 4362–4367. 86. Trottier MD Jr., Palian BM, Reiss CS (2005). Virology 333, 215–225. 87. Petersen JM, Her LS, Varvel V, Lund E, Dahlberg JE (2000). Mol Cell Biol 20, 8590–8601. 88. Lyles DS, McKenzi MO, Ahmed M, Woolwine SC (1996). Virology 225, 172–180. 89. Ahmed M, McKenzie MO, Puckett S, Hojnacki M, Poliquin L, Lyles DS (2003). J Virol 77, 4646–4657. 90. Faria PA, Chakraborty P, Levay A, Barber GN, Ezelle HJ, Enninga J, Arana C, van DJ, Fontoura BM (2005). Mol Cell 17, 93–102. 91. Lichty BD, McBride H, Hanson S, Bell JC (2006). J Gen Virol 87, 3379–3384. 92. Balachandran S, Roberts PC, Kipperman T, Bhalla KN, Compans RW, Archer DR, Barber GN (2000). J Virol 74, 1513–1523. 93. Lichty BD, Power AT, Stojdl DF, Bell JC (2004). Trends Mol Med 10, 210–216. 94. Megyeri K, Orosz L, Kemeny L (2007). Biomed Pharmacother 61, 254–260. 95. Sharif-Askari E, Nakhaei P, Oliere S, Tumilasci V, Hernandez E, Wilkinson P, Lin R, Bell J, Hiscott J (2007). Virology 365, 20–33. 96. Gaddy DF, Lyles DS (2007). J Virol 81, 2792–2804.

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97. Lallemand C, Blanchard B, Palmieri M, Lebon P, May E, Tovey MG (2006). Oncogene 26, 328–338. 98. Reiss CS (1993). Viruses and the Cellular Immune Response, pp. 337–361. 99. Zhang W, Young AC, Imarai M, Nathenson SG, Sacchettini JC (1992). Proc Natl Acad Sci USA 89, 8403–8407. 100. Ciavarra R, Forman J (1982). J Exp Med 156, 778–790. 101. Stroynowski I, Orn A, Goodenow RS, McMillan M, Forman J, Brayton PR, Frelinger J, Hood L (1984). Immunogenetics 20, 141–154. 102. Murre C, Choi E, Weis J, Seidman JG, Ozato K, Liu L, Burakoff SJ, Reiss CS (1984). J Exp Med 160, 167–178. 103. Murre C, Reiss CS, Bernabeu C, Chen LB, Burakoff SJ, Seidman JG (1984). Nature 307, 432–436. 104. Murre C, Parker KC, Reiss CS, Driller JP, Wiley DC, Burakoff SJ, Seidman JG (1986). Mol Cell Biol 6, 1315–1319. 105. Reiss CS, Evans GA, Margulies DH, Seidman JG, Burakoff SJ (1983). Proc Natl Acad Sci USA 80, 2709–2712. 106. Reiss CS, Evans GA, Murre C, Margulies DH, Seidman JG, Burakoff SJ (1984). Fed Proc 43, 271–275. 107. Reiss CS, Oien SA, Bartido SM (1996). Viral Genome Methods, pp. 185–200. 108. Lefrancois L, Lyles DS (1983). J Immunol 130, 394–398. 109. Bartido SM, Diment S, Reiss CS (1995). Eur J Immunol 25, 2211–2219. 110. Petrarca MA, Reiss CS, Diamond DC, Boni J, Burakoff SJ, Faller DV (1988). Microb Pathog 5, 319–332. 111. Reiss CS, Chen SS, Huang AS, Doherty R (1986). Microb Pathog 1, 261–267. 112. Zinkernagel RM, Althag, A, Holland J (1978). J Immunol 121, 744–748. 113. Burkhart C, Freer G, Steinhoff U, Li Y, Bishop DH, Zinkernagel RM, Hengartner H (1994). Viral Immunol 7, 103–111. 114. Kagi D, Seile, P, Pavlovic J, Ledermann B, Burki K, Zinkernagel RM, Hengartner H (1995). Eur J Immunol 25, 3256–3262. 115. Browning M, Reiss CS, Huang A (1990). J Virol 64, 3810–3816. 116. Browning MJ, Huang AS, Reiss CS (1990). J Immunol 145, 985–994. 117. Reiss CS, Gapud CP, Keil W (1992). Cell Immunol 139, 229–238. 118. Schluns KS, Williams K, Ma, Zheng XX, Lefrancois L (2002). J Immunol 168, 4827–4831.

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119. Turner DL, Cauley LS, Khanna KM, Lefrancois L (2007). J Virol 81, 2039–2046. 120. Roost HP, Charan S, Zinkernagel RM (1990). Eur J Immunol 20, 2547–2554. 121. Brundler MA, Aichele P, Bachmann M Kitamura D, Rajewsky K, Zinkernagel RM (1996). Eur J Immunol 26, 2257–2262. 122. Beebe DP, Coope NR (1981). J Immunol 126, 1562–1568. 123. Gobet R, Cerny, Ruedi E, Hengartner H, Zinkernagel RM (1988). Exp Cell Biol 56, 175–180. 124. Mills BJ, Beebe DP, Cooper NR (1979). J Immunol 123, 2518–2524. 125. Fehr T, Bachmann MF, Bluethmann H, Kikutani H, Hengartner H, Zinkernagel RM (1996). Cell Immunol 168, 184–192. 126. Ochsenbein AF, Pinschewer DD, Odermatt B, Carroll MC, Hengartner H, Zinkernagel RM (1999). J Exp Med 190, 1165–1174. 127. Barber SA, Herbst DS, Bullock BT, Gama L, Clements JE (2004). J Neurovirol 10 Suppl 1, 15–20. 128. Majid AM, Ezelle H, Shah S, Barber GN (2006). J Virol 80, 6993–7008. 129. McKenna PM, McGettigan JP, Pomerantz RJ, Dietzschold B, Schnell MJ (2003). Curr HIV Res 1, 229–237. 130. Johnson JE, Nasar F, Coleman JW, Price RE, Javadian A, Draper K, Lee M, Reilly PA, Clarke DK, Hendry RM et al. (2007). Virology 360, 36–49. 131. Gaddy DF, Lyles DS (2007). J Virol 81, 2792–2804. 132. Stojdl DF, Lichty BD, tenOever BR, Paterson JM, Power AT, Knowles S, Marius R, Reynard J, Poliquin L, Atkins H et al. (2003). Cancer Cell 4, 263–275. 133. Shinozaki K, Ebert O, Woo SL (2005). Int J Cancer 114, 659–664. 134. Lun X, Senger DL, Alain T, Oprea A, Parato K, Stojdl D, Lichty B, Power A, Johnston R, Hamilton M et al. (2006). J Natl Cancer Inst 98, 1546–1557. 135. Reiss CS, Chesler DA, Hodges J, Ireland DD, Chen N (2002). Curr Top Microbiol Immunol 265, 63–94. 136. Reiss CS (ed.) (2008). Neurotropic Virus Infections. Cambridge University Press, Cambridge, pp. 265–291. 137. Forger JM. III, Bronson RT, Huang AS, Reiss CS (1991). J Virol 65, 4950–4958. 138. Herrera RA, Oved J, Reiss CS (2008) Viral Immunol 21, 141–152.

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CHAPTER 11

Hantavirus Infection and Innate Immunity Nandini Sen, Adrish Sen, Peter Alff, Irina Gavrilovskaya & Erich R. Mackow

ABSTRACT Pathogenic hantaviruses replicate within human endothelial cells and cause two diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In order to replicate in human endothelial cells, pathogenic hantaviruses inhibit the early induction of type I interferon (IFN) and establish an IFN resistant state within infected cells. In contrast, the non-pathogenic hantavirus, PHV, induces an early high level IFN response and fails to replicate within human endothelial cells. However, PHV replicates within IFN deficient Vero E6 cells and presumably regulates cellular IFN responses within host endothelial cells. Pathogenic hantavirus Gn proteins have been shown to regulate early innate cellular responses by blocking IFN induction at the level of the TBK1 complex. The cytoplasmic tail of pathogenic hantavirus Gn proteins also contains a degron which directs protein ubiquitination and degradation. Since the activation of TBK1-TRAF3 complexes is regulated by the state of TRAF3 ubiquitination, these findings tie the regulation of TBK1-directed IFN responses to Gn degradation. The mechanism by which hantaviruses regulate TBK1 complex formation and IFN induction is only beginning to unfold. However, it is clear that IFN regulation by the Gn-tail is required for hantavirus success within human endothelial cells. As a result, IFN regulatory elements within the Gn-tail are determinants of hantavirus pathogenesis and viable targets for attenuating pathogenic hantaviruses. 247

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1. INTRODUCTION 1.1 Hantavirus Structure and Genome More than 20 hantaviruses have been classified based on antigenic, genetic or serological characteristics.1,2 Hantaviruses define a unique genus and are the only members of the Bunyaviridae family that are not arthropod borne. Hantaviruses are enveloped viruses (100 nm) with a spherical or ovoid shape and possess two surface glycoproteins Gn and Gc.2 Hantaviruses contain three negative-sense RNA segments designated small (S), medium (M) and large (L) that encode four viral proteins. The 6.6 kb L segment encodes the viral RNA-dependent RNA polymerase while the S segment (1.7–2.1 kb) encodes a 428 amino acid nucleocapsid (N) protein.2 The hantavirus N-protein is the predominant viral antigen and contains cross-reactive epitopes which permit hantavirus recognition.2 The M genomic segment (3.6 kb) encodes a single 1139 residue polyprotein which is co-translationally cleaved into the N-terminal 70 kDa G1 or Gn protein (amino acid 1–652) and the C-terminal 58 kDa G2 or Gc protein (amino acid 653–1139).1,2 Gn and Gc proteins form heterodimers which oligomerize to form the virion surface and are recognized by the neutralizing antibodies. Gn and Gc are trafficked to the cis-Golgi and assembled onto hantaviruses by viral budding into the lumen of the Golgi.2,3 The exit of hantavirus from cells is consistent with an aberrant secretory process involving vesicular trafficking to the cell surface. Gn and Gc presumably direct cell attachment and membrane fusion. Gc contains a putative type 2 fusion domain with structural homology to the Tick Borne Encephalitis Virus (TBEV) E protein.4 Until recently, non-structural proteins were not identified for hantaviruses. However recently two hantaviruses, Tula and Puumala, were reported to contain NSs proteins although potential NSs ORFs are truncated in most hantaviruses.5

1.2 Hantavirus Hosts and Transmission Each pathogenic hantavirus persistently infects a primary small mammal host where it causes no apparent disease.1,2 Although most of these animals

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are termed mice, phylogenetically these field species are more closely related to hamsters or voles than to laboratory mice, and hantaviruses do not infect and are not carried by lab mice (Mus musculus). Hantaviruses appear to have co-evolved with their primary hosts since nearly identical phylogenetic trees can be constructed from host mitochondrial DNA sequences and viral RNA sequences.6,7 Transmission of hantaviruses from hosts to humans occurs primarily by inhalation of virus-containing aerosols of rodent excreta. Although human infection is normally a dead end for hantaviruses, person-to-person transmission of ANDV has been reported.1,2,8,9

1.3 Hantavirus Disease and Epidemiology Hantaviruses infect endothelial cells and cause two vascular permeabilitybased diseases, Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS), with acute pulmonary edema or hemorrhage as their primary manifestations. Hantaviruses replicate predominantly in pulmonary and renal capillary bed endothelial cells.10–13 Endothelial cells are not lysed by hantavirus infection and virus can be passaged with endothelial cells in tissue culture. In humans, hantaviruses cause acute disease following a 7–14 days incubation period.10,14 An increase in vascular permeability and acute thrombocytopenia are common to HFRS and HPS infections. However the mechanisms by which hantaviruses direct vascular permeability or cause specific respiratory (HPS) or renal disease (HFRS) syndromes are unknown.2,11,13 In contrast to pathogenic hantaviruses, Prospect Hill virus (PHV) and Tula virus (TULV) are hantaviruses that are not associated with any human disease and hence are considered to be non-pathogenic hantaviruses.1,2,12,15 Both of these non-pathogenic hantaviruses still infect human endothelial cells and determinants of hantavirus pathogenesis are only beginning to be uncovered.

1.3.1 Hemorrhagic fever with renal syndrome HFRS-causing hantaviruses are primarily present in Eurasia and include Hantaan (HTNV), Seoul (SEOV), Puumala (PUUV) and Dobrava (DOBV)

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viruses which have a 0.1–5% mortality rate. In Asia approximately 150 000–200 000 HFRS cases requiring hospitalization are reported each year.1 Hantaan virus (HTNV) is the prototypic HFRS-causing virus that was first isolated in Korea as the cause of Korean Hemorrhagic fever and Hantaan is the namesake of the genus.16,17 HFRS includes microvascular hemorrhage, thrombocytopenia, hypotension, shock, and in some cases renal failure.1 The mechanism by which HFRS viruses cause vascular hemorrhage is unclear although pathogenic hantaviruses use and dysregulate β 3 integrins which are linked to hemorrhagic disease. Circulating immune complexes are also evident in HFRS patients and may contribute to the disease.18 The severity of HFRS has been shown to vary with the type of infecting hantavirus strain, with Hantaan and Dobrava virus infections usually associated with severe disease.1,2

1.3.2 Hantavirus pulmonary syndrome Hantavirus pulmonary syndrome (HPS) or an alternate name, hantavirus cardiopulmonary syndrome (HCPS) is an acute respiratory disease syndrome with a high mortality rate (35–40%).1,14,19,20 The main clinical manifestations of HPS cases are increased vascular permeability, thrombocytopenia and severe pulmonary edema.11,13,21 Patient edema is primarily or exclusively exudative in nature22 although some immunoblasts are reported in edematous autopsy tissue.11 HPS-causing hantaviruses were first identified in 1993 as the cause of an outbreak of acute respiratory disease in the Four Corners region of North America and dubbed Sin Nombre virus (SNV).14,23 Since recognition of the disease in 1993, CDC has confirmed 465 HPS cases from 30 states with a 30–40% mortality rate and identified many different HPS-associated hantaviruses and their hosts.24 HPS-associated hantaviruses include New York 1 (NY-1), Black Creek Canal, and Bayou in North America; Andes (ANDV) and Laguna Negra, in South America. HPS is characterized by a febrile illness associated with severe pulmonary edema leading to respiratory distress and cardiogenic shock. ANDV infection of Syrian hamsters results in a fatal HPS-like disease, closely mimicking the course and symptoms of HPS patients. ANDV infection of Syrian hamsters is also the primary lethal animal model of hantavirus

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disease.25 Like HPS, disease in Syrian hamsters includes a long incubation period followed by pulmonary edema, pleural effusions and rapidly progressive respiratory distress.

1.4 Hantavirus-Cell Interactions Hantaviruses replicate predominantly within pulmonary and renal capillary bed endothelial cells. Both pathogenic and non-pathogenic hantaviruses infect endothelial cells12,26 suggesting that specific cellular responses are central to the development of HPS and HFRS diseases. The endothelium forms the primary fluid barrier within the vasculature and the permeability of the endothelium is determined by a combination of immune responses, cytokine and chemokine induction, and cellular receptors that mediate endothelial cell movement and repair. β 3 integrins have been shown to be the key regulators of vascular permeability and β 3 dysfunction is a known cause of several hemorrhage and edematous diseases.27–29 Hantaviruses do not lyse endothelial cells but the mechanism by which pathogenic hantaviruses cause vascular permeability is incompletely understood. Interestingly, the entry of pathogenic but not nonpathogenic hantaviruses into endothelial cells is mediated by viral binding to α vβ 3 integrins.30–32 Pathogenic hantaviruses bind to the β 3 integrin PSI domain which is present at the apex of inactive α vβ 3 conformers and pathogenic hantaviruses block α vβ 3 integrin function late in infection.33 The use and dysregulation of β 3 integrins by only pathogenic hantaviruses which can cause vascular disease provide a compelling role for the involvement of β 3 integrins in HPS and HFRS diseases. Recently, it was demonstrated that pathogenic hantaviruses hypersensitize endothelial cells to vascular endothelial cell growth factor (VEGF) which was originally called vascular permeability factor.34 These findings are directly related to α vβ 3 dysfunction, which mimic the responses of β 3−/− cells in response to VEGF27,28 and suggest a mechanism for hantavirus-directed permeability. In contrast to pathogenic hantaviruses, the entry of non-pathogenic hantaviruses is independent of α vβ 3 and consistent with the use of α 5β 1 integrins. Non-pathogenic PHV and TULV do not regulate the function of

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α vβ 3 integrins or enhance endothelial cell permeability in response to VEGF.30,31,34 As a result, pathogenic hantavirus interactions with α vβ 3 integrins are likely to play a central role in vascular permeability defects that contribute to hantavirus pathogenesis. In addition to viral entry into endothelial cells, all hantaviruses must successfully replicate within human endothelial cells in order to be pathogenic.35 This means that pathogenic hantaviruses must bypass innate cellular responses that are designed to detect and limit viral replication. Recent studies suggest that pathogenic hantaviruses are capable of regulating the cellular induction of IFN at the early stages after infection and that this regulation permits viral replication within human endothelial cells. Additionally, hantaviruses appear to be resistant to the effects of IFN at later times after infection, suggesting that viral products generate an IFN resistant state within infected cells. Here we will review the means by which pathogenic hantaviruses engage cellular pathways in order to successfully negotiate IFN responses and replicate within human endothelial cells.

2. HANTAVIRUS REGULATION OF CELLULAR INTERFERON RESPONSES Cells elicit early antiviral responses to infection which play a critical role in both the outcome of infection and the pathogenicity of the infectious agent. IFNs are a family of inducible cytokines that serve as components of our native immunity36–38 and type I IFNs (IFNα /β ) are secreted in response to viral infection by almost all cell types.39 Regulating cellular IFN responses is fundamental to viral success and determines the ability of the virus to replicate in specific hosts, tissues, or cells. Invading viruses are detected by pattern recognition receptors (PRRs) and direct host cell responses which serve to limit viral replication and spread. PRRs include cytoplasmically located retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and membrane bound Toll-like receptors (TLRs).40–43 PRRs transmit IFN activation signals via multiple transcription factors including IRF3, NF-κ B, and c-jun/ ATF-2 that triggers the induction of type I IFNs.36,44–49 Secreted IFNs affect autocrine and paracrine cellular responses by binding to IFN receptors

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(IFNAR). The receptor-ligand interaction triggers activation of the Jak-STAT signaling pathway leading to further IFN induction as well as the induction of several interferon-stimulated genes (ISGs) that have antiviral functions.39 The cumulative effect of IFN signaling establishes a cellular defense program that innately restricts the spread of invading viral pathogens. Within virus-infected cells, viral products are recognized, signaling pathways are activated and IFN are directed to be produced. DsRNA or RNA containing 5′ triphosphates are detected by cellular RNA helicases, RIG-I and MDA5.50 These PRRs then interact via the caspase recruitment domain (CARD) with the mitochondrially located adaptor protein, MAVS/ IPS-1/CARDIF/VISA that leads to the recruitment of signaling complexes that activate both the IRF3 and NF-κ B activation pathways.51–54 To negotiate IFN induction, viruses have evolved different mechanisms to antagonize IFN responses. Viruses encode one or in some case multiple proteins that either block IFN induction or IFNAR signaling responses.36 Hantaviruses exhibit a very limited genetic repertoire, encoding only four structural proteins that could potentially regulate cellular IFN responses during infection. Until very recently little was known regarding hantavirus regulation of endothelial cell signaling and cellular defense responses. Several groups have recently reported studies of IFN regulation by hantaviruses5,35,55–57 and these studies are gradually increasing our understanding of hantavirus regulation of innate host immune responses.

2.1 Interferon Secretion During Hantavirus Infection A recent study reported that systemic levels of IFNα and IFNβ remain unchanged during acute and convalescent phases of infection in Puumala virus-infected HFRS patients.58 IFNγ levels are also reported to be elevated in serum during hantavirus infection. Consistent with observations from infected patients,59,60 very low levels of IFN production have been shown from in vitro studies of pathogenic hantavirus infected endothelial cells. Hantavirus-infected endothelial cells were initially shown to secrete IFNβ by demonstrating that neutralizing antibodies to IFNβ blocked the production of hantavirus-induced ISGs.35 HTNV and TULV were shown to secrete IFN > 2 days postinfection, coinciding with mRNA increases > 2 days

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postinfection.35,56 HTNV- and ANDV-infected human umbilical vein endothelial cells (HUVECs) were also shown to induce low levels of IFNβ mRNA but no increase in IFNα or IFNλ mRNA.58 PHV-directed IFN production and transcriptional responses have been shown to be highly induced at early times after infection.61 Hence, while both pathogenic and non-pathogenic hantaviruses eventually induce IFN, pathogenic hantaviruses appear to regulate the early induction of IFN responses in infected cells.

2.2 Differential Responses of Pathogenic and Non-pathogenic Hantavirus to IFNs Vero E6 cells are the most commonly used cell line for hantavirus propagation. Pathogenic and non-pathogenic hantaviruses replicate to the same titers in Vero E6 cells.35 Interestingly, Vero E6 cells reportedly lack the type I IFN locus and are deficient in IFN production but are responsive to IFN addition.62 Several studies have also reported that hantavirus replication can be blocked by pretreating cells with IFN.26,35,63,64 A recent study by Alff et al. demonstrates that pretreatment of HUVECs with IFNα completely inhibits hantavirus replication.35 A similar inhibition is observed when IFNα is added to human endothelial cells up to 6 to 12 hours postinfection. The non-pathogenic hantavirus PHV appears to trigger a selflimiting IFN response in infected endothelial cells leading to little or no replication. In contrast, viral titers for pathogenic hantaviruses increase from one to five days postinfection in HUVECS as is evident from increased mRNA and nucleocaspsid protein levels in NY-1V- and HTNVinfected endothlial cells. Consistent with the absence of PHV replication in HUVECs, a decrease in S-segment RNA and nucleocapsid protein levels was observed two to five days post PHV infection. Alff et al. also reported that addition of IFNα 15 to 24 hours postinfection had little effect on the replication NY-1V and HTNV.35 Therefore, hantavirus replication correlates inversely with the early induction of IFN in endothelial cells. This observation along with the fact that pathogenic hantaviruses induce high ISG levels at late times postinfection suggests that pathogenic hantaviruses have evolved mechanisms to circumvent the early induction of IFN responses and that later IFN responses do not restrict hantavirus replication.

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Interestingly, another non-pathogenic hantavirus, TULV, is capable of replicating within human endothelial cells similarly to HTNV.56 IFN responses of TULV-infected endothelial cells are reportedly identical to HTNV one day postinfection but induced two to five days postinfection, although at lower levels than HTNV.56 However, it is reported that TULV induces very low levels of MxA 16 to 24 hours postinfection and that MxA levels are dramatically enhanced two to three days postinfection. Although it is unclear how MxA is induced in the absence of IFN one day postinfection it is possible that the response is at a subthreshold level for regulating TULV replication or that 16–24 hours postinfection TULV is resistant to the effects of MxA similar to NY-1V and HTNV.

2.3 Induction of Interferon Stimulated Genes During Hantavirus Infection IFN-dependent cellular effects are mediated by transcriptional induction of IFN-stimulated genes (ISGs). MxA is used as a marker for ISG induction and IFN responses, while ISG56 may be stimulated directly and independent of IFNs during virus infection.65 Interestingly, DNA microarray analysis of hantavirus-infected endothelial cells revealed striking differences in the induction of ISGs by pathogenic and nonpathogenic hantaviruses.56,61 The non-pathogenic hantavirus PHV directs the high level induction of many ISGs one day after infection. In contrast, virtually no ISG responses were detected by the pathogenic strains NY-1V (HPS) or HTNV (HFRS) one day postinfection61 suggesting a difference in IFN production and ISG induction between pathogenic and non-pathogenic hantaviruses at early times postinfection. DNA microarray analysis indicated that the ISG, MxA is induced 161-fold by PHV one day postinfection compared to 3.7 and < 2-fold by NY-1V and HTNV, respectively.61 However, MxA is induced about 200-fold by all hantaviruses at late times postinfection, along with a variety of additional ISGs.61 Using Real Time PCR it was reported that PHV infection of endothelial cells directed a 539-fold increase in MxA mRNA (one day postinfection), while pathogenic NY-1V or HTNV induced a substantially smaller 9- to 31-fold increase in MxA mRNA levels, respectively. Similarly, ANDV has been shown to stimulate MxA expression in HUVECs

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48 hours after infection.66 MxA induction by hantaviruses can be blocked by using IFN-β , but not IFN-α , neutralizing antibody, indicating IFN-β dependent MxA stimulation.35 Similar to MxA induction, the kinetics of ISG56 induction by nonpathogenic PHV was > 225-fold compared to a 6-fold induction in NY-1V infected endothelial cells at early times postinfection.35 Therefore, increased IFN secretion by PHV at early times after infection directs transcription of IFN stimulated genes which inhibit PHV replication in endothelial cells. On the other hand, by delaying the onset of early IFN responses, pathogenic hantaviruses are able to suppress the early induction of ISGs, thereby evading the host defense mechanisms that would limit their successful replication in human endothelial cells.

2.4 MxA-dependent Inhibition of Hantavirus Replication The antiviral effects of type I IFNs (IFNα /β ) are mediated by at least three protein systems, 2′,5′-oligoadenylate synthetase (OAS)/RNase L, dsRNA activated protein kinase (PKR), and Mx proteins.67 MxA activation cannot be triggered directly by virus infection but is solely dependent on IFN production. Human MxA is a cytoplasmic 76-kDa protein that has been shown to inhibit several negative-stranded RNA viruses. Normally only a small amount of MxA protein is detectable in cells but stimulation with type I IFNs directs the cytoplasmic accumulation of MxA protein within cells. MxA is a protein that belongs to the superfamily of dynaminlike GTPases and its intrinsic GTPase activity is required for antiviral activity.36,39,68 Studies by Kanvera et al. and Frese et al. have shown that constitutively expressed MxA protein has the capacity to inhibit both hantavirus protein expression and RNA accumulation in virus-infected cells.69,70 The accumulation of PUUV, TULV, and HTNV nucleocapsid protein was considerably reduced but not completely abolished in MxA-positive cell lines at late times of infection. Despite the low level synthesis of N protein, the production of infectious virus in MxA positive cell lines diminished almost 100-fold as compared to controlled cell lines. Northern blot analysis revealed the lack of detectable hantavirus RNA in infected MxA expressing cells.69,70 This result suggested that pathogenic and non-pathogenic

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hantaviruses are similarly susceptible to the antiviral action of pre-existing MxA, which acts at an early step of the virus replication cycle.

2.5 MxA-independent Inhibition of Hantavirus Infection A recent study by Oelschlegel et al. has shown that endogenously expressed MxA in A549 cells fails to regulate HTNV infection.71 In this study, HTNV infection was shown to be blocked by the prior addition of either type I or type II IFNs although only IFNα but not IFNγ was shown to induce MxA expression in human A549 cells. Further, IFNα could successfully block production of HTNV-N protein and infectious virions in the presence of MxA siRNA. In these studies the authors were unable to detect MxA expression in Vero E6 cells after IFNα addition although a dose-dependent decrease in HTNV replication was observed in the presence of both IFNα and IFNγ .71 From their observations, the authors have suggested an MxA-independent mechanism of IFN regulation of hantavirus replication. However, further studies are required to identify IFNstimulated but MxA-independent factors that could inhibit the replication of pathogenic and non-pathogenic hantaviruses.

2.6 Mechanism of MxA-mediated Inhibition Although hantavirus replication is sensitive to the presence of MxA within the cell, the mechanism by which MxA inhibits hantavirus replication is poorly understood. The MxA protein has been shown to form a complex with the N proteins of other members of the Bunyaviridae family like the La Crosse and Thogoto viruses.72,73 It was postulated that MxA soaks up the cytoplasmic N protein and redistributes it to the perinuclear region thus affecting the availability of N protein required for viral replication. For hantaviruses it has been reported that MxA co-localizes with the nucleocapsid protein.66 Co-localization data has been used to suggest that MxA forms a complex with hantavirus N protein which may interfere with N protein accumulation and intracellular localization. However these findings do not explain the resistance of hantaviruses to IFN addition at late times after infection or the ability of hantaviruses to replicate at late times after infection in the presence of highly induced MxA and additional

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ISGs. In fact, these findings could suggest that the abundantly expressed nucleocapsid protein could serve to soak up MxA and prevent MxA restriction of hantavirus replication at late times postinfection.

3. HANTAVIRUS ENCODED INTERFERON ANTAGONISTS 3.1 Role of Hantavirus G1 Cytoplasmic Tail in Interferon Regulation Several lines of evidence indicate that pathogenic and non-pathogenic hantaviruses differ in their ability to regulate IFN production early after infection.56,57,61 However, until recently there was no information on the hantaviral protein that participates in IFN regulation of infected cells or the mechanism by which pathogenic hantaviruses modulate the IFN pathway. Recently, Alff et al. have demonstrated that the C-terminal cytoplasmic domain of pathogenic hantavirus G1 proteins dramatically inhibit (30–50 fold) IFN transcriptional responses.35 Further, the G1 protein from the non-pathogenic hantavirus, PHV, failed to regulate IFN responses. This finding is consistent with DNA microarray data indicating distinct IFN regulation by pathogenic but not non-pathogenic hantaviruses.61 In infected cells, the hantavirus G1 protein localizes to the cis-Golgi with translocation of its N-terminal domain into the lumen of the Golgi. This leaves a 142 amino acid long C-terminal domain within the cytoplasm, termed the G1-tail.1,2 As a result, the G1-tail is one of the only three cytoplasmic hantavirus proteins. The G1-tail is presumed to be multifunctional since it is highly conserved and present in the mature virion yet the 142 residue length of the tail suggests that it could provide matrix protein or IFN regulatory functions that are normally ascribed to non-structural proteins.74 The G1 protein cytoplasmic tails from both pathogenic and nonpathogenic hantaviruses harbor two potential protein–protein interaction domains. A membrane proximal zinc finger domain is conserved among hantaviruses and a hydrophobic domain is present at the extreme G1 Cterminus.75 The hydrophobic domain of pathogenic hantavirus G1-tails contains a degron that directs proteasomal degradation of the protein.

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In addition, HPS-causing hantaviruses like NY-1V and ANDV contain an ITAM motif that is found in B-cell and T-cell receptors where they function to bind critical Src and Syk family of tyrosine kinases.76 The presence of important protein-interaction motifs, ubiquitination and degradation directing domains, and its cytoplasmic location within the host cell makes the G1-tail a very strong candidate for regulating host cell responses that originate within the cytoplasm.

3.2 Pathogenic Hantavirus G1-Tails Block RIG-I and TBK1-directed Transcriptional Responses Retinoic acid inducible gene-1 (RIG-I) is a recently described viral pathogen recognition receptor that senses viral RNA within the cytoplasm. RIG-I is a DexD/H box-containing RNA helicase which detects viral RNA while interactions of the RIG-I CARD mediate the activation of downstream signaling pathway effectors which direct IFN transcriptional responses.43 RIG-I activation stimulates IRF3 and NF-κ B transcriptional responses. Studies with the G1-tails of pathogenic (NY-1V) and nonpathogenic (PHV) hantaviruses indicate that only the G1-tail of NY-1V suppresses IFNβ and ISRE transcriptional responses triggered by RIG-I. RIG-I-induced ISRE transcription was inhibited by > 90% in the presence of the NY-1V G1 cytoplasmic tail.35 A dose-dependent inhibition of RIG-I-mediated ISRE activation was observed in the presence of increasing amounts of the G1-tail, indicating that the pathogenic hantavirus G1-tail specifically inhibits IFN activation. Therefore, the G1-tail disrupts cellular IFN signaling responses that limit viral replication, and at one level this G1tail regulatory function is a determinant of hantavirus pathogenesis. TBK1 is a downstream effector of RIG-I activation, and TBK1 is the IRF3 kinase required for ISRE and IFNβ transcriptional responses.36 TBK1 is a serine threonine kinase that is critical for IRF3 phosphorylation and IFN induction. Activated TBK1 phosphorylates IRF3 which dimerizes and translocates to the nucleus where it is required for transcriptional responses from IFNβ and ISRE promoters.77,78 TBK1-directed ISRE and IFNβ transcriptional responses were inhibited by > 90% when the NY-1V G1-tail was co-expressed in cells.35 In contrast, co-expressing the nucleocapsid protein or the PHV G1-tail did not inhibit TBK1-directed transcriptional responses.

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A constitutively active mutant of IRF3, IRF3-5D, activates ISRE transcriptional responses downstream of TBK1. However, ISRE activation by IRF3-5D was not inhibited by co-expressing the pathogenic NY-1V G1tail.35 Thus the G1-tail of pathogenic hantaviruses is incapable of inhibiting phospho-IRF3-directed transcriptional responses. This indicates that the NY-1V G1 tail acts upstream of IRF3 phosphorylation and suggests that G1-tail regulation occurs at the level of the TBK1 complex. Collectively, these findings indicate that the pathogenic hantavirus G1-tail suppresses IFN transcriptional responses by blocking IRF3 phosphorylation. Consistent with this, Spiropoulou et al. (2007) reported that ANDV infection could inhibit IRF3 dimerization and nuclear localization in HMVECs.57 Interestingly, this same report suggested that transfecting cells with the hantavirus M-segment failed to block IRF3 translocation to the nucleus directed by a subsequent Sendai virus infection.57 There are a variety of possibilities for how this negative result might occur including insufficient protein expression, infection versus transfection comparisons, virals compartmentalization, analysis of controls, and others. However, hantavirus regulation of IFN responses may simply be insufficient to block Sendai virus-directed IFN responses or IFN directed by viruses that replicate to very high levels. Even late in hantavirus infection there does not appear to be any regulation of cellular IFN responses.61 This suggests that the early regulation of IFN responses is a transient effect that may only be sufficient below a specific threshold of viral replication. Consistent with the inability of PHV to grow in IFN competent endothelial cells, the PHV G1-tail failed to inhibit RIG-I- and TBK1directed ISRE and IFNβ transcription.35 PHV infection of endothelial cells resulted in IRF3 phosphorylation, dimerization, and nuclear translocation, indicating a lack of regulation of the IFN pathway. These findings demonstrate that the PHV G1-tail lacks IFN regulatory functions of pathogenic hantavirus proteins.

3.3 Mechanism of Interferon Regulation by the G1 Cytoplasmic Tail Recently we have shown that the G1-tail of pathogenic NY-1V, ANDV, and HTNV, but not PHV, contains a C-terminal degron that targets the

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protein for proteasomal degradation.75 The degron is a structural motif whose function can be abolished by mutating four non-linear residues in NY-1V G1 tail.75 A comparative domain swap analysis of the G1-tails of NY-1V and PHV revealed that the hydrophobicity of the C-terminal hydrophobic domain in the G1-tail is important for degron function. Similar to other degrons, the G1-tail degron is an autonomous domain and when fused to these proteins the G1 degron targets stable fluorescent reporters (GFP and RFP) for degradation.75 The pathogenic NY-1V G1tail inhibits IFN induction and is also ubiquitinated and degraded by the proteasome. In contrast, the G1-tail of non-pathogenic PHV is stably expressed but unable to inhibit the IFN induction. These findings link the degradation of the G1-tail to the regulation of cellular IFN responses and suggest that the G1 degron may be a determinant of hantavirus pathogenesis. One possibility is that the pathogenic hantavirus G1-tail binds components of the TBK1 complex and targets them for degradation. Degrons with a role in antagonizing IFN induction were described for Sendai viruses in which the virally encoded C protein inhibits IFN pathway activation by directing STAT1 degradation.79 Whether the G1-tail behaves in a similar manner remains to be investigated. Since the NY-1V G1 tail fails to inhibit IFN transcription by activating p-IRF3 it appears that the G1 tail does not target p-IRF3 for degradation or modulate its function. The fact that NY-1V G1-tail blocks TBK1-mediated transcriptional response indicates that inhibition occurs at the level of the TBK1-TRAF3 complex. Since the NY-1V G1-tail is degraded, one possibility is that the G1-tail regulates IFN transcription by directing the degradation of TBK1, TRAF3 or other regulatory components of the TBK1 complex. Although no viral proteins have been implicated in degradation of TBK1, the HCV NS3 protein has been shown to bind to TBK1 and thus block downstream transmission of IFN signaling responses.80 The Epstein Bar virus LMP1 protein, which is proteasomally regulated like NY-1V G1 tail, has been shown to facilitate TRAF3 ubiquitination and its recruitment to lipid rafts.81,82 Although LMP1 does not direct TRAF3 degradation, TRAF3 is required for IFN induction and altering TRAF3 ubiquitination could regulate pathway activation.83,84 In fact, a recent paper indicates that the state of TRAF3 ubiquitination regulates TBK1 complex formation required for IFN transcription.83

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TRAF3 ubiquitination is regulated by DUBA which when associated with TRAF3, deubiquitinates the protein and prevents its activation.83 The NY1V G1 tail could enhance DUBA’s association with TRAF3 or disrupt TRAF3-TBK1 complex formation. This suggests that the degron within the G1-tail could alter the ubiquitination, degradation and regulation of components within the TBK1 complex. Further analysis of G1-tail regulation of TBK1 complexes and TRAF3 interactions are required to understand pathogenic hantavirus regulation of cellular IFN responses.

3.4 Alternate Theories of Hantavirus Regulation of the IFN Pathway Various alternate hypotheses of IFN regulation by hantaviruses have been reported from several groups. Prescott et al. postulated that IFN responses during infection with the HPS-causing SNV occurs independent of virus entry through recognition by the characteristic pattern recognition receptors (PRRs) and IRF3 activation.85 The authors have suggested that ISG56 transcription is induced when cells are exposed to hantavirus particles but they indicated that this response does not require binding of viral nucleic acids or proteins to PRRs like RIG-I or toll-like receptors. ISG56 induction was also shown to be independent of IRF3 or IRF7 activation and virus entry thus suggesting that cellular responses to the virus are transduced by a new undefined mechanism emanating from the plasma membrane. This study has hypothesized that an undefined PAMP-PRR pathway triggered by purified SNV directs ISG transcription in the absence of cellular transcriptional factors like IRF3. However, sucrose cushion pelleted viral preps used for these studies are complex and it remains unexplained as to how a PAMP-independent mechanism is induced or directed. Further understanding of a new PAMP-independent mechanism of IFN induction requires more extensive study especially since several groups have shown that pathogenic and non-pathogenic hantaviruses differentially stimulate and regulate IFN responses in infected cells through conventional IFN signaling pathways. One paper also indicates that hantaviruses regulate STAT phosphorylation following addition of exogenous IFN to cells.57 This finding suggests that pathways downstream of the IFN receptor are regulated by hantaviruses.

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However, this paper also indicates that pathogenic hantavirus induction of IFN is restricted upstream of IRF3 phosphorylation, thus restricting IFN production. Further, hantaviruses do not block ISG induction in response to IFN addition and it is unclear how ISG induction occurs in the presence of STAT regulation. The high level induction of MxA and other ISGs by all hantaviruses suggest that IFN receptor-directed responses are not regulated by hantaviruses and that late in infection, hantaviruses are resistant to the effects of induced ISGs. A recent finding has implicated a role for hantavirus encoded NSs in regulation of the IFN pathway.5,55 The bunyavirus NSs protein has been shown to inhibit the host cell IFN response.74 However, unlike other members of the Bunyaviridae family,74 hantavirus NSs proteins have not been identified during infection. It was recently reported that pathogenic Puumala virus and non-pathogenic Tula virus express NSs proteins during infection that participate in IFN regulation.5,55 However, potential NSs proteins have not been identified in HTNV, ANDV, NY-1V and other pathogenic hantaviruses and the ORF reported in the PUUV is disrupted in these viruses. Further, IFN regulation, suggested to be associated with PUUV and TULV NSs expression, results in only a 30% decrease in IFN transcriptional responses,5 suggesting at best a minor secondary role for this protein in IFN regulation by hantaviruses. Thus it is not clear whether a hantavirus NSs protein significantly regulates cellular IFN responses.

4. CONCLUSION All hantaviruses replicate successfully in host endothelial cells, and thus they all possess the ability to regulate IFN responses. This suggests that the subtle differences in host and human IFN pathway proteins are likely to determine whether a hantavirus has the potential to be pathogenic. Nonpathogenic TULV still replicates within human endothelial cells suggesting that there are additional determinants of hantavirus pathogenesis beyond IFN regulation, although IFN regulation is a required primary determinant of hantavirus pathogenesis. Interestingly, all hantaviruses induce high level of IFN responses late in infection. These findings and the resistance of hantaviruses to the later addition of IFN suggest that the early regulation of IFN responses is

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necessary only until the virus makes a product that permits viral replication in the presence of cellular ISG responses. There is little understanding of what switches this regulatory response or how hantaviruses effect a late IFN resistant state. The cytoplasmic tails of pathogenic hantaviruses appear to block IFN induction by regulating responses at the level of the TBK1 complex. The TBK1-TRAF3 complex has recently been shown to be regulated by the state of TRAF3 ubiquitination and the G1-tails of pathogenic hantaviruses are ubiquitinated and degraded. Although specific interactions of G1-tails and their degrons have not been studied, these findings suggest that the G1-tail has the potential to alter TBK1 complex formation, activation or regulation in a variety ways. These interactions are just beginning to be investigated and are important since the G1-tail is a determinant of hantavirus pathogenesis that can be potentially manipulated to attenuate pathogenic hantaviruses.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants R01AI47873, PO1AI055621, R21AI075000 and U54AI57158 (Northeast Biodefense Center- Lipkin) and by a Veterans Affairs Merit Award.

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28. Hynes RO, Hodivala-Dilke KM (1999). Thromb Haemost 82, 481–485. 29. Joutsi-Korhonen L, Preston S, Smethurst PA, Ijsseldijk M, SchaffnerReckinger E, Armour KL, Watkins NA, Clark MR, de Groot PG, Farndale RW, Ouwehand WH, Williamson LM (2004). Thromb Haemost 91, 743–754. 30. Gavrilovskaya IN, Brown EJ, Ginsberg MH, Mackow ER (1999). J Virol 73, 3951–3959. 31. Gavrilovskaya IN, Shepley M, Shaw R, Ginsberg MH, Mackow ER (1998). Proc Natl Acad Sci USA 95, 7074–7079. 32. Mackow ER, Gavrilovskaya IN (2001). Curr Top Microbiol Immunol 256, 91–115. 33. Raymond T, Gorbunova E, Gavrilovskaya IN, Mackow ER (2005). Proc Natl Acad Sci USA 102, 1163–1168. 34. Gavrilovskaya I, Gorbunova E, Mackow N, Mackow ER (2008). J Virol 82, 5797–5806. 35. Alff PJ, Gavrilovskaya IN, Gorbunova E, Endriss K, Chong Y, Geimonen E, Sen N, Reich NC, Mackow ER (2006). J Virol 80, 9676–9686. 36. Randall RE, Goodbourn S (2008). J Gen Virol 89, 1–47. 37. Isaacs A, Lindenmann J (1957). Proc R Soc Lond B Biol Sci 147, 258–267. 38. Pestka S, Langer JA, Zoon KC, Samuel CE (1987). Annu Rev Biochem 56, 727–777. 39. Samuel CE (2001). Clin Microbiol Rev 14, 778–809, Table of contents. 40. Boehme KW, Compton T (2004). J Virol 78, 7867–7873. 41. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S (2006). Nature 441, 101–105. 42. Loo YM, Fornek J, Crochet N, Bajwa G, Perwitasari O, Martinez-Sobrido L, Akira S, Gill MA, Garcia-Sastre A, Katze MG, Gale M, Jr. (2008). J Virol 82, 335–345. 43. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T (2004). Nat Immunol 5, 730–737. 44. Maniatis T, Falvo JV, Kim TH, Kim TK, Lin CH, Parekh BS, Wathelet MG (1998). Cold Spring Harb Symp Quant Biol 63, 609–620. 45. Du W, Maniatis T (1992). Proc Natl Acad Sci USA 89, 2150–2154. 46. Lenardo MJ, Fan CM, Maniatis T, Baltimore D (1989). Cell 57, 287–294. 47. Lin R, Heylbroeck C, Pitha PM, Hiscott J (1998). Mol Cell Biol 18, 2986–2996.

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48. Sato M, Tanaka N, Hata N, Oda E, Taniguchi T (1998). FEBS Lett 425, 112–116. 49. Wathelet MG, Lin CH, Parekh BS, Ronco LV, Howley PM, Maniatis T (1998). Mol Cell 1, 507–518. 50. Andrejeva J, Childs KS, Young DF, Carlos TS, Stock N, Goodbourn S, Randall RE (2004). Proc Natl Acad Sci USA 101, 17264–17269. 51. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, Ishii KJ, Takeuchi O, Akira S (2005). Nat Immunol 6, 981–988. 52. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, Tschopp J (2005). Nature 437, 1167–1172. 53. Seth RB, Sun L, Ea CK, Chen ZJ (2005). Cell 122, 669–682. 54. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB (2005). Mol Cell 19, 727–740. 55. Jaaskelainen KM, Plyusnina A, Lundkvist A, Vaheri A, Plyusnin A (2008). J Virol 5, 3. 56. Kraus AA, Raftery MJ, Giese T, Ulrich R, Zawatzky R, Hippenstiel S, Suttorp N, Kruger DH, Schonrich G (2004). J Virol 78, 6143–6150. 57. Spiropoulou CF, Albarino CG, Ksiazek TG, Rollin PE (2007). J Virol 81, 2769–2776. 58. Stoltz M, Ahlm C, Lundkvist A, Klingstrom J (2007). J Virol 81, 8685–8691. 59. Krakauer T, Leduc JW, Morrill JC, Anderson AO, Krakauer H (1994). Viral Immunol 7, 97–101. 60. Ahlm C, Linden C, Linderholm M, Alexeyev OA, Billheden J, Elgh F, Fagerlund M, Zetterlund B, Settergren B (1998). J Infect 36, 149–155. 61. Geimonen E, Neff S, Raymond T, Kocer SS, Gavrilovskaya IN, Mackow ER (2002). Proc Natl Acad Sci USA 99, 13837–13842. 62. Emeny JM, Morgan MJ (1979). J Gen Virol 43, 247–252. 63. Tamura M, Asada H, Kondo K, Takahashi M, Yamanishi K (1987). Antiviral Res 8, 171–178. 64. Temonen M, Vapalahti O, Holthofer H, Brummer-Korvenkontio M, Vaheri A, Lankinen H (1993). J Gen Virol 74 (Pt 3), 515–518. 65. Sarkar SN, Sen GC (2004). Pharmacol Ther 103, 245–259. 66. Khaiboullina SF, Rizvanov AA, Deyde VM, St Jeor SC (2005). J Med Virol 75, 267–275. 67. Sen GC, Ransohoff RM (1993). Adv Virus Res 42, 57–102. 68. Horisberger MA (1992). J Virol 66, 4705–4709.

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69. Frese M, Kochs G, Feldmann H, Hertkorn C, Haller O (1996). J Virol 70, 915–923. 70. Kanerva M, Melen K, Vaheri A, Julkunen I (1996). Virology 224, 55–62. 71. Oelschlegel R, Kruger DH, Rang A (2007). Virus Res 127, 100–105. 72. Frese M, Kochs G, Meier-Dieter U, Siebler J, Haller O (1995). J Virol 69, 3904–3909. 73. Kochs G, Janzen C, Hohenberg H, Haller O (2002). Proc Natl Acad Sci USA 99, 3153–3158. 74. Weber F, Bridgen A, Fazakerley JK, Streitenfeld H, Kessler N, Randall RE, Elliott RM (2002). J Virol 76, 7949–7955. 75. Sen N, Sen A, Mackow ER (2007). J Virol 81, 4323–4330. 76. Geimonen E, LaMonica R, Springer K, Farooqui Y, Gavrilovskaya IN, Mackow ER (2003). J Virol 77, 1638–1643. 77. Hiscott J, Grandvaux N, Sharma S, Tenoever BR, Servant MJ, Lin R (2003). Ann NY Acad Sci 1010, 237–248. 78. Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J (2003). Science 300, 1148–1151. 79. Garcin D, Marq JB, Iseni F, Martin S, Kolakofsky D (2004). J Virol 78, 8799–8811. 80. Otsuka M, Kato N, Moriyama M, Taniguchi H, Wang Y, Dharel N, Kawabe T, Omata M (2005). Hepatology 41, 1004–1012. 81. Rothenberger S, Burns K, Rousseaux M, Tschopp J, Bron C (2003). Oncogene 22, 5614–5618. 82. Rothenberger S, Rousseaux M, Knecht H, Bender FC, Legler DF, Bron C (2002). Cell Mol Life Sci 59, 171–180. 83. Kayagaki N, Phung Q, Chan S, Chaudhari R, Quan C, O’Rourke KM, Eby M, Pietras E, Cheng G, Bazan JF, Zhang Z, Arnott D, Dixit VM (2007). Science 318, 1628–1632. 84. Oganesyan G, Saha SK, Guo B, He JQ, Shahangian A, Zarnegar B, Perry A, Cheng G (2006). Nature 439, 208–211. 85. Prescott JB, Hall PR, Bondu-Hawkins VS, Ye C, Hjelle B (2007). J Immunol 179, 1796–1802.

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CHAPTER 12

Measles Virus Captures Specific Host Machineries to Cause Immunosuppression and Disease Bumsuk Hahm & Michael B. A. Oldstone

ABSTRACT The highly contagious human pathogen, measles virus (MV) continues to infect 30 million individuals worldwide leading to ∼350 000 deaths annually, despite the availability of protective vaccines. MV induces profound suppression of the immune system, rendering infected persons vulnerable to secondary microbial invasion. In rare cases, MV persistently infects the central nervous system (CNS) causing subacute sclerosing panencephalitis (SSPE), a fatal neurodegenerative disease. Two cellular receptors, human signaling lymphocyte activation molecule (SLAM; CD150) and CD46, were identified as proteins that bind MV hemagglutinin, allowing the virus to enter target cells. Investigators created transgenic mice bearing MV receptor either SLAM or CD46, which have greatly advanced the study of MV pathogenesis. Here, we dissected the interplay between MV and host innate and adaptive immune responses by introducing research results especially from molecular and cellular mechanistic studies performed with transgenic mice models. These results showed that (i) transgenic mice engineered to express MV receptors ubiquitously or specifically on neurons, T cells, or dendritic cells (DCs) were permissive to the virus infection; (ii) following MV infection, these models manifested the cardinal features of MV-induced

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immunosuppression, MV persistence, and SSPE; (iii) CD4+ T lymphocytes were crucial elements controlling primary MV infection of the CNS, assisted by either CD8+ T cells or B cells; (iv) MV suppressed DC development via type I IFN-mediated STAT1-independent, but STAT2specific signaling; (v) MV targeted TLR4 signaling on DCs to interfere selectively with the synthesis of IL-12. This review incorporates the outcomes of research on animal models as well as clinical and experimental studies of humans for the purpose of better understanding the immunobiology of MV infection. Additionally, we address prospects for the development of new therapeutics that target viral mechanisms to regulate host immune-signaling responses.

1. INTRODUCTION Following development of an effective and efficient attenuated vaccine by John Enders and his colleagues, measles virus (MV) lost its reputation as a notoriously dangerous agent of infection that causes a high rate of morbidity and mortality. However, despite the presence of this vaccine, outbreaks of MV infection have occurred continuously worldwide, causing havoc and perpetuating the virus’ existence. Eliminating this harmful pathogen would be difficult, considering that approximately 95% herd immunity is required to block MV transmission and that the virus is, characteristically, extremely contagious. Persons infected with MV can transmit the virus several days before the clinical manifestations of upper respiratory infection and rash appear. Moreover, the heat- and light-sensitive properties of the vaccine raise concerns about its effectiveness in tropical areas such as parts of Africa, where the infection remains rampant. Additionally, the measles vaccine is not effective in treating newborns up to nine months of age due to their enduring maternal antibodies. Not only is the kinetics of antibody’s disappearance unpredictable in newborns before vaccine administration but also prolonged nursing remains important in Third World countries. Furthermore, no treatment for a MV-caused SSPE is available. This pathogen’s long-term circulation and widespread infection of humans has left a number of fundamental questions unanswered. How does MV suppress the host’s innate and adaptive immune responses? Why

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are specific cellular proteins selected as receptors for MV’s entrance? How does MV establish years-long persistence in both neurons and lymphocytes in SSPE patients? How does MV trigger the progressive neuronal disease SSPE? What are the defensive responses by host cells against MV? This review focuses on recent developments leading to a better understanding of mechanisms that enable MV to exert immunosuppression and cause CNS diseases.

2. CELLULAR RECEPTORS FOR MV Multiple viruses initiate their contact with a host via direct association with specific proteins called “virus receptors” expressed on the surfaces of target cells. Through the efforts of virologists,1–3 human CD46 (membrane cofactor protein) was identified as a molecule that binds the hemagglutinin (HA) glycoprotein of MV allowing the virus to enter cells that express CD46 (Table 1). Molecular analysis revealed that MV HA interacts with two short consensus repeats (SCRs) 1 and 2 of the four SCRs belonging to CD46 molecules, specifically two domains in SCR1 (amino acids 37–56) and SCR2 (amino acids 85–104).4,5 Clinical isolates of wildtype (wt) MV, which efficiently infect lymphoid cell lines like B95-8 cells, hardly infect CD46-expressing epithelial cells or require multiple passages through the cells to enhance viral titers or cause MV-specific syncytia to form. This difference is caused mainly by the deletion of SCR1 in B95-8 cells and by the higher affinity of MV for the second receptor Signaling Lymphocyte Activation Molecule (SLAM; CD150). Vaccine strains of MV adapted to epithelial cells use the CD46 receptor to infect CD46-expressing cells. This result initially indicated that MV acquired high binding affinity to CD46 through cell culture adaptation and that wt MV might interact with another receptor expressed on lymphoid cells. Indeed, investigators found SLAM receptors expressed on B95-8 cells mediating wt MV infection6 (Table 1). Accordingly, MV isolates from specimens of infected patients efficiently infected modified epithelial cell lines like Vero cells (African green monkey kidney cells) or CHO cells (Chinese hamster ovary cells) engineered to express SLAM. Although controversy remains about the binding of wt MV to CD46 receptors,7,8 it is likely that wt MV has strong binding affinity to SLAM

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Receptor

Interaction Domain with MV

Expression Pattern

Other Microbes that Use the Protein as a Receptor

Cellular Functions

Canine distemper virus Rinderpestvirus

T cells stimulation Th1/Th2 cytokine regulation (IL-4) TLR4-mediated cytokine production Inhibition of macrophage function SLAM-SAP-Fyn T signaling

CD46

SCR1 and SCR2 of CD46 with HA of vaccine MV (weak binding with several wt MV)

All nucleated cells

Human herpesvirus 6, Bovine viral diarrhea virus, Certain serotypes of adenovirus (Ad3, Ad11, Ad35, and Ad37), Streptococcus pyrogenes, Neisseria

Regulation of complement pathwayreceptor for C2b/C2b T cell differentiation Alteration of T cell polarity for antigen presenting cells

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SLAM

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Table 1. Cellular receptors for measles virus.

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receptors but a weak binding capacity for CD46. Multiple passages through CD46-receptor-expressing cells in vitro have likely enabled vaccine strains of MV to develop a strong affinity for CD46 while retaining SLAM-binding ability. While CD46 receptors are expressed on all nucleated cells,9,10 SLAM has been detected solely on certain immune cells including immature thymocytes, memory T cells, activated T cells, B cells, dendritic cells (DCs), monocytes, and macrophages.11,12 The expression profiles of these two receptors suggest that they have different roles in MV pathogenesis. Expression of CD46 or other unknown receptors (Workshop on Negative Strand RNA Viruses 2007) on cells that do not express SLAM (e.g., CNS) may account for MV’s invasion of neurons and epithelial cells. It is still not clear why vaccine strains of MV are less adept at causing immunosuppression and virulence in humans than wt strains of MV. The difference in the degree of immunosuppression may be due to the differential ability of the virus replication in specific immune cells, the function of wt MV proteins controlling host immunity, and/or differential receptor usages. Possibly the vaccine strains of MV interact strongly with CD46 receptor-expressing cells in the initial infection route rather than rapidly infecting lymphocytes and secondary lymphoid organs expressing the SLAM receptor. If so, early and strong host immune responses might be induced without compromising lymphoid cells. Although wt MV is thought to infect and spread in humans via the same route as vaccine strains, wt virus might search for SLAM-expressing immune cells (e.g., DCs) during an early phase of invasion. Thus, wt MV could efficiently migrate into a major site for the induction of adaptive immune responses, which can strongly impair T lymphocyte responses. Syncytia formation in lymphoid areas has been a hallmark of MV infection. It is also plausible that wt MV attacks innate immune cells expressing SLAM thereby altering the innate immune responses, whereas vaccine strains initially contact CD46-expressing epithelial cells in the respiratory tract. Such contact (MV-CD46 on epithelial cells) could induce the release of inflammatory cytokines/chemokines and viral components, which might help in the stimulation of host innate and adaptive immunity. Further investigation is required to determine whether different receptor usages are critical for the attenuation of MV’s virulence. Also, since the initial phase of wt MV

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infection involves epithelium of the upper respiratory tract, the role of SLAM is not yet clear, since that receptor is absent from such cells. In this regard, several reports imply that some cellular receptors for MV are still unknown, because the virus infects cells that express neither CD46 nor SLAM.13–15 Further, molecular pattern recognition receptors of DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DCSIGN)16 and toll-like receptor (TLR)217 were proposed to interact with MV. Although these molecules do not act as receptors for viral entrance into cells, the attachment of MV to specific cell surface proteins is likely to affect the pathogenesis of MV infection. In fact, the transport of human immunodeficiency virus (HIV)-attached to DC-SIGN on migrating DCs into CD4+ T cell-resident secondary lymphoid organs has explained the efficient spreading of HIV and HIV-induced immune suppression.18 Perhaps MV uses attachment proteins in a similar way to efficiently transmit MV particles to the secondary lymphoid tissues. Alternatively, MV glycoprotein’s interaction with DC-SIGN or TLR2 induces DC maturation, which subsequently elevates the level of SLAM receptor expression allowing MV entrance through a SLAM-MV HA interaction. Consequently, SLAM receptors newly induced on maturing DCs might be available for free MV particles or for virus already attached to DC-SIGN or TLR2. However, how SLAM is expressed near the site of DC-SIGN or TLR2 is not known. MV appears to spread in neurons through a different pathway than in other cells. Initial MV infection of neurons is dependent on a cellular receptor, likely to be CD46, but continued neuron-to-neuron spread necessitates neither MV-CD46 interaction nor syncytium formation.19 Instead, MV spreads via cell-to-cell contact in terminally differentiated, non-dividing neurons. Recently, investigators found that once MV infects neurons, it utilizes neurokinin-1 for efficient trans-synaptic spread.20 Collectively, these data suggest that MV has diverse strategies for the use of cellular receptors and attachment proteins in their survival depending both on the course of infection and the site of viral localization. Among the numerous cellular proteins, why does MV choose SLAM and CD46 as receptors? Canine distemper virus (CDV) and rinderpestvirus (RPV) along with MV belong to the Morbillivirus genus of the Paramyxoviridae family. Along with MV, CDV and RPV use canine and bovine SLAM as receptors, respectively, for their cellular entry.21 CD46

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also serves as a receptor for several other pathogens22 including human herpesvirus 6,23 bovine viral diarrhea virus,24 certain serotypes of adenovirus groups B and D (Ad3, Ad11, Ad35, and Ad37),25,26 Streptococcus pyrogenes27 and piliated pathogenic Neisseria.28 Do CD46 and SLAM act only as receptors allowing the initial entrance of pathogens? Are cellular functions of CD46 and SLAM disrupted as well by the interaction with MV for the virus’ survival? Conceivably MV perturbs the unique immune regulatory function of SLAM and/or CD46. Initially, SLAM was isolated as a co-stimulatory molecule for T cell proliferation in a CD28-independent manner.11 Genetic deletion of SLAM in a mouse system revealed that SLAM is important for regulating Th1/Th2 responses required for efficient IL-4 expression and inflammatory cytokine production associated with TLR4 ligation.29 Interestingly, MV specifically suppressed TLR4mediated IL-12 synthesis on DCs via SLAM-MV interaction.30 An issue that should be examined now is whether or how MV impairs SLAM signaling to interfere with TLR4-triggered IL-12 expression. Besides a regulatory role of complement activation,31 CD46 was newly defined as a co-stimulatory molecule for T cell activation,32 inducing Th1 type cells or regulatory T cells secreting IL-10 depending on the stimulatory signals. In addition, CD46 ligation was reported to impair lymphocyte polarization toward antigen-presenting cells.33 Understanding the mechanism of MV-receptor interaction and its effect on host immune responses should assist in the development of novel therapeutics that block specific viral mechanisms for receptor interaction and/or receptor-mediated signaling pathways.

3. MV-INDUCED IMMUNOSUPPRESSION MV was known to suppress its host’s immune system from clinical observations recorded by Clements von Pirquet, twelve years before MV was isolated and over 85 years before HIV was isolated and reported to cause immunosuppression. During MV infection, the microbes in an arrested state like those causing tuberculosis and syphilis in humans, become reactivated and spread. In addition, MV predisposes infected individuals to other microbial invasions, as their immune system becomes crippled. The immunosuppression lasts for several weeks to months after acute MV

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infection, raising the possibility that MV persists locally in certain tissues for a longer period than is expected despite the loss of viremia.34 Why this immunosuppression lasts after MV clearance and why immunosuppression is eventually resolved are not clear; however, the immunosuppression associated with secondary pathogenic infection is responsible for most measles-related fatalities. Nevertheless, the mechanisms engaged by MV for immune suppression and for enhanced susceptibility of the host to secondary microbial infections remain poorly understood.

3.1 Inhibition of the T Cell Response When T cells become infected directly with MV or even when they come in contact with MV-infected cells, mitogen unresponsiveness is induced. Blocking of T cell stimulation by antigen-presenting DCs does not require direct MV infection of T cells (as discussed in Section 3.2). Yet, stimulation of T cells with mitogens is required for efficient MV replication and production of infectious MV in human lymphoid cells.35,36 These observations suggest that activated T cells are one of the major targets for MV infection, perhaps because of the enhanced expression of SLAM receptor or other cellular proteins on activated T cells. However, following the infection, probably after MV amplification, MV inhibits progression of T lymphocytes’ progression through the cell cycle, which is arrested at G0/G1.37,38 At this point, the cell-cycle regulatory proteins, cyclins D3 and E, which positively regulate entry into S phase from G1, are significantly downregulated.38 Instead, a cell-cycle (S phase) inhibitory molecule, Cdk inhibitor p27, is maintained at a high level in G0/G1-blocked subpopulations of MV-infected cells compared to proliferating MV-infected cells. Indeed, progressive retinoblastoma protein activation was not observed in MV-infected lymphocytes. Thus, MV manipulates cell cycle machinery in favor of stimulating further MV replication while altering the function of T cells. On the viral side of immunosuppression, the MV glycoproteins HA and fusion protein display inhibitory activity with respect to T cell proliferation.39,40 Even without direct infection of T cells, MV glycoproteins simply in contact with uninfected T cells can block their proliferation. Furthermore, MV was reported to interact with glycosphingolipid-enriched

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membrane microdomains (lipid rafts) on T cells, altering cellular signaling pathway of phosphatidylinositol 3-kinase (PI3K)41 and impairing IL-2dependent Akt kinase activation.42 During and after MV infection, Th2 type cytokine responses were skewed43 and described as an explanation for the inhibition of Th1mediated cellular immunity and possibly enhanced susceptibility to secondary microbial invasion. Although the Th1 cytokines IFN-γ and IL-2 are only transiently produced by T cells in children with measles, their plasma levels of IL-4 and IL-10 are significantly elevated and maintained even when analyzed one month after the onset of rash and long after its disappearance.44 Currently, there is no clear evidence that direct MV infection of T cells promote a biased cytokine response. Suppression of IL-12 synthesis from monocytes45 and DCs46 was proposed as a mechanism for inhibition of the Th1 type cellular immune response. Interestingly, the MV receptor SLAM was reported to regulate Th1/Th2 cytokine production of T cells. When a SLAM deficiency was induced in mice by genetic modification, IL-4 synthesis from CD4+ T cells was significantly inhibited upon primary and secondary T cell receptor (TCR) stimulation.29 By contrast, SLAM-deficient T cells produced a higher level of IFN-γ than wt cells, indicating a key role of SLAM for Th1/Th2 polarizing cytokine production. MV may alter a SLAM-mediated signaling pathway on T cells upon infection to regulate cytokine responses. Importantly, SLAM proteins on T cells were downregulated upon MV infection. In transgenic mice whose CD4+ and CD8+ T cells expressed human SLAM, but not CD46, the amount of SLAM receptor on T cells correlated directly with cellular susceptibility to wt MV infection.47 Upon infection, the SLAM receptor was downregulated on the surfaces of both CD4+ and CD8+ T cells, with a loss of 50 to 80% of SLAM expression. Downmodulation of SLAM expression was detected in both MV(+) and MV(−) populations among MVtreated T cells (B. Hahm, unpublished data), suggesting that SLAM expression on uninfected cells might be downregulated by contact with MV HA expressed on MV-infected cells. Indeed, expression of the HA protein of MV was sufficient for the downregulation of SLAM.48,49 MV HA was shown to induce downregulation of SLAM via its interaction with the endoplasmic reticulum or receptor-mediated binding at the host cell surface. Further studies should disclose the physiological meaning of

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SLAM downmodulation in MV pathogenesis. Similarly, expression of the CD46 receptor is downregulated by MV infection50 allowing complement-mediated lysis of infected cells.51 SLAM signaling on T cells is, in part, governed by SLAM-Associated Protein’s (SAP) transmission of the signal to src kinase FynT.52,53 Thus, it is of interest to investigate how MV HA docking to SLAM on T cells modulates SLAM-SAP-FynT signaling and its final effect on T cell activation or T cell polarization. Investigating the viral mechanism that regulates specific T cell signaling and the related consequences should help us to understand MV immunobiology and pathogenesis.

3.2 Disruption of the Antigen-Presenting DC Network DCs are the most potent antigen-presenting cells (APCs) and they signal the host immune system upon pathogenic invasion. MV appears to have developed several strategies to alter DC responses that contribute to both innate and adaptive immunity (Figures 1 and 2). MV was initially reported to infect human DCs obtained from cultures of blood CD34+ cells, monocytes, and Langerhans cells in vitro.54–56 MV infection increased the rate of DC apoptosis and inhibited CD40 liganddependent terminal differentiation of DCs.57 Co-culture of MV-infected DCs with T lymphocytes also enhanced virus production and Fas-mediated apoptosis of DCs.58 MV-infected DCs lost their efficient allostimulatory capacity in mixed lymphocyte reactions and actively inhibiting proliferation of naïve and activated T cells in response to mitogenic stimulation. Research with transgenic mice bearing the MV receptor human SLAM (hSLAM) on DCs (DC-hSLAM Tg) reproduced the previous findings in studies with human DCs and further revealed novel MV-DC interplay via MV binding to hSLAM.30,59–61 In this model, hSLAM protein was expressed on CD11c+ DCs under transcriptional control of a CD11c promoter. After incorporating just one molecule of hSLAM protein, murine DCs became susceptible to wt MV infection (Figure 1A). MV infected bone marrow (BM)-derived DCs and DCs in the lung (B. Hahm, unpublished data) and spleen from DC-hSLAM Tg mice in vitro and in vivo via the interaction with its cognate receptor hSLAM protein. Interestingly,

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Figure 1. MV suppresses DC responses via MV-hSLAM interaction. A. MV infects CD11c+ DCs expressing hSLAM receptors in vivo. DC-hSLAM Tg mice were either mock-infected (filled histogram) or infected with the JW strain of wt MV (open histogram) at 6 × 106 TCID50. Three days after infection, their splenocytes were isolated and analyzed by flow cytometry for the expression of MV proteins on the surfaces of CD4+ and CD8+ T cells and CD11c+ DCs. Percentages of MV(+) cells are shown. B. MV inhibits DC development. BM cells from hSLAM Tg mice were cultured for two days in a GM-CSF-supplemented complete medium to deplete cells attached to the bottom of the plates and provide signals for DC commitment. The floating cells were mock infected or treated with MV JW at 0.3 multiplicity of infection (MOI) and re-cultured using the GM-CSF-dependent DC development system. After four days of culture, the cells were analyzed for the expression of CD11c and MHC class II (I-A/I-E). Percentages of DCs defined as CD11c+MHC-II+ cells are depicted in the boxes. C. TLR4-mediated IL-12 suppression of MV-infected DCs expressing hSLAM. BM-derived DCs were mock infected (open bars) or infected with MV-JW (filled bars). Following treatment with poly(I:C), LPS, loxoribirine, or CpG as ligands for TLR3, TLR4, TLR7 and TLR9, respectively, DCs expressing IL-12p40/p70 were examined by FACS and are represented in the graph. D. MV-infected DCs actively impair mitogen-induced proliferation of CD4+ and CD8+ T cells. Mock-infected (left panels) or MV-infected DCs (right panels) from DC-hSLAM Tg mice were mixed with CFSE-labeled CD4+ T cells (upper panels) or CD8+ T cells (lower panels) from wt mice in the presence of PMA and ionomycin. CFSE fluorescence from T cells was traced from day 0 (d0, filled histogram) to day 5 (d5, open histogram) to determine T cell proliferation in response to mitogenic stimulation.

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Figure 2. MV attacks the DC network and T cells to induce immunosuppression. Schematic diagram represents a model for diverse strategies of MV to utilize or disrupt host immune responses of DCs and T cells. [1] MV infects or attaches to immature DC (iDC) in peripheral tissues (probably respiratory tissues) and induces maturation phenotypically, which enhances the expression of SLAM receptors and migratory signals on mature DC (mDC) and [2] allows efficient migration into secondary lymphoid organs such as the mediastinal lymph nodes (MLN). [3] MV transferred into the major site for T cell clonal expansion begins to infect DCs resident in that tissue, which further blocks DC activation. [4] MV-infected DCs strongly alter DC-mediated T cell stimulation. [5] Also, MV infects T cells directly, amplifies MV progeny, interferes with cell-cycle progress, and regulates cellular signaling pathways for T cell activation and T cell polarization. On the other hand, MV-infected DCs in the periphery or bone marrow (BM) secrete type I IFNs, which mediate both antiviral and immunosuppressive activity. Although type I IFN blocks MV spread, [6] a type I IFN-induced unique signaling pathway dependent on STAT2, but not on STAT1, suppresses DC development from its precursors in BM hematopoietic stem cells (HSC). [7] Upon infection by a secondary microbe containing the TLR4L motif, induction of an effective host immune response is impaired, as MV inhibits the innate immune response of DCs in the periphery to synthesize IL-12 selectively via TLR4 signaling. References related to Figure 2 are found in the Section 3.

MV infection significantly inhibited the development of DCs from hematopoietic BM stem cells; such inhibition occurred in either a granulocyte macrophage colony-stimulating factor (GM-CSF) (Figure 1B) or a fms-like tyrosine kinase 3 ligand (Flt3-L)-supplemented DC development system in vitro and ex vivo.60 Limitation of DCs expressing high levels of MHC molecules (MHC class I and II) and co-stimulatory molecules such

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as B7-1, B7-2, and CD40 likely contributes to MV-induced suppression of immune responses and increases host susceptibility to secondary microbial infections. Yet, no blockade in DC generation caused by MV infection was observed when DC precursors from Tg mice were deficient in type I IFN receptor. Thus, these data indicate that virus-induced type I IFN perturbed DC precursors’ capacity to differentiate into DCs. The result was recapitulated by a direct treatment of the culture with recombinant (r) IFN-β , demonstrating type I IFN-mediated DC suppression. MVinduced type I IFN interfered with both proliferation and differentiation of DC progenitors/precursors into CD11c+MHC-II+ DCs. These results imply that MV utilizes type I IFN to suppress the DC commitment required for inducing adaptive immune responses. The molecular mechanism of type I IFN-mediated DC inhibition was investigated with the use of mice genetically modified to become specifically defective in a gene involved in the type I IFN signaling cascade. The classical type I IFN signaling pathway involves activation of both STAT1 and STAT2 to induce transcriptional activation of multiple type I IFNinducible genes.62 Unlike this typical IFN signaling pathway, MV-induced type I IFN depends on the expression of STAT2 but is independent of either STAT1, STAT4, or STAT6 for the inhibition of DC development. Thus, MV-induced type I IFNs transmit the signaling through STAT2 without the help of STAT1, resulting in suppression of DC development (Figure 2). This STAT2-mediated DC inhibition was reproduced with direct treatment of rIFN-β , proving the existence of a cell type specific IFN signaling pathway. In DC precursor cells, type I IFN-induced STAT2 signaling, even in the absence of STAT1 expression, led to activation of an IFN stimulation response element (ISRE)-mediated gene transcription. Currently, it is unclear why the function of STAT1-mediating type I IFN signaling is nullified in DC precursors. The mechanism by which STAT2 transmits a type I IFN signal in DC precursors remain to be explored. Since STAT1independent, but STAT2-dependent, type I IFN signaling provides a uniquely effective way of inhibiting the development of DC, the therapeutic potential of targeting STAT2-selective signaling to modulate IFN’s function has a clear advantage for immunoregulatory drug development. Type I IFN is a well-recognized inhibitor of viral replication and spread.63,64 Through the JAK/STAT signaling cascade, multiple antiviral

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proteins are produced to suppress viral replication. To confront this potent host innate immune response, viruses have devised several strategies to block type I IFN’s antiviral activities, which include inhibiting type I IFN production, blocking JAK/STAT signaling that leads to activation of ISRE-mediated gene transcription, and suppressing the function of specific antiviral proteins induced by type I IFN. MV was reported to inhibit type I IFN signaling in several cell lines via perturbation of STAT1 activation,65–68 which is an essential component for the typical signal transduction pathway of both type I IFN and type II IFN (IFN-γ ).69,70 Although the action mechanism is controversial, MV V protein was identified as a potent inhibitor of STAT1 activation. Conceivably, MV inhibits the antiviral actions of type I IFNs in the infected cells when the virus wants to or needs to amplify itself. However, MV-induced type I IFN suppresses DC development, suggesting that MV may enhance its own survival by utilizing type I IFN’s immunomodulatory ability. Indeed, previous reports demonstrated that type I IFN could be harmful to the host.71 For instance, the level of IFN-α is elevated in the sera of patients with systemic lupus erythematosus, and IFN-α therapy exacerbates the disease.72 Virusinduced type I IFN was also found to be detrimental to the host when blocking this cytokine’s activity decreased liver cell necrosis and enhanced the survival of neonatal mice infected with lymphocytic choriomeningitis virus (LCMV) in vivo, a condition that otherwise results in death.73 Thus, vicious viruses might block the ordinarily beneficial role of type I IFN inhibiting viral replication on one hand and intensify its detrimental actions on the other. MV was previously reported to induce the phenotypic maturation of DCs generated from human blood cells in a GM-CSF-mediated culture system, as judged by expression levels of DC marker proteins including the co-stimulatory molecules B7-1, B7-2, and CD40. Results were similar when DCs derived from the BM of DC-hSLAM Tg mice were infected with MV (B. Hahm, unpublished data). However, MV downregulated the expression of those co-stimulatory molecules and MHC class I and II proteins on the surfaces of CD11c+ DCs from spleens of the Tg mice, indicating that MV suppressed maturation of splenic DCs. These results suggest that MV may have different tactics in altering DCs depending on their localization and the status of DC differentiation. Also possible is that

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MV-induced DC maturation in the periphery helps DCs to convey MV from the primary site of MV infection (respiratory tract epithelium) to secondary lymphoid tissues (Figure 2), where MV is particularly adept at disrupting the activation of resident DCs and T cells in the lymphoid organ. For efficient detection of microbial pathogens, DCs are equipped with molecular sensors such as Toll-like receptors (TLRs) that recognize molecular signatures including proteins and nucleic acids.74–76 TLR signaling activates DCs by inducing the production of inflammatory cytokines and chemokines, and affecting their ability to present antigens.77 To survive better in the infected host, viruses may have evolved multiple strategies to evade or suppress TLR responses.78 Indeed, MV activated TLR2 signaling on monocytes, inducing the expression of IL-6 and SLAM,17 thereby suppressing TLR7- and TLR9-stimulated production of type I IFNs from human plasmacytoid DCs.79 Investigators showed that MV inhibited the production of IL-12 from lipopolysaccharide (LPS)stimulated monocytes and CD40 ligand-stimulated DCs.45,57 Roles for CD46 and MV nucleoprotein were proposed to explain MV-induced IL12 suppression. When DCs from DC-hSLAM tg mice were infected via MV-hSLAM interaction, the production of IL-12 by DCs was suppressed in response to LPS activation of TLR4, but not to the agonists of TLR2, TLR3, TLR7 or TLR930 (Figure 1C). Since other cytokines, i.e., tumor necrosis factor (TNF)-α and IL-6 were not suppressed on MV-infected DCs in the ligation of TLRs including TLR4, the inhibition of IL-12 was a specific event. TLR4-mediated IL-12 suppression was observed on MVinfected DCs even in the presence of other TLR activation. TLR7- or TLR9-triggered IL-12 synthesis was actively suppressed on MV-infected DCs by concomitant treatment with TLR4 ligand, indicating that TLR4mediated IL-12 suppression is negatively dominant over other TLR activation. Currently, no TLR signaling mechanisms have been identified to satisfactorily explain the specificity of TLR4-mediated IL-12 suppression. MV did not impair the level of TLR4 expression on the surfaces of MVinfected DCs with or without treatment by the TLR4 agonist LPS. IL-10 is an immunosuppressive cytokine and counter-regulates IL-12 expression. However, antibodies against IL-10 or IL-10 receptor could not restore TLR4-activated IL-12 expression (B. Hahm, unpublished data).

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Commercial inhibitors that block the activation of cellular signaling components for mitogen-activated protein kinase (MAPK), e.g., extracellular signal-regulated kinase,14 also failed to reverse MV’s inhibition of IL-12 (B. Hahm, unpublished data). Since IL-6 and TNF-α were not suppressed upon TLR4 stimulation of MV-infected DCs, it is likely that MV inhibition occurs via specific signaling for the late stage of IL-12 gene expression, without generalized suppression of TLR4 signaling. It is also imaginable that TLR4 activation, but not other TLR ligations, induces the expression of some unknown molecule(s) in MV-infected DCs to suppress IL-12 synthesis. Utilization of a MV reverse genetic system allowed investigators to examine a possible role of viral V or C protein. V or C gene-deficient MV displayed a similar IL-12-inhibitory activity as did wt MV, indicating that V and C proteins are not responsible for IL-12 suppression. Interestingly, UV-inactivated, replication-defective MV suppressed TLR4-mediated synthesis of IL-12 from hSLAM(+) DCs in a situation wherein this virus did not impair IL-12 production from hSLAM(−) DCs. This result suggests that a MV HA interaction with hSLAM contributes to TLR4mediated IL-12 inhibition. In support, IL-12 inhibition occurred in both MV(−) and MV(+) populations among MV-treated DCs, demonstrating no requirement for direct viral infection for the IL-12 inhibition. However, considering that MV nucleoprotein and MV-CD46 interaction were reported to be important for blocking IL-12 expression,45,46 MV may utilize several viral and cellular components to maximize IL-12 suppression. MV-induced inhibition of IL-12 synthesis may explain, in part, a biased cytokine response of the Th2 phenotype observed in MV-infected patients, since IL-12 is a pivotal cytokine in pushing Th1 type immune responses. Additionally, profound IL-12 inhibition upon TLR4-activated secondary microbial infection, even in the presence of other TLR activation, could worsen MV infection in patients. The ability of MV to prohibit T lymphocytes from proliferating in response to stimuli by recalled antigen, mitogen, and allogen has been documented. MV infected DCs expressing hSLAM receptors from DChSLAM Tg mice and aborted DCs’ allo-stimulatory capacity when examined in mixed lymphocyte reactions. Indeed, DCs infected by MV failed to upregulate the expression of T cell activation markers (CD44, CD25,

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and CD69) on allogeneic T cells, leading to suppression of MHC class IIdependent T cell expansion.59 In addition, MV-infected DCs actively blocked mitogen-stimulated T cell amplification (Figure 1D). Utilization of gene-deficient mice allowed further examination of the molecular mechanisms involved.59 MV-infected DCs inhibited mitogen-induced T cell proliferation regardless of the expression of hSLAM, type I IFN receptor, TNF-α , lymphotoxin (LT)-α , or LT-β on the surfaces of T cells. Individual expression of those immunomodulatory molecules on T cells was not essential for inhibition of mitogen-dependent T cell proliferation. Precisely how MV-infected DCs inhibit T cell proliferation needs further elucidation at the molecular level.

4. CNS DISEASE CAUSED BY MV INFECTION MV causes encephalomyelitis in 1 per 1000 MV-infected persons and subacute sclerosing panencephalitis (SSPE) in rare (∼ 1/500 000) cases. SSPE is a fatal neurodegenerative disease with clinical signs appearing years (on the average of seven years) after the primary infection. Symptoms include progressive dementia, ataxia, and seizures leading to death. Related studies of pathogenesis and molecular mechanisms were hampered by a lack of suitable animal models until discovery of the MV receptor CD46. Cells in the CNS do not express SLAM, but do express CD46. Incorporation of human CD46 into murine neurons made them permissive to MV infection.80,81 MV infection via intracerebral inoculation of mouse neurons bearing CD46 resulted in a neurologic disease manifested by frequent seizures, paralysis, and death of the animals. As in humans, neonates were much more susceptible to MV infection and subsequent disease than adult mice. T and B cell-deficient RAG knockout mice (RAG ko) crossed with CD46 Tg mice became even more susceptible to the infection, indicating that adult mice possessing a competent immune system were protected from MV-caused neuronal disease.82 MV persisted in the CNS of RAG ko X CD46 Tg adult mice, until they died, on average of 45 days after infection, although some of these mice lived for 90 days or longer. The long-term persistence of MV (> 200 days) in the CNS was observed when the immune systems of CD46 Tg mice were suppressed by LCMV Cl 13 infection before MV inoculation.83

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The Cl 13 variant of LCMV induces immunosuppression in mice by inhibiting the function of DCs and subsequently blocking the generation of cytotoxic T lymphocytes (CTLs). This dual viral-hit model displays the cardinal features of human SSPE including presence of MV antigens in neurons, infiltration of lymphocytes into the CNS, high level of antiMV antibodies in the bloodstream, and biased hypermutation (A to G or U to C) in the RNA genome mainly encoding MV matrix protein.83 Thus, this model provides a great opportunity for dissecting the molecular mechanisms of MV persistence in neurons and pathogenesis for the progressive disease SSPE. Recombinant MV containing a hypermutated M gene, which came from Biken strain SSPE MV, was generated with a reverse genetics system and examined for infectivity and persistence in the CNS.84 This recombinant MV infected neurons from Tg mice expressing CD46 in vivo caused a chronic progressive CNS disease leading to death with slower kinetics than that caused by the Edmonston strain of MV.84 Thus, it is likely that hypermutation in the MV genome actively contributes to viral persistence and the prolonged neurodegenerative disease it engenders. A host enzyme, adenosine deaminase acting on RNA (ADAR) 1a (L form), is thought to mediate the mutation from adenosine (A) to guanosine (G) via the catalysis of A-to-inosine (I) deamination. Specifically induced by IFN, ADAR1a binds to doublestranded (ds) RNA and introduces into viral genes mutations that decrease stability of the viral genome and impair production of viral proteins.85 Interestingly, ADAR1a was recently reported to bind antiviral protein dsRNA-activated protein kinase (PKR), inhibit the kinase activity, and consequently enhance host susceptibility to vesicular stomatitis virus (VSV) infection.86 Therefore, ADAR1a retains both proviral and antiviral activity. The roles of ADAR1a in MV replication, MV-induced immunosuppression and MV pathogenesis are of great importance. The creation of mice deficient in ADAR1a should provide much-needed information in further investigations. Animal models in which CD46 Tg mice are crossed with RAG ko mice for studies of MV persistence and disease87 have facilitated detection of molecular and cellular mechanisms of MV clearance. Reconstitution experiments with specific T cells and B cells demonstrated that CD4+ T cells were critical components for immune protection from MV invasion

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but required help from either CD8+ T cells or B cells.87 In contrast, in the absence of CD4+ T cells, CD8+ T cells and B cells could not block viral spread. IFN-γ in combination with TNF-α provided protection for the host from MV-induced death to a greater extent than IFN-γ per se, whereas neither perforin nor TNF-α alone did so. Downstream molecular mechanisms should be uncovered to determine particular signaling mediators critical for triggering the MV-induced neuronal disease process.

5. CONCLUSION Exploring the mechanisms used by MV for immune regulation, persistence in its host, and causation of CNS diseases is a worthy endeavor from the standpoint of clinical application as well as biological knowledge. Recent findings from research into MV biology, especially innate immunity, MV-DC interaction, and receptor usage have drawn the attention of virologists and immunologists. Creation of small animal models and advanced reverse genetics systems greatly assists in the dissection of molecular mechanisms for MV-induced immunosuppression and pathogenesis. Based on the recent dramatic increase in scientific information about virus-specific signaling mechanisms during virus-host interplay, a desirable objective is to develop therapeutics that target specific molecular signals in that sequence. Investigators in medicinal biological chemistry with effective screening systems for identifying drug candidates should keep track of the biological answers to eventually help victims of such damaging pathogens as MV.

ACKNOWLEDGMENTS This is publication No 18990 from Department of Immunology and Microbial Science, and Department of Infectology, The Scripps Research Institute. B.H. is supported by Departments of Surgery and Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology, University of Missouri-Columbia School of Medicine. This work was supported in USPHS grants AI36222, AI09484 and AI45927.

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SECTION III

Positive Single-Stranded RNA Virus

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CHAPTER 13

Severe Acute Respiratory Syndrome Coronavirus Induces Differential Host Gene Expression Responses Associated with Pathogenesis Vincent T. K. Chow & W. F. Leong

ABSTRACT The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) exhibits unique characteristics with respect to its molecular biology, replication, transcription and assembly. Several cell types including Vero E6 cells are permissive for SARS-CoV replication with resultant cytopathic effects. Microarray analyses have elucidated the cellular expression patterns of human genes in response to SARS-CoV infection. These studies have revealed alterations in the transcription and translation of genes belonging to various functional groups including cell cycle, apoptosis, signal transduction, transcriptional regulation, host translation, protein modulators, protein trafficking, cytoskeletal network, cellular metabolism, and antiviral resistance. There is significant induction of heat shock proteins that are crucial to the immune response mechanism. Specific immune-related genes are upregulated, coinciding with the high cytokine profiles in SARS patients which incite proinflammatory responses. Modified levels of transcripts mediating proinflammatory and anti-inflammatory processes illustrate the balance between opposing forces during SARS pathogenesis. Another interesting phenomenon is the differential expression of genes that support both anti-apoptotic and proapoptotic processes. Thus, anti-apoptotic

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mechanisms facilitate the initial viral multiplication, whereas at later infection phase, apoptosis results in cell lysis to release viral progeny. Transcriptomic and proteomic analyses provide new insights into the host-pathogen interactions and pathophysiology of SARS-CoV infection. These critical interactions involve an elaborate interplay between various mechanisms to favor virus propagation before frank apoptosis and the triggering of specific pathways in host cells that attempt to eliminate the pathogen.

1. SEVERE ACUTE RESPIRATORY SYNDROME (SARS): A NEWLY EMERGING LETHAL VIRAL DISEASE In November 2002, a novel infectious agent causing severe acute respiratory syndrome (SARS) emerged in Guangdong province, China. A global outbreak alert was declared by the World Health Organization (WHO) in March 2003. When WHO finally declared the end of the worldwide outbreak in July 2003, SARS had spread to about 30 nations, resulting in 8098 cases with either atypical pneumonia or respiratory distress syndrome and 774 fatalities. In Singapore alone, there were 238 cases with 33 deaths, accounting for a mortality rate of ∼14%. Strikingly, the epidemic was the first to demonstrate the wide-spreading effect of air travel, and was controlled by aggressive quarantine measures. During March 2003, a number of laboratories independently reported the isolation of a novel coronavirus (CoV) from SARS patients.1–3 Electron microscopy revealed viral particles similar to the morphology of CoV. Sequence analyses demonstrated significant similarity of SARS-CoV to the family Coronaviridae. SARS-CoV may represent a new antigenic group of viruses, or it may be a related member (early split-off) of group 2 CoVs.4 Another new human group 1 CoV, HCVNL63, was isolated from an infant suffering from bronchiolitis and conjunctivitis. Two more novel human CoV, HCV-HKU1 and HCV-NH, are associated with pneumonia and respiratory tract disease in infants and young children.5

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Fever, dry cough, dyspnea, myalgia and lymphopenia are common characteristic features of SARS patients,6 although these are also symptomatic of other respiratory infections. Gastrointestinal symptoms and diarrhea have also been reported.3 Airborne droplets from patients may constitute the main route of transmission. SARS-CoV replicates in peripheral blood mononuclear cells from patients,7 and may be detected in stools of patients for more than ten weeks after the onset of symptoms.8 Blood and feco-oral transmission may thus be possible. Interestingly, a virus related to SARS-CoV was isolated from palm civets and raccoon dogs (known delicacies in southern China) as well as from horseshoe bats, thus raising the possibility that the virus may have jumped recently from these mammals to humans.9,10

2. SARS CORONAVIRUS BIOLOGY, REPLICATION AND PATHOGENESIS The Coronaviridae constitute a diverse group of large, enveloped, positive-stranded RNA viruses with non-segmented genomes of about 27–32 kb — the largest among the RNA viruses. The reference type virus for the genus Coronavirus is avian infectious bronchitis virus. The name “coronavirus” is derived from the crown or solar corona-like appearance of the virus particles in negatively-stained electron micrographs, resulting from prominent surface projections known as spike proteins. They are pleomorphic, usually roughly spherical with a diameter of approximately 80–120 nm, and possess helical nucleocapsids. Currently, there are three groups in the genus, i.e., groups 1 and 2, comprising mammalian CoVs, and avian CoVs belonging to group 3. CoVs have narrow host ranges and are fastidious in cell culture. They usually infect only cells of their natural host species, based on the ability of their spike proteins to interact with their respective receptors on cell membranes. CoVs are responsible for a number of economically important diseases in animals. Humans suffer from two different CoVs, HCV-229E and HCV-OC43, which account for ∼30% of mild upper respiratory tract infections.5

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2.1 Genome Organization of SARS Coronavirus Sequence analysis of SARS-CoV reveals a genome of 29 740 nucleotides with characteristic features of CoVs. A total of 14 open reading frames (ORFs) are identified: (a) 2 large 5′-terminal ORFs, 1a and 1b, encompassing about two-thirds of the genome that constitute the replicase gene which encodes proteins required for viral RNA synthesis; (b) 4 ORFs encoding structural proteins in this gene order, i.e., spike (S), envelope (E), membrane (M) and nucleocapsid (N), which are important for viral replication efficiency; (c) 8 ORFs encoding accessory proteins that are unlikely to be essential in cell culture but may provide a selective advantage in the infected host (Figure 1).11–13 Preceding the 5′ untranslated region (UTR) is a predicted 72nucleotide RNA leader sequence which is also found in a nested set of eight subgenomic (sg) mRNAs. Due to ribosomal frameshifting into the −1 reading frame occurring just upstream of the ORF1, a stop codon, polyprotein (pp) 1a can be extended with the ORF1b-encoded sequences to form pp1ab.14,15 The pp1a and pp1ab are extensively cleaved by viral proteinases, papain-like cysteine protease (PLpro) and 3C-like cysteine protease (3CLpro), into individual polypeptides necessary for viral RNA replication and transcription. SARS-CoV has a PLpro2 ortholog to process the N-proximal regions of the replicative polyproteins.4 The narrow substrate specificity and deubiquitinating activity of SARSCoV PLpro offers the potential for identifying selective inhibitors.16 3CLpro processes the central and C-terminal regions and releases the key viral replicative functions, e.g., RNA-dependent RNA polymerase (RdRp) and helicase with ATPase and DNA/RNA duplex unwinding activity.14 Four novel coronavirus RNA-processing activities are conserved in all CoVs, thereby supporting their essential roles in the coronavirus life cycle.4 These include 3′ to 5′ exonuclease, uridylatespecific endoribonuclease, S-adenosylmethionine-dependent 2-O-ribose

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Genome organization of SARS coronavirus and proteolytic processing of polyprotein 1a and 1ab.11

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methyltransferase, and ADP-ribose 1-phosphatase. Together with host factors, some of these viral proteins may constitute the viral replicationtranscription machinery associated with the membranous structures in infected cells.17 The remaining 3′ part of the genome encodes the four structural proteins and accessory proteins, and possesses another UTR followed by a poly(A) tract.

2.2 Coronavirus Entry via Membrane Fusion A crucial entity to the life cycle of CoVs, the S protein consists of three domains: (a) external conserved N-terminal S1 and S2 subdomains; (b) transmembrane domain; and (c) short cytoplasmic C-terminal domain. Angiotensin-converting enzyme 2 (ACE2), a zinc metalloprotease, binds efficiently to the S1 domain of the SARS-CoV spike protein, and is also the functional receptor for SARS-CoV.18 This binding is further corroborated by the observation that NIH-3T3 cells that express ACE2 can be infected with SARS pseudovirus.19 Furthermore, human monoclonal antibodies against S1 protein block the ACE2 receptor association, and enable potent neutralization of SARS-CoV.20 The interaction of the receptor-binding domain within the S1 domain with the cellular receptor marks the first process of virus entry. The S2 domain mediates the fusion of the viral envelope with the host cell membrane through its heptad repeat 1 and 2.21–22 CD209L (L-SIGN) has also been reported to be a receptor for SARS-CoV.23

2.3 Coronavirus Cap-dependent Initiation of Translation After fusion, the RNA genome is then released into the cytoplasm where replication proceeds, possibly through the dephosphorylation of N protein that promotes its dissociation from the RNA. CoV genome expression begins with the cap-dependent translation of the overlapping ORF1a and ORF1b by a ribosomal frameshifting mechanism to produce pp1a and pp1ab. Cleavage of the polyproteins by the PLpro and 3CLpro yields the necessary components to assemble the multisubunit viral replication-transcription complex including the RdRp.17

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2.4 Coronavirus Genome Replication and Transcription Several mechanisms of replication and transcription are proposed. A “discontinuous transcription” strategy during negative-strand synthesis is widely accepted to produce a nested set of sg mRNAs. In the replicon model, sg positive-strand mRNA is generated via “leader-primed transcription”. Nested sg positive-strand mRNA serves as template for the replication of sg negative-strand RNA, which is in turn used for the synthesis of more sg positive-strand mRNA. The “discontinuous extension of negativestrand RNA” model is more attractive than the replicon model because it utilizes transcription-regulating sequences downstream of the 5′ leader sequence as replication signals. The sg negative-strand RNAs are synthesized directly from the genome in a discontinuous fashion, and serve as templates for continuous sg positive-strand mRNA replication.24 In SARS-CoV, the sg RNAs are either functionally monocistronic or bicistronic.4,14

2.5 Coronavirus Virion Assembly Once synthesized, phosphorylated N protein and genomic RNA assemble in the cytoplasm to form the helical nucleocapsid through the RNAbinding site on the N protein. For SARS-CoV, it is observed to assemble in the rough endoplasmic reticulum (rER) which gradually loses the ribosomes and swell to become virus morphogenesis matrix vesicae.22 SARS-CoV N protein has a putative nuclear localization signal (NLS), suggesting its ability to enter the nucleus and arrest the cells in G2-M phase to allow for maximum translation of viral mRNAs.25,26 CoVs acquire their membrane envelope by budding into the pre-Golgi and Golgi compartments. CoV M protein interacts with the packaging signal in mRNA 1 to form a M protein-nucleocapsid complex near the budding site, and targets the nucleocapsid for packaging into virus particles. M protein can form complexes with either S or HE proteins, most probably on rER or the budding compartment. However, HE, N and S proteins are not essential for virus assembly as M and E proteins alone are able to form virus-like particles (VLP). M protein thus plays a key role as the central organizer in the assembly of these complexes, and in ensuring that S and HE proteins are assembled into virions.5

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E protein alone is sufficient for VLP production, and it may also have a morphogenetic role in virus assembly by positioning within the lattice of membrane proteins to generate the required membrane curvature for budding. When E and M proteins are co-expressed, they colocalize in the Golgi complex near the budding site.5 During the transport of the virions through the Golgi apparatus, both M and S proteins undergo glycosylation, with the S protein being heavily glycosylated from the 120 kDa precursor to the 180 kDa polypeptide.27 SARS-CoV S protein can be cleaved into S1 and S2 domains, but this cleavage is dispensable for the function of S protein.28 Any S protein that is not incorporated into the virions is transported to the cell surface. From the Golgi apparatus, vesicles containing the virions bud off towards the cell surface. Finally, the virus is released from the host cell by fusion of virion-containing vesicles with the plasma membrane.

2.6 Pathogenesis and Treatment of SARS Coronavirus Infection The mechanisms of tissue injury caused by SARS-CoV infection are not fully established, and a better understanding of the molecular pathogenesis of SARS will facilitate the design of treatment strategies.29 Tsui et al.30 proposed a disease model consisting of three phases: (a) viral replication; (b) immune hyperactivity; and (c) pulmonary destruction. SARS pathology of the lung is associated with diffuse alveolar damage, epithelial cell proliferation, and increased infiltration of macrophages. Lymphopenia, hemophagocytosis in the lung, and white-pulp atrophy of the spleen are also observed in SARS patients.31,32 Strikingly, the presence of hemophagocytosis supports a cytokine dysregulation.33 Proinflammatory cytokines released by stimulated macrophages in the alveoli may play a role in the pathogenesis of SARS. Based on this cytokine dysregulation hypothesis, treatment of SARS patients has included the administration of corticosteroids to modulate the exacerbated cytokine response, but such treatment has been ineffective.30 The antiviral potential of interferon (IFN) α , β and γ has been assessed, and IFN-β is a potent inhibitor of SARSCoV when used alone or in combination.34–36 Glycyrrhizin, a component of liquorice roots, also has activity against SARS-CoV.37

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Viral enzymes such as helicase, PLpro and RdRp are promising targets for antiviral drugs, while the M, N and S proteins are candidate vaccine targets. Cellular proteins that are essential for virus replication may also be considered as possible targets. Small interfering RNAs (siRNAs) may have considerable potential for blocking replication but the development of efficacious delivery systems is greatly desired. However, the antiviral efficacy of siRNAs needs to be proven using animal models of infection.38 Antibodies that are able to neutralize viral infection represent a highly effective strategy to prevent the viral disease. Monoclonal antibodies from immortalized B-lymphocytes of SARS convalescent patients can neutralize virus infection in vitro, and prevent virus replication in animal models of SARS-CoV infection.39,40 Post-exposure treatment with monoclonal antibody reduces SARS viral burden in golden Syrian hamsters.41 Candidate vaccines based on killed virus have been tested for prophylaxis and their efficacy proven in monkeys and in mice.42,43 In general, humoral response is necessary to prevent recurrence of viral replication after T-cell mediated clearance in the early acute phase, while cellular immune response is required for host defense and viral clearance.44 Both T- and B-cell epitopes have also been mapped to various CoV proteins. However, immune responses may also contribute to pathogenesis in certain CoV infections. Humoral immune response to feline CoV may contribute to host pathology,45 while T-cells appear to be involved in the induction of inflammation and demyelination of the central nervous system in murine CoV-infected mice.46

3. TRANSCRIPTOMICS OF SARS CORONAVIRUS INFECTION IN VERO E6 AND OTHER CELLS Chest radiographs of severely ill SARS patients display rapid progression of unilateral peripheral air-space consolidation to bilateral patchy consolidation usually within a week.47 How this novel human CoV causes such extensive damage compared with other known human CoVs that are common etiological agents of relatively mild upper respiratory illness remains to be fully elucidated. Interestingly, animal CoVs generally cause more severe diseases, and the civet cat is currently the prime suspect as the

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animal source of SARS-CoV that may have crossed the species barrier to humans. To understand the pathophysiology of SARS-CoV infection and host cellular responses, cDNA microarray, RT-PCR and cDNA sequencing analyses were employed to investigate the gene expression changes during the infection process.48 Similar studies have previously been conducted on other viral infections.49–52 Vero E6 served as the host cells for infection as this cell line is the most permissive for SARS-CoV replication.1–3 SARS-CoV-infected Vero E6 cells exhibit visible cytopathic effect and undergo lysis that mimic the apoptosis observed in the respiratory epithelial cells of severely ill SARS patients.53 The infection process is very rapid such that virus particles are internalized into vacuoles within ten minutes postinfection, extracellular virus particles exist in ∼30% of the cells by six hours postinfection, and numerous large vacuoles containing mature virus are present in the cytoplasm of infected cells from 12–21 hours postinfection,22 as evident from the ∼105-fold increase in the level of SARS-CoV transcripts by real-time RT-PCR analysis. Expression of the SARS-CoV protein 3a is detectable in infected Vero E6 cells at 8–12 hours postinfection, and also in the pneumocytes of a SARS patient’s lung.54 Furthermore, the incubation period of SARS is variously reported to be 6.4 days,55 5 days,56 with a range of 3–8 days.57 Therefore, 12 hours after the infection of Vero cells represents an appropriate timepoint that allows for a larger population of cells to be infected so that significant changes representative of the pathophysiological process of SARS-CoV infection can be elucidated. In view of the relatively small differences between the genomes of humans and primates,58 Vero E6 cells derived from African green monkey represent a viable alternative model for investigating cellular gene responses to SARS-CoV in lieu of a human cell line. This was further substantiated by sequencing of the genes selected for real-time analyses, which revealed sequence differences of only 1–5%.48

3.1 Dysregulation of Genes Leading to Growth Inhibition During infection, certain cellular genes are dysregulated, possibly to limit cell growth, and to channel resources for viral replication. Cathepsin L

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(CTSL) is a lysosomal cysteine proteinase that plays a major role in intracellular protein catabolism. Suppression of CTSL expression in A549 lung cancer cells leads to growth inhibition, which is compatible with CTSL downregulation during SARS-CoV infection. However, this growth inhibition is partially compensated by upregulation of interleukin-8 (IL-8) production.59 In addition, CTSL secreted from human fibroblasts in response to external stimuli, plays an important role in the processing of IL-8 to mature form in inflammatory sites.60 Interestingly, SARS-CoV utilizes CTSL to infect ACE2-expressing cells.61 Tissue inhibitor of metalloproteinase-2 (TIMP2) activates Ras through a PKA-mediated pathway, leading to the formation of the Ras/PI3K complex, with roles in proliferation, differentiation, and membrane ruffling.62 The downregulation of TIMP2 in SARS-CoV infection may thus be involved in restricting cell growth. Certain transcripts involved in the cell cycle and development (e.g., DUSP1 and FANCC) are upregulated. Dual specificity phosphatase 1 (DUSP1) plays an important role as a protein phosphatase in human cellular response to environmental stress, and as a negative regulator of cellular proliferation by inactivating mitogen-activated protein kinases (MAPK).63 Noteworthy is the upregulation of KLF5, a member of the Kruppellike factor subfamily of zinc finger proteins. Conflicting reports indicate that KLF5 inhibits cell proliferation,64 but its overexpression in NIH3T3 fibroblasts positively regulates cellular proliferation.65 Thus, the elevated level of KLF5 transcripts in SARS-CoV infection warrants further investigation, e.g., its expression at protein level and whether overexpression of a dominant-negative KLF5 construct in SARS-CoV-infected cells affects the infection process.

3.2 Differential Expression of Apoptotic and Anti-apoptotic Genes TIMP2 also plays a role in the prevention of apoptosis, and thus reduced TIMP2 expression in SARS-CoV infection is predicted to promote apoptosis of infected cells. From the transcriptional analysis, elevated expression of MGAT5 may contribute to the initiation of GlcNAc β 1,6 branching on N-glycans, thereby increasing N-acetyllactosamine, the ligand for galectins. In turn, galectins modulate T-cell proliferation as well as

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enhance apoptosis at the site of antigen presentation.66 Fas apoptotic inhibitory molecule (FAIM), an anti-apoptotic gene, is downregulated in SARS-CoV infection. The susceptibility of primary B-cells to Fas-mediated apoptosis is modulated by FAIM and NF-κ B.67 Thus, the lower expression of FAIM and the higher expression of NFKBIA which inhibits NF-κ B are likely to favor Fas-mediated apoptosis in SARS-CoV infection. The elevated expression of NFKBIA, an inhibitor of the NF-κ B complex, may attenuate the effect of NF-κ B.68 A critical regulator of apoptosis, NF-κ B is commonly involved in the suppression of programmed cell death via the transactivation of anti-apoptotic gene expression.69 NF-κ B also plays a key role in controlling cell proliferation as evidenced by the deregulated expression of its constitutively active form in certain cancers.70 These transcriptional aberrations may explain the phenomenon of apoptosis in SARS-CoV-infected cells. In contrast, the expression of other genes may also be modified as a possible means of subverting apoptosis. Insulin-like growth factor binding protein 3 (IGFBP3) is essential for apoptosis induced by tumor necrosis factor α (TNFα ),71 but downregulation of its transcript level in SARS-CoV-infected cells may imply a mechanism to avert virus-induced apoptosis, thus favoring viral replication. Increased transcript level of the hIAP1 apoptotic inhibitor72 during SARS-CoV infection mirrors that observed in dengue virus infection.49 Although infected Vero cells eventually lyse at the late phase of infection, hIAP1 may attenuate apoptosis at the early phase, thereby allowing the virus to replicate to high titer. SARS-CoV-infected Vero E6 cells also exhibit upregulation of the combined transcripts of the anti-apoptotic DENN isoform and the proapoptotic IG20 isoform that both interact with TNF receptor 1 and participate in the MAPK pathway.73,74 Therefore, the interplay between, and altered expression of apoptosis-related genes in SARS-CoV infection may help to achieve viral replication before cell death (Figure 2).

3.3 Upregulation of a p53 Splice Variant in SARS-CoV Infection In SARS-CoV-infected Vero E6 cells, an additional larger transcript of the p53 tumor suppressor gene is expressed, arising from the use of an

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Differential expression of apoptosis-related genes in SARS-CoV infection.

alternative 5′ donor splicing site with the resultant loss and modification of the C-terminus.75,76 The p53 C-terminus plays a pivotal role in regulating the activity of the wild-type molecule. Compared to the regularly spliced form of p53, the C-terminally altered p53 protein inhibits both p53-dependent apoptosis and transactivation; binds more efficiently to DNA in a sequence-specific manner; is more efficient in concentrationdependent transcriptional repression of the promoter of p21 cyclindependent kinase inhibitor gene; and it associates with and interferes more efficiently with the binding of TATA-binding protein to a TATA-containing DNA sequence.77,78 The presence of this p53 isoform with a truncated C-terminus is therefore expected to modify the activity of the wild-type p53, and exert a role in the pathogenesis of SARS. Compared to wild-type p53, the expression of this alternate transcript is relatively lower but it does not possess ubiquitination sites and may thus be more stable, allowing it to function longer in infected cells. The product of this splice variant forms a complex with wild-type p53, and enhances the transcriptional activity of wild-type p53 on the BAX promoter but it does not significantly increase p53-mediated apoptosis in H1299 cells.76 Nevertheless, it may modulate the biological

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activity of p53, and may influence p53-mediated apoptosis during SARSCoV infection.

3.4 Downregulation of Host Translational Genes Among the most distinguishable transcripts downregulated by infection are those involved in the host translational machinery. The genes include those encoding 40S ribosomal proteins (RPS2, RPS3, RPS10, RPS15, RPS18, RPS19), 60S ribosomal proteins (RPL3, RPL13, RPL18, RPL18A, RPL27A, RPL36), isoleucine tRNA synthetase (IARS), modifying enzyme (TRIT1), and eukaryotic translation elongation factors (EEF1A1 and EEF1G). Such host translational shutoff is frequently observed, as evident in other viral infections.79–81 However, unlike poliovirus infection which often results in selective translation of uncapped viral mRNAs, CoVs give rise to mRNAs that are structurally similar to their eukaryotic hosts. This allows CoVs to parasitize upon the host machinery to translate the viral mRNA. At high multiplicity of infection, murine CoV inhibits host protein synthesis during the very early stage of infection, suggesting that the increased number of viral mRNAs produced during the later phase of infection compete with cellular mRNAs for cellular ribosomes.82

3.5 Upregulation of Genes Instigating Proinflammatory Responses Several genes pertaining to proinflammatory responses are upregulated during SARS-CoV infection. The upregulation of several heat shock proteins (HSPs) is not surprising given that elevation of HSP expression represents a crucial response towards external stimuli or stresses such as infections. Noteworthy is the strong induction of HSPA1A which is widely involved in translocation of membrane proteins, and acts as a scavenger of degraded peptides for antigen presentation. During viral infections, a rise in the level of secreted cytokines such as IL-2 is often encountered. Besides inducing the proliferation of T-lymphocytes, IL-2 also induces the expression of HSPA1A and HSPCA. In certain inflammatory lung diseases with marked accumulation of eosinophils, free

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oxygen radicals are produced which induce alveolar macrophages to synthesize antioxidants such as HSPs.83 Such a phenomenon may operate in the atypical pneumonia characteristic of SARS patients who show significantly elevated plasma levels of IFN-γ , IL-1, IL-6, IL-8, IL-12, monocyte chemoattractant protein-1, and IFN-gamma-inducible protein-10.84 IL-8 is a proinflammatory cytokine with roles in chemotaxis and activation of monocytes, selective chemotaxis of memory T-cells, and induction of neutrophil infiltration in vivo. In dengue virus infection, IL-8 transcription is controlled by NF-κ B which activates many immunoregulatory genes in response to proinflammatory stimuli.85 In SARS-CoV infection, the upregulation of IL8RA (a low affinity receptor for IL-8) implies more active IL-8 signal transduction at the site of inflammation where the relative concentration of IL-8 is high.86 Altered transcription of signal transduction molecules is also observed in SARS-CoV-infected cells. Signal transducing adaptor molecule 1 (STAM1) protein contains a SH3 domain and an immunoreceptor tyrosine-based activation motif (ITAM). STAM1 associates with JAK3 and JAK2 kinases via its ITAM region, and is phosphorylated by the JAK kinases upon cytokine stimulation, thus acting as an adaptor molecule involved in the downstream signaling of cytokine receptors.87 STAM1 upregulation suggests its enhanced role in the downstream signaling of cytokine receptors during SARS-CoV infection. Inhibition of STAM1 via siRNA may confirm whether this signaling cascade is involved in SARS. Many studies generally document increased inflammatory responses in SARS, but an exaggerated induction is detrimental to the tissues involved. Mx proteins comprise a group of antiviral GTPases that play an important role in IFN-induced antiviral defenses. They also act to sequester viral nucleocapsids and limit their accessibility for viral replication.88 The marked elevation of Mx1 expression (∼60-fold increase) suggests a highly notable antiviral response to SARS-CoV pathogenesis, implying the utility of IFN treatment.34 SARS-CoV may have developed a strategy to prevent IFN-β induction by blocking the early nuclear transport of IRF-3, which is essential for IFN-β promoter activity.89 Certain SARS patients exhibit patchy changes in their lungs associated with pulmonary fibrosis. This is consistent with the downregulation of genes related to fibrinolysis in SARS-CoV infection. Annexin A2

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(ANXA2), a fibrinolytic receptor, binds plasminogen and tissue plasminogen activator independently at the surface of monocytes and macrophages, thereby enhancing the catalytic efficiency of plasmin production,90 limiting pulmonary fibrosis. The downregulation of ANXA2 in SARS-CoV infection is expected to decrease fibrinolysis, compatible with the events of hypercoagulation and hypofibrinolysis in SARS patients.91,92 The TIMP gene family encodes natural inhibitors of matrix metalloproteinases (MMPs), a group of peptidases involved in degradation of the extracellular matrix. The downregulation of TIMP2 in SARSCoV infection implies an increase in the level of MMPs which contribute to the pathogenesis of tissue destruction processes in a wide variety of diseases including lung diseases, particularly pulmonary fibrosis (Figure 3).93,94

3.6 Downregulation of Cytoskeleton-associated Genes SARS-CoV-infected cells also exhibit diminished expression of genes related to the maintenance of cytoskeletal structure. Cytoplasmic linker 2 (CYLN2) mediates the interaction between specific membranous

Figure 3. Upregulation of genes mediating pro-inflammatory responses in SARSCoV infection.

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organelles and microtubules, and may be an anticatastrophic factor.95 CLASP1 binds CYLN proteins and microtubules, co-localizing at the distal ends to provide stabilizing effects to the microtubules.96 The microtubule network plays an important role in viral replication and viral protein trafficking. Thus, disrupting the network may initially result in less virus production to a point at which the cells are overwhelmed by virus multiplication and then lyse to release virus particles. By exposing peritoneal macrophages to cytochalasin B, murine CoV infection changes from an acute cytopathology to a persistent type.97 In SARS-CoV infection, these microtubule networks may become disrupted, making it conducive for persistent viral infection and subsequent release. The actin assembly may also be greatly affected by downregulation of TMSB4X and TMSB10, which are actin-sequestering proteins. Indeed, actin mRNAs are reduced in murine CoV infection. 5 Furthermore, reduction of TMSB4X transcripts may disrupt cellular functions, since TMSB4X may have a unique integrative function that links the actin cytoskeleton to important immune and cell growthsignaling cascades.98

3.7 Roles of SARS-CoV Proteins in Host-Pathogen Interactions and Pathogenesis SARS-CoV spike (S) glycoprotein interacts with DC-SIGN, DC-SIGNR, pulmonary surfactant protein D, activates macrophages, induces ER stress and upregulates expression of chemokines such as IL-8.99–102 SARS-CoV nucleocapsid (N) protein is capable of self-association,103 and has a putative NLS, implying its ability to enter the nucleus.25,104 The N protein is phosphorylated, localizes in the cytoplasm via 14-3-3mediated translocation,105 and evokes changes in host responses probably at transcriptional level. In the absence of growth factors, SARS-CoV N protein induces actin reorganization and apoptosis in COS-1 cells.26,106 SARS-CoV 3a protein is a minor structural protein that interacts with viral genomic RNA,107 M, E and S proteins,108 and is assembled into VLP with M and E proteins.109 Furthermore, 3a protein upregulates the expression of fibrinogen in lung epithelial cells, which may contribute to

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SARS-CoV pathogenesis via excessive formation of fibrin.108 The 3a protein can also induce apoptosis and cell cycle arrest.110,111 SARS-CoV 7a protein inhibits cellular protein synthesis and cell cycle progression.112,113 SARS-CoV 3a, 7a and non-structural protein 1 contribute to chemokine dysregulation via NF-κ B activation.114,115

4. CONCLUSIONS AND FUTURE PROSPECTS Although Vero cells are deficient in IFN response, alterations in transcription of genes that are conserved between African green monkeys and humans provide a better understanding of the biology of SARS-CoV infection. Transcriptomic analyses offer a global view of the cascade of host transcriptional changes culminating from infection with SARS-CoV. Such molecular insights into the pathophysiological mechanisms may lead to novel and viable strategies for intervention of the infection process.116 In infected cells, there is generally downregulation of host translational components, inhibition of cell proliferation together with delayed onset of apoptosis. Another feature is the dysregulation of cytoskeleton-related genes that destabilizes or rearranges the microtubule network to support viral replication. Differential expression of genes that mediate both anti-apoptotic and proapoptotic processes may be a mechanism by which anti-apoptosis initially facilitates virus multiplication, while at the later stages, apoptosis operates to lyse the cells to release viral progeny.117 There is a general trend of increased expression of inflammatory response genes that is often attributed as a major pathophysiologic mechanism of SARS-CoV infection, and which correlates strongly with the high cytokine profiles in SARS patients.118–121 An interesting feature is that an array of inflammation and immune-related genes is upregulated in SARS-CoV infection which incites overt proinflammatory responses in different cell types in vitro86,122–125 and in vivo,126 whereas in enterovirus 71 infection, such genes are downregulated possibly to evade immune defenses.51 The counterbalancing of several anti-inflammatory and proinflammatory pathways, together with the variable expression of apoptosis-

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related genes, underpin the mechanisms that ensure cell survival during the early phase of SARS-CoV infection, to allow rapid multiplication of progeny virus before the cytopathic effects occur.127 Whether these phenomena are the direct or indirect effects of the infection or viral proteins merits further experiments since these cellular genes also have physiologic and homeostatic functions in various tissue types. Through in vitro manipulation of these differentially expressed genes either by overexpressing or knocking down expression via mutation or siRNA, one can observe their corresponding effects on the process and outcome of SARS virus infection. The availability of suitable animal models for the study of SARS-CoV infection also permits testing of the effects of such manipulations in an in vivo environment,126 e.g., whether these relieve clinical manifestations. A cautionary note in targeting pathways such as the inflammatory response, either by blocking or enhancing a particular process during infection is that the intervention may not always be feasible as it may benefit virus survival and/or enhance host pathologic effects. For example, the prolonged inhibition of cell growth that leads to the induction of massive apoptosis may cause organ destruction, or the excessive induction of immune-related responses may provoke severe inflammatory reactions. Another viable treatment strategy is to target the viral replication process, especially the proteins that mediate replication and those that utilize the nucleo-cytoplasmic transport mechanism. Proteomic alterations in SARS patient samples identify upregulated and downregulated proteins that lend insights into pathogenesis. There is significant correlation between specific clinico-pathologic parameters and the levels of proteins mediating inflammation, innate and adaptive immune responses, lung protection and other critical processes. Such novel protein signatures may serve as useful biological markers for diagnosis and disease progression, and to improve the clinical management of SARS.128–130 In conclusion, transcriptomic and proteomic technologies are valuable for elucidating the critical groups of host genes and proteins that are intimately involved in SARS, and for acquiring important leads to better understand molecular mechanisms or to explore new hypotheses that may culminate in novel strategies for alleviating the devastating effects of the disease.

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ACKNOWLEDGMENTS The SARS research work in the laboratory is supported by the National University of Singapore and the Biomedical Research Council, Singapore.

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CHAPTER 14

Characterization of Signaling Pathways in Cells Infected with SARS-CoV Tetsuya Mizutani

ABSTRACT Severe Acute Respiratory Syndrome (SARS) is a recently discovered infectious disease caused by a previously unknown human coronavirus, the SARS coronavirus (SARS-CoV). Signal transduction in cells infected with SARS-CoV has been studied to understand the mechanisms of pathology in SARS. Generally, two competing cellular programs, apoptotic signaling pathways promoting apoptosis of virusinfected cells and survival signaling pathways delaying apoptosis of virus-infected cells, are observed. Mitogen-activated protein kinases (MAPKs) are the key participants in the determination of death and survival of virus-infected cells. Akt, p90 ribosomal S6 kinase (p90RSK), and signal transducers for the activation of transcription 3 (STAT3) signaling pathways involve phosphorylation and dephosphorylation by viral infection. Expression studies of proteins of SARSCoV show that these viral proteins can activate signaling pathways and that some proteins have the ability to induce apoptosis. Understanding signaling pathways in virus-infected cells is important for the development of anti-SARS-CoV drugs. This review highlights the recent progress in characterizing signal transduction in cells infected with SARS-CoV.

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1. INTRODUCTION Severe acute respiratory syndrome (SARS) was the first pandemic emerging infectious disease of the 21st century. A respiratory illness with variable flu-like symptoms and pneumonia was first recognized in China in November 2002 and subsequently spread to 29 other countries by infected travelers. This new syndrome was designated “severe acute respiratory syndrome” (SARS). The New England Journal of Medicine website published two articles regarding clusters of SARS patients,1,2 and three laboratories reported a novel coronavirus (SARS-CoV) as the etiological agent of SARS.3–5 The World Health Organization (WHO) reported a total of 8098 cases of SARS in 29 countries, of which 774 (9.57%) resulted in death (http://www.who.int/csr/sars/country/table2004_04_21/ en/index.html). SARS-CoV causes severe, rapidly progressive atypical pneumonia with fever and diarrhea. SARS-CoV belongs to the Coronaviridae family (order Nidovirales) of enveloped, single, positive-stranded RNA viruses.6–8 The site of viral replication is the cytoplasm. The genome is approximately 30 kb in length, which is the longest known RNA viruses. The genomic RNA has a cap structure and a poly A tail at the 5′-end and 3′-end, respectively (Figure 1A). Spike (S) protein binds to the virus receptor and is then internalized into the cytoplasm by endocytosis. The viral genome is initially translated into viral polymerase. Full genome-sized negative-stranded RNA is transcribed from genomic positive-stranded RNA which act as a template. At least 9 mRNAs were detected in cells infected with SARS-CoV (Figures 1A and 1B). All mRNAs have a unique “nested set” structure, 5′non-translated leader sequences (approximately 70 bases) and 3′ poly A tails (Figure 1A). The leader RNA is thought to be transcribed from the 3′-end of full genome-sized negative-stranded RNA and bound to intergenic sequence on negative-stranded RNA. Fourteen open reading frames (ORF) are identified in the viral genome, but eight proteins were unknown in coronaviruses. Approximately two-thirds of the viral genome encode non-structural proteins which are essential for viral replication, as large overlapping replicase polyproteins, 1a and 1ab (Figure 1C). These polyproteins are processed into 16 non-structural proteins by viral proteases, chymotrypsin-like cysteine protease (3CLpro) and papain-like

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Figure 1. Genome organization of SARS-CoV. (A) Northern blot analysis of viral mRNA in infected Vero E6 cells. Dig probe was used at the region of mRNA 9. (B) Viral mRNAs in infected cells.(C) Viral proteins in infected cells.

protease (PLpro). The virus particle consists of four structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) (Figure 1C). To understand the pathological mechanisms of SARS, cultured cells that are sensitive to viral infection have been used. As apoptotic cell death is observed in these cells after SARS-CoV infection, it is clear that various signaling pathways are activated in SARS-CoV-infected cells (for a review, see Ref. 9). Among them, in the most common signaling pathways, mitogen-activated protein kinases (MAPKs) are thought to be the key proteins responsible for apoptosis (for a review, see Ref. 10). MAPKs are highly conserved in a wide range of species from yeast to mammals. Three major MAPKs, NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK have been characterized in mammals. MAPK is

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activated by MAPK kinase (MAPKK), and MAPKK is activated by MAPKK kinase (MAPKKK). MAPKs, which are a superfamily of protein kinases, play a number of important roles in living cells. MAPKs have crucial roles in signaling pathways from the surface to the nucleus of a cell. Regulation of cell death or cell survival is one of the roles of MAPKs. Generally, the ERK signaling pathway promotes cell survival and proliferation, while JNK and p38 MAPK induce apoptosis. Signal transduction studies using SARS-CoV-infected cells indicate that some viral proteins have the ability to induce activation or phosphorylation of MAPKs. However, because the roles of these MAPKs signaling pathways in cell death and survival are thought to be complicated even in cultured cells, as of which protein actually has the ability to phosphorylate them is still not clear. This review highlights the recent progress in characterizing signal transduction, especially MAPKs, in cells infected with SARS-CoV.

2. p38 MAPK SIGNALING PATHWAY IN VIRUS-INFECTED CELLS The p38 MAPK signaling pathway has been shown to be activated and phosphorylated by several viruses, including SARS-CoV, in infected cells. However, the precise role of p38 MAPK in viral replication remains unclear because of its complicated signaling pathway. p38 MAPK is stimulated by environmental stress, UV irradiation, proinflammatory cytokines, and oxidative stimuli. It has at least four isoforms: p38α , p38β , p38γ , and p38δ .11–13 The p38α and p38β MAPKs share >70% similarity at the amino acid level, and their functions are inhibited by the pyridinylimidazole SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4pyridyl) imidazole). The p38γ and p38δ MAPKs have 60% similarity to p38α , but they are not inhibited by SB203580. They may have different functions because p38α and p38β MAPKs are widely expressed in tissues, whereas p38γ and p38δ MAPKs expression is tissue-specific. Upstream of p38 MAPK, there are the MAPKKs, MKK3 and MKK6. Furthermore, TGF-β -activating kinase (TAK1), apoptosis signal-regulatory kinase (ASK1), and MAPKKK4 are known to be MAPKKKs of MKK3 and MKK6. Mixed lineage kinase 3 (MLK3) may be one of the MAPKKKs of MKK3 and MKK6, but this is still controversial.14,15 On the

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other hand, many downstream targets of p38 MAPK are known. Mitogen and stress-activated protein kinase 1 (MSK1), MAP kinase-interacting kinase 1 (MNK1), and MAPK-activated protein kinase 2 and 3 (MAPKAPK 2 and 3) are kinase substrates of p38 MAPK.16 MAPKAPK2 activates heat shock protein 27 (HSP27), cAMP response element-binding protein (CREB), and transcription factor-1 (ATF-1).17,18 MNK1 activates the eukaryotic translation initiation factor 4E (eIF4E).19 The p38 MAPK is one of the well-studied signaling pathways in virus-infected cells. However, as described below, the role of the p38 MAPK signaling pathway in cellular responses is diverse, depending on the cell type and stimulus. Although the p38 MAPK signaling pathway seems to primarily induce apoptosis in virus-infected cells, p38 MAPK is also able to promote both cell death and cell survival. Many reports have been published regarding the p38 MAPK signaling pathway in virus-infected cells. Mouse hepatitis virus (MHV)-3, which is known to induce fulminant hepatitis and depend on macrophage activation and expression of the specific prothrombinase fgl-220 in mice, induces activation of ERK and p38 MAPK in macrophages. The fgl-2 mRNA level is increased by activation of p38 MAPK.21 p38 MAPK activation in other MHV A59 strain-infected cells increases production of IL6.22 Interestingly, p38 MAPK inhibitors also suppress viral mRNA and protein production. eIF4E is increased in virus-infected cells, and MHV may utilize eIF4E phosphorylation by activating p38 MAPK to promote virus-specific protein synthesis. Viral proteins sometimes negatively regulate p38 MAPK. Varicella-Zoster virus (VZV), which is a neurotropic alphaherpesvirus, transcribes immediate early (IE) genes following entry into the host cell. Among these IE genes, open reading frame (ORF) 61 is known to repress phosphorylation of p38 MAPK.23 The ORF61 may be a negative regulator of cellular proinflammatory responses via repression of p38 MAPK activation. A recent study indicates that PKR interacts with and activates p38 MAPK.24 MKK6 has an affinity for PKR in the presence of double-stranded RNA (dsRNA), poly(rI:rC), but not MKK3. PKR is capable of activating MKK6 and interacts strongly with p38 MAPK in hepatitis C virus-core-expressing cells.25 The core protein induces deregulation of the mitotic checkpoint at the boundary of G2 phase of cell cycle and mitosis by the interaction between p38 MAPK and PKR. On the other

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hand, human immunodeficiency virus (HIV)-1 gp120 induces neuronal dysfunction and death by activation of p38 MAPK.26 The p38 MAPK inhibitor, RWJ67657 (4-(4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4pyridyl)-1H-imidazol-2-yl)-3-butyn-1-ol), inhibits HIV-1 replication in both T-cell and monocyte cell lines, suppresses both reverse transcriptase and protease-resistant escape mutant viruses, and inhibits HIV-induced T-cell apoptosis.27 p38 MAPK is known to enhance transcription of CHOP, which is a protein homologous to C/EBP as growth arrest and DNA damage-inducible gene 153 (GADD153).28 Japanese encephalitis virus (JEV) induces apoptosis under conditions of ER stress, and treatment with a p38 MAPK inhibitor partially blocks this apoptosis.29 Thus, activated p38 MAPK induces apoptosis via activation of the CHOP pathway. Activation status of NF-κ B and p38 MAPK in H5N1 and H1N1 subtypes of influenza virus was compared.30 Although NF-κ B signaling pathways are activated in both viruses, only the H5N1 virus induces high levels of p38 MAPK. TNF-α expression is inhibited by treatment with SB203580. Thus, p38 MAPK induced by H5N1 viral infection plays important regulatory roles in deregulation of cytokines. This phenomenon may be reflected in the pathogenesis of disease caused by H5N1. p38 MAPK signaling pathway has a variety of roles in virus-infected cells, such as in apoptosis and cell cycle.

3. p38 MAPK SIGNALING PATHWAY IN SARS-CoV-INFECTED CELLS Vero and Vero E6 cells, which are monkey kidney cells, are widely used in SARS-CoV research due to their high susceptibility to infection as they lack interferon genes. Using these cell lines, apoptosis has been shown to be inducible by infection with SARS-CoV.31,32 The amount of phosphorylated p38 MAPK increases in SARS-CoV-infected Vero E6 cells30 (Figure 2). Phosphorylation of p38 MAPK is not transient and it reached a maximal level at 18 h.p.i. The p38 MAPK is activated in MHVinfected cells, and MHV replication is necessary for p38 MAPK activation.22 However, it seems that SARS-CoV replication does not require p38 MAPK phosphorylation. In general, activated p38 MAPK induces apoptosis in virus-infected cells. Cytopathic effects (CPE) caused by SARS-CoV

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SARS-CoV

MEK1/2 ERK1/2

MKK3/6

MKK4/7

p38 (MAPK)

JNK eIF4E

MAPKAPK-2

Cell death

HSP-27

MSK-1

Anti-apoptosis? Elk-1

ATF-2

CREB

Transcription

Figure 2. Upstream and downstream components of MAPK pathways. After SARSCoV-infection of Vero E6 cells, MAPKKs of ERK1/2, JNK and p38 MAPK are activated. Although activation of p38 MAPK induces cell death, activated downstream targets of p38 MAPK have anti-apoptotic roles.

are due to focal cell rounding. At 24 h.p.i., typical DNA fragmentation of apoptosis is observed. Caspase-3, which is one of the main effector caspases that become activated in response to both intracellular and extracellular death signals, is activated by SARS-CoV infection as a peak at 24 h.p.i. Thus, SARS-CoV induces apoptosis in infected cells. Although both CPE and apoptosis are thought to be linked, CPE caused by SARS-CoV infection is slightly inhibited by SB203580, whereas DNA fragmentation is not inhibited. It is known that CPE can be caused by either virusinduced apoptosis or cell necrosis; but the CPE due to SARS-CoV infection of Vero E6 cells is induced by apoptosis, rather than by necrosis.32 These observations at least show that activation of p38 MAPK promotes cell death of Vero E6 cells by SARS-CoV infection. In addition, p38 MAPK plays an important role in cell death in co-infection with Mycoplasma in Vero E6 cells.33

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Investigation of the p38 MAPK upstream and downstream targets is important to understand the mechanisms of cell death induced by SARSCoV infection. The translation initiation factor, eIF4E enhances translation rates of cap-containing mRNAs.19 In the case of MHV, eIF4E phosphorylation is utilized to promote virus-specific protein synthesis. The level of phosphorylated eIF4E is also increased by SARS-CoV infection.31 However, activated eIF4E is not advantageous for viral protein synthesis, as demonstrated by the similar kinetics of viral protein accumulation in infected Vero E6 cells, in the presence and absence of SB203580. MAPKAPK-2, which is a downstream target and become activated in response to stress and growth factors,16,34 is phosphorylated in SARS-CoV-infected Vero E6 cells. One of the substrates of MAPKAPK-2, HSP-27, is also phosphorylated in the SARS-CoV-infected cells. As phosphorylation of HSP-27 is inhibited by SB203580, Hsp-27 is specifically phosphorylated by p38 MAPK in viral-infected cells. Hsp-27 does not promote cell death in SARS-CoV-infected cells due to what is known as an anti-apoptotic protein that inhibits apoptosome formation.17 cAMP response element-binding protein (CREB), which is a substrate of MSK-1, is phosphorylated in SARS-CoV-infected cells. MSK-1 is thought to be a substrate of p38 MAPK and/or ERK 1/2. CREB is also known to mediate an important survival signal under various conditions.18,35,36 Nucleocapsid (N) protein is able to induce phosphorylation of Hsp-27 and CREB in transfected cells.37 These phosphorylated proteins may induce an antiapoptotic environment in SARS-CoV-infected cells. As p38 MAPK is able to promote both cell death and survival,38 there are other substrates of p38 MAPK that are inducible on cell death of Vero E6 cells caused by SARS-CoV infection. There are reports regarding phosphorylation of p38 MAPK by overexpression of viral proteins. The N protein of SARS-CoV is able to induce phosphorylation of p38 MAPK in COS-1 cells in the absence of serum.37 Moreover, activation of the p38 MAPK pathway induces actin reorganization in cells devoid of growth factors. The N protein is able to induce phosphorylation of Hsp-27 and CREB in transfected cells.37 The 7a protein of SARS-CoV induces apoptotic cell death and phosphorylation of p38 MAPK into 293T cells.39 However, SB203580 does not prevent cell rounding, apoptosis, and chromatin condensation induced by the 7a protein.

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4. p90 RSK IS DOWNSTREAM OF p38 MAPK The p90 ribosomal S6 kinases (RSK) are a family of serine/threonine kinases. These kinases are known to be substrates of ERK.40 As members of the p90RSK family, four isoforms, RSK1, 2, 3, and 4, and two structurally related RSK-like protein kinases, (RLPK/MSK1) and RSK-B (MSK2), have also been reported.40–43 Among these, p90RSK1 is expressed more strongly than p90RSK2 in Vero E6 cells while p90RSK3 and 4 are not detected in Vero E6 cells. p90RSK is thought to have multiple functions. The p90RSK activates NF-κ B by phosphorylation and phosphorylates the transcription factors c-Fos and cAMP-response elementbinding protein (CREB).40 p90RSK also phosphorylates Bad44,45 and C/EBPβ ,46 which protects cells against apoptosis. Thus, p90RSK plays key roles in regulating cellular functions in the ERK signaling pathway. Recent studies have clarified the mechanisms of activation of p90RSK. p90RSK1 is phosphorylated at Thr-573 in the activation loop of the C-terminal kinase domain by ERK.47,48 This activation of the C-terminal kinase domain induces autophosphorylation at Ser-380 in the linker region, and PDK1 then phosphorylates at Ser-221 in the activation loop of the Nterminal kinase domain.49–51 In the case of Vero E6 cells, p90RSK phosphorylation at Thr-573 and Ser-380 are increased weakly and strongly respectively, by decreasing cell density.52 On the other hand, the level of PDK-1 phosphorylation is also dependent on cell densities and Ser-221 of p90RSK is also phosphorylated similarly. Thus, the phosphorylation level of Ser-221 of p90RSK is not influenced by the status of cell proliferation. Interestingly, although both Thr-573 and Ser-380 of p90RSK in Vero E6 cells are phosphorylated early after EGF treatment, the phosphorylation level of p90RSK Ser-221 is not altered by EGF treatment. When SARSCoV infects confluent Vero E6 cells, no significant differences were observed in phosphorylation levels of PDK-1 or p90RSK at Ser-221. Phosphorylation of Thr-573 is not upregulated by viral infection. However, Ser-380 of p90RSK is phosphorylated in virus-infected confluent cells. Thus, phosphorylation of p90RSK Ser-380 is upregulated without upregulation of Thr-573 in SARS-CoV-infected cells. Which signaling pathway regulates phosphorylation of Ser-380 of p90RSK in SARS-CoV-infected cells? The ERK signaling pathway is not important

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for phosphorylation of Ser-380 in viral infected cells because Ser-380 is phosphorylated without phosphorylation of Thr-573. On the other hand, the phosphorylation of Ser-380 is decreased in SB203580-treated viralinfected cells. Thus, p38 MAPK can induce phosphorylation of Ser-380. There has been at least one report that indicates that p90RSK phosphorylates CREB.40 Phosphorylation of CREB is regulated by p38 MAPK in SARS-CoV-infected Vero E6 cells.31 After p38 MAPK activation by viral infection, Ser-380 of p90RSK is strongly phosphorylated by the activated p38 MAPK; and in turn, CREB is thought to be phosphorylated by the activated p90RSK. From the above results, p90RSK should be expected to have anti-apoptotic activity in SARS-CoV-infected cells.

5. STAT3 IS DOWNSTREAM OF p38 MAPK Signal transducer and activator of transcription (STAT) proteins, which are transcription factors, are induced by phosphorylation of a single tyrosine residue, leading to dimerization via an intermolecular SH2 phosphotyrosine interaction.53–56 Among the STATs, STAT3 is known to be activated in response to interleukin-6 (IL-6) and IL-10. Due to inhibition of STAT3 signaling by dominant negative and antisense STAT3 inhibitors that decreases cell viability and subsequent apoptosis,57–59 STAT3 is thought to act as an anti-apoptotic transcription factor. In Vero E6 cells, STAT3 is constitutively phosphorylated at Tyr-705 and is slightly phosphorylated at Ser-727, in manners similar to those which are observed in breast carcinoma cell lines.60 When SARS-CoV infects Vero E6 cells, the total amount of STAT3 does not change until 24 h.p.i., but Tyr-705phosphorylated STAT3 is not detected after 18 h.p.i.60(Figure 3). Thus, STAT3 Tyr-705 is dephosphorylated from 18 to 24 h.p.i. On the other hand, Ser-727-phosphorylated STAT3 increased slightly at the same points in time. As Tyr-705 phosphorylation of STAT is necessary for its activation,53–56 SARS-CoV infection leads to a decrease of STAT3 activation. In the case of IL-6 stimulation, Janus kinases (JAK1 and 2) and Tyk2 are phosphorylated at tyrosine residues through a conserved membraneproximal binding domain;61 and dimeric STAT3 Tyr-phosphorylated by JAKs and Tyk2 migrates to the nucleus, where STAT3 activates transcription of specific genes. JAK1, JAK2, and Tyk2 were phosphorylated at low

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Cell membrane

STAT3 Activation of p38 MAPK P S

Infection of SARSSARS-CoV

S P nucleus

P

Y

YP

P S

S P

transcription

c-myc, Bcl-xL, survivin

Genomic DNA

Figure 3. Dephosphorylation of STAT3 by activation of p38 MAPK. Tyr705-phosphorylated STAT3 is localized primarily in the nucleus in mock-infected cells. After activation of p38 MAPK by infection of SARS-CoV, STAT3 migrates from nucleus to cytosol. STAT3 does not activate transcription in the host genome, resulting in anti-apoptotic molecules decreasing in cells.

levels in mock-infected Vero E6 cells, even though the phosphorylation level of JAKs did not change after virus infection. Therefore, there is a possibility that Tyr-705 dephosphorylation of STAT3 in virus-infected cells occurs independent of its upstream kinases. Almost all of the STAT3 including both phosphorylated and dephosphorylated molecules are located primarily in the cytosol, and also in membranes, organelles and the nuclear fraction; whereas Tyr-705-phosphorylated STAT3 appears primarily in the nuclear fraction in mock-infected Vero E6 cells. Tyr-705phosphorylated STAT3 clearly disappears from the nuclear fraction after SARS-CoV infection. Thus, STAT3 does not act as a transcriptional enhancer in SARS-CoV-infected Vero E6. Although inhibitors of MEK and JNK have no effect on the phosphorylation status of STAT3 in virusinfected cells, two inhibitors of p38 MAPK (SB203580 and SB202190) partially inhibit dephosphorylation of STAT3 at Tyr-705. This result clearly indicates that p38 MAPK signaling pathway is upstream of Tyr705 dephosphorylation of STAT3 in SARS-CoV-infected Vero E6 cells.

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In addition, Tyr-705 dephosphorylation and Ser-727 phosphorylation exhibit almost the same timing in SARS-CoV-infected cells. In SARSinfected Vero E6 cells, STAT3 dephosphorylation via p38 MAPK activation leads to cell death. However, it is still unclear whether p38 MAPK dephosphorylates Tyr-705 of STAT3 directly or indirectly. Suppressors of cytokine signaling-3 (SOCS3) mRNA are found to be suppressed in SARS-CoV-infected Caco-2 cells.62 This suppression may lead to continuous activation of STAT3 in Caco-2 cells. Kinetics of STAT3 by SARSCoV infection may be different among cells. Thus, activation of p38 MAPK signaling pathway results in CPE (cell death) in SARS-CoV-infected cells, which is possible due to inactivation of STAT3. STAT3 decreases anti-apoptotic activity in the cells as a downstream target of p38 MAPK (Figure 3). At the same time, activation of the p38 MAPK signaling pathway induces anti-apoptotic events by HSP-27, p90RSK and CREB in the cells.31,52 However, it is still unclear whether the observation that the p38 MAPK signaling pathway promotes both cell death and cell survival applies to one individual cell or whether cell death is promoted in some cells and cell survival in other cells. Other STAT signal transduction pathways in SARS-CoV infection have been reported. Signal transducing adaptor molecule 1 (STAM1), which is known to be associated with Jak2 and 3 via the immunoreceptor tyrosine-based activation motif, is upregulated in SARS-CoVinfected Vero E6 cells.63 STAT1 translocation is inhibited in 293T cells expressing ORF6 protein, but phosphorylation of STAT1 is not inhibited by the protein.64

6. ACTIVATION OF ERK1/2 BY SARS-CoV INFECTION ERK1/2 is observed to be phosphorylated in SARS-CoV-infected Vero E6 cells60 (Figure 2). MEK1/2-specific inhibitor-treated (PD98059) SARSCoV-infected Vero E6 cells exhibit no significant changes in activated caspase-3 or caspase-7. Thus, activation of ERK1/2 is not sufficient to prevent apoptotic cell death by SARS-CoV infection. Interestingly, phosphorylation kinetics between ERK1 and ERK2 in EGF-treated and SARSCoV-infected cells are different.52 The phosphorylation level of ERK1 in EGF-treated cells is lower than that of ERK2, while the phosphorylation

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level of ERK1 in SARS-CoV-infected cells is similar to that of ERK2 in SARS-CoV-infected Vero E6 cells. The phosphorylation level of ERK1 is sometimes observed to be higher than that of ERK2 in viral-infected cells. What is the role of ERK1 and ERK2 in SARS-CoV-infected cells? There are reports that indicate ERK2 phosphorylates p90RSK65 and then inhibits glycogen synthase kinase 3β (GSK-3β ).66 However, our experiments indicate that the ERK signaling pathway is not important for phosphorylation of p90RSK Ser-380 in viral-infected cells because Ser-380 is phosphorylated without phosphorylation of Thr-573.52 Further research regarding the roles of ERK1 and ERK2 in SARS-CoV-infected cells needs to be done. Viral proteins are able to induce phosphorylation of ERK1/2. Phosphorylation of ERK1/2 is downregulated in N protein-expressing COS-1 cells in the absence of serum.37 Phosphorylation of ERK1/2 (and JNK) increases in the presence of S protein in HEK293T cells, and S proteininduced ERK phosphorylation depends on PKC.67 The S-induced PKC/ ERK signaling pathway promotes NF-κ B binding to cyclooxygenase-2 (COX-2) promoter. Similar results have been reported using the N protein of SARS-CoV.68 S protein expression induces release of IL-8 via ERK and p38 MAPK signaling pathways including AP-1 in A549 cells, but not JNK.69 S and N proteins may cause inflammation of the lungs by activating COX-2 gene expression. Activation of ERK1/2 is not necessary to establish persistent infection of SARS-CoV in Vero E6 cells.71

7. JNK ACTIVATION FOR PERSISTENT INFECTION JNK is phosphorylated in SARS-CoV-infected Vero E6 cells at least 12 h.p.i.60,70 (Figure 2). Apoptotic signals, such as cleaved caspase-3 and DNA fragmentation, are detected at 18 and 24 h.p.i. Cells begin to show rounding at 24 h.p.i. and persistently infected cells are observed after 48 h.p.i.71 At 50 h.p.i., JNK, Akt and p38 MAPK are phosphorylated in viral-infected cells. Treatment with SP600125, as an inhibitor of JNK, and LY294002, as an inhibitor of phosphatidylinositol 3-kinase (PI3K), after one hour of virus incubation, inhibits the establishment of persistence; whereas PD98059, as an inhibitor of MEK1/2, and SB203580, as an inhibitor of p38 MAPK, does not.71 Thus, the two signaling pathways of JNK and PI3K/Akt are important for the establishment of persistence in Vero

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Vero E6

SARS-CoV infection

Akt JNK ERK p38

2 days < 5%

JNK PI3K/Akt inhibitor

JNK inhibitor

JNK BclBcl-xL BclBcl-2

persistence

Apoptotic cell death

Figure 4. Mechanism of persistent infection of SARS-CoV. Most Vero E6 cells died after 48 h.p.i. and only < 5% of viral-infected cells remained alive. PI3K/Akt and JNK inhibitors were able to inhibit this survival. The surviving cells died when JNK inhibitor was added, but not when PI3K/Akt inhibitor was added.

E6 cells (Figure 4). When cells are treated with inhibitors (SP600125 and LY294002) at 50 h.p.i., SP600125 kills the cells completely, whereas LY294002 does not.72 Thus, activation of PI3K/Akt is essential for the establishment of persistent infection with SARS-CoV prior to cell death, whereas activation of JNK is required at the time of establishment of persistence. Furthermore, N-expressing Vero E6 cells induce phosphorylation of JNK and Akt.72 Thus, JNK and Akt signaling pathways are key factors for understanding persistence of SARS-CoV. As for other roles of JNK, the S protein of SARS-CoV induces CREB binding to COX-2 promoter, mediated via the PI3K/PKC/JNK pathway in HEK293T cells.73 Phosphorylation of JNK increases in N-expressing COS-1 cells in the absence of serum.67 The phosphorylation level of Jun, which is reflected by activation of JNK, also increases in the absence of serum. N protein can activate AP-1, which is composed of homodimers and heterodimers of Fos, Jun, CREB, and ATF subunits, in Vero and Huh7 cells.74 Thus, the N protein is able to induce phosphorylation (activation) of JNK in several cell lines. Accessory proteins, 3a and 7a, phosphorylate JNK1 and JNK3 in HEK293T cells.75

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8. ROLE OF AKT IN APOPTOSIS IN VIRUS-INFECTED CELLS Akt, which is also known as protein kinase B (PKB), is phosphorylated at both Ser-473 and Thr-308 residues by the PI3K signaling pathway, on stimulation by growth factors, insulin, and hormones.76–79 Activation of PI3K results in local accumulation of phosphatidylinositol (PtdIns)-3,4,5triphosphate (P3) at the plasma membrane recruiting both PDK-1 and Akt, and Akt is then autophosphorylated at Ser-473. After PDK-1 activation on the plasma membrane, PDK-1 phosphorylates Akt on Thr-308, and then Akt shows a high level of activity. The main role of Akt is inhibition of apoptosis via phosphorylation of the forkhead transcription factor (FKHR) family, GSK-3β, caspase-9, and Bad.80–82 Ser-473 of Akt is phosphorylated at 8 h.p.i. and maximal phosphorylation is observed at 18 h.p.i. in SARS-CoV-infected Vero E6 cells.83 SARS-CoV infection is necessary for phosphorylation of Ser-473 because the UV-inactivated virus fails to induce serine phosphorylation. Thr-308 phosphorylation has not been detected in Vero E6 cells. The phosphorylation of Ser-473 of Akt by viral infection is inhibited by LY294002, which is an inhibitor of the PI3K signaling pathway. Thus, PI3K is activated in virus-infected cells. Phosphorylation levels of PTEN and PDK-1, which are upstream kinases, are not significantly altered in SARS-CoV-infected Vero E6 cells. Therefore, Thr-308 of Akt is not phosphorylated by virus infection. In vitro kinase activity assay of Akt in SARS-CoV-infected cells indicates that Akt is highly phosphorylated only at serine residues by SARS-CoV infection, but the level of activity of Akt is low. Actually, the level of phosphorylation of GSK-3β (Ser9), which is a proapoptotic signaling molecule and is inactivated by phosphorylation of the N-terminal serine residue Ser-9,84 is slightly increased in virus-infected cells, while phosphorylated Bad and FKHR are not detected. In addition, DNA fragmentations in infected Vero E6 cells at 30 h.p.i. are similar in the presence and absence of LY294002. Therefore, weak activation of Akt cannot prevent apoptosis induced by SARS-CoV infection in Vero E6 cells. G418selected clones established from parent Vero E6 cells are transfected with a plasmid containing the neomycin resistance gene. When these clones are infected with SARS-CoV, they result in a potential cell population capable

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of persistence. At 20 h.p.i., phosphorylated Akt is not detectable in virusinfected clone cell lines, which cannot establish persistence; whereas phosphorylated Akt is detectable in viral-infected parental Vero E6 and clone cell lines, which can establish persistence. Therefore, at least, the phosphorylation of Akt in viral-infected cells is necessary to establish persistence (Figure 4). In addition, Akt is not phosphorylated after establishing persistent cell lines.71,72 Activation of PI3K/Akt is essential for the establishment of persistent infection with SARS-CoV before cell death. The phosphorylation of Akt is not necessary to maintain persistence after establishment. N-protein-expressing Vero E6 cells induce phosphorylation of Akt.72 The kinetics of N protein accumulated in infected Vero E6 cells is similar in the presence and absence of LY294002. Akt serine phosphorylation induced by SARS-CoV infection has no effect on viral replication. The M protein of SARS-CoV induces apoptosis in both HEK293T cells and transgenic Drosophila.85 The M protein-induced apoptosis involves mitochondrial release of cytochrome c protein. As PDK-1 is a dominant suppressor of M-induced apoptotic cell death, apoptosis is induced through modulation of the PI3K/Akt signaling pathway. The above characterizations of Akt in SARS-CoV-infected Vero E6 cells are mainly applicable to experiments using confluent cells. Although phosphorylation followed by dephosphorylation of Akt occurs during the course of virus infection, it is still unclear whether virus infection is actually responsible for phosphorylation and/or dephosphorylation of Akt. When subconfluent Vero E6 cells are infected by SARS-CoV, cell proliferation is inhibited.86 Ser-473 of Akt is highly phosphorylated in subconfluent mock-infected cells. The amount of phosphorylated Akt of mock-infected subconfluent cells is 4.8-fold higher than that of SARS-CoV-infected confluent cells. GSK3β is also dephosphorylated in SARS-CoV-infected subconfluent cells. SARS-CoV infection induces dephosphorylation of a serine residue of Akt in subconfluent cultures, without tentative upregulation of phosphorylation prior to dephosphorylation. Thus, activation of Akt in subconfluent Vero E6 cells plays an important role in cell proliferation, while downregulation of Akt activity in SARS-CoV-infected cells prevents cell proliferation.

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The protein kinase C (PKC) superfamily, which is a major cellular mediator of biological activity, is divided into sub-superfamilies: conventional PKC (cPKC α , β I, β II, γ ), novel PKC (nPKC δ, ε, η, θ ), atypical PKC (aPKCζ, ι/λ ), PKCµ /PKD, and PKCυ.76 Among them, PKCζ can interact with Akt.87 PKCζ (Thr-410) is phosphorylated in SARS-CoVinfected Vero E6 cells, possibly as an anti-apoptotic response to virus infection.83 PKCθ is also slightly phosphorylated by virus infection. PKCα /β II and δ (Ser643) are always phosphorylated in both virusinfected and mock-infected cells. The interaction of phosphorylation of PKCζ with Akt may protect against apoptosis by SARS-CoV infection. Another report indicates that PKCα and PKCε play important roles in the activation of COX-2 by the S protein of SARS-CoV in HEK293T cells.67 PKCα is the upstream kinase for S-induced ERK/NF-κ B activation and PKCε is the upstream kinase for JNK/CREB activation. Thus, S-activated COX-2 expression is mediated via two signaling pathways led by PKCα /ERK and PKCε /JNK.

9. CONCLUSIONS AND PROSPECTS As described in front, many proapoptotic and anti-apoptotic signaling pathways are activated in cultured cells after infection with SARS-CoV (Figure 5). Generally, it is thought that both proapoptotic and prosurvival signaling pathways are activated during viral replication. The balance between activation of proapoptotic and anti-apoptotic signaling pathways may determine whether cells die or survive. However, we do not really know whether both signaling pathways are activated in a single cell. Masses of Vero E6 cells have two populations: one has the potential to permit persistent infection of SARS-CoV, whereas the other does not. Therefore, in the future, the analysis of individual cells may be necessary to determine the correct reaction of signal transduction to viral infection. In acute SARS-CoV-infected Vero E6 cells, proapoptotic (or cell death) signaling pathways, such as p38 MAPK, may be stronger than anti-apoptotic signaling pathways, such as Akt, p90RSK, and STAT3, including Bcl-xL. However, only a part of the activated signaling pathway by SARSCoV infection is known. For example, the entire signaling pathway of p38 MAPK after viral infection is still not known. Furthermore, NF-κ B

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Anti-Apoptosis

GSK3β Anti-apoptosis?

PKCζ Akt

CREB

PI3K

Cell proliferation Persistent

ERK1/2

Anti-apoptosis ?

p90RSK

SARS-CoV Infection Anti-apoptosis?

MAPKAPK-2 Hsp-27 CREB ATF-1 eIF4E STAT3

p38 MAPK

JNK

Persistent

Caspase 3, 6, 7

Apoptosis, CPE

Figure 5. Activated and inactivated signaling pathways in infection of SARS-CoV. As each report in this chapter used different cultured cells, this figure is shown based on our experiments using Vero E6 cells.

is thought to play an important role in SARS-CoV-infected cells because N protein inhibits interferon production in 293T cells via inhibition of NF-κ B.64 To understand the mechanisms of pathology by SARS-CoV infection, the manners in which viral proteins interact with cellular proteins in signaling pathways must be further clarified. We do not know which viral proteins are necessary and sufficient to fully activate signaling pathways. It is possible that virus replication in a cell is necessary to fully activate signaling pathways. One of the goals of understanding signal transduction in the field of virology is the development of therapeutic reagents that can inhibit the pathways of apoptotic cell death by viral infection. In cells infected with SARS-CoV and many other viruses, p38 MAPK promotes cell death. Therefore, chemical inhibitors of p38 MAPK have been developed for therapeutic purposes. RNAi techniques to block p38 MAPK will also be

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useful. A better understanding of which signaling pathways are activated and which are inactivated in specific tissues in SARS patients will allow the development of improved targeted therapies.

ACKNOWLEDGMENTS I am grateful to Dr. Shuetsu Fukushi, Dr. Masayuki Saijo, Dr. Momoko Ogata, Dr. Kouji Sakai, Dr. Ichiro Kurane, and Dr. Shigeru Morikawa (National Institute of Infectious Diseases) for their comments on this manuscript. This work was supported, in part, by a grant-in-aid from Ministry of Health, Labor, and Welfare of Japan and Japan Society for the Promotion of Science.

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10. Garrington TP, Johnson GL (1999). Curr Opin Cell Biol 11, 211–218. 11. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME (1999). Science 286, 1358–1362. 12. Lee CH, Chen RF, Liu JW, Yeh WT, Chang JC, Liu PM, Eng HL, Lin MC, Yang KD (2004). J Immunol 172, 7841–7847. 13. Platanias LC (2003). Pharmacol Ther 98, 129–142. 14. Teramoto H, Coso OA, Miyata H, Igishi T, Miki T, Gutkind JS (1996). J Biol Chem 271, 27225–27228. 15. Tibbles LA, Ing YL, Kiefer F, Chan J, Iscove N, Woodgett JR, Lassam NJ (1996). EMBO J 15, 7026–7035. 16. Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J, Saklatvala J (1994). Cell 78, 1039–1049. 17. Garrido C, Schmitt E, Candé C, Vahsen N, Parcellier A, Kroemer G (2003). Cell Cycle 2, 579–584. 18. Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ (1996). EMBO J 15, 4629–4642. 19. Gingras AC, Raught B, Sonenberg N (1999). Annu Rev Biochem 68, 913–963. 20. Fingerote RJ, Abecassis M, Phillips MJ, Rao YS, Cole EH, Leibowitz J, Levy GA (1996). J Virol 70, 4275–4282. 21. McGilvray ID, Lu Z, Wei AC, Dackiw AP, Marshall JC, Kapus A, Levy G, Rotstein OD (1998). J Biol Chem 273, 32222–32229. 22. Banerjee S, Narayanan K, Mizutani T, Makino S (2002). J Virol 76, 5937–5948. 23. Rahaus M, Desloges N, Wolff MH (2005). J Med Virol 76, 424–433. 24. Silva AM, Whitmore M, Xu Z, Jiang Z, Li X, Williams BR (2004). J Biol Chem 279, 37670–37676. 25. Spaziani A, Alisi A, Sanna D, Balsano C (2006). J Biol Chem 281, 10983–10989. 26. Singh IN, El-Hage N, Campbell ME, Lutz SE, Knapp PE, Nath A, Hauser, KF (2005). Neuroscience 135, 781–790. 27. Muthumani K, Wadsworth SA, Dayes NS, Hwang DS, Choo AY, Abeysinghe HR, Siekierka JJ, Weiner DB (2004). AIDS 18, 739–748. 28. Wang XZ, Lawson B, Brewer JW, Zinszner H, Sanjay A, Mi LJ, Boorstein R, Kreibich G, Hendershot LM, Ron D (1996). Mol Cell Biol 16, 4273–4280. 29. Su HL, Liao CL, Lin YL (2002). J Virol 76, 4162–4171.

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30. Lee DC, Cheung CY, Law AH, Mok CK, Peiris M, Lau AS (2005). J Virol 79, 10147–10154. 31. Mizutani T, Fukushi S, Saijo M, Kurane I, Morikawa S (2004). Biochem Biophys Res Commun 319, 1228–1234. 32. Yan H, Xiao G, Zhang J, Hu Y, Yuan F, Cole DK, Zheng C, Gao GF (2004). J Med Virol 73, 323–331. 33. Mizutani T, Fukushi S, Kenri T, Sasaki Y, Ishii K, Endoh D, Zamoto A, Saijo M, Kurane I, Morikawa S (2007). Arch Virol 152, 1019–1025. 34. Foltz IN, Lee JC, Young PR, Schrader JW (1997). J Biol Chem 272, 3296–3301. 35. Ginty DD, Bonni A, Greenberg ME (1994). Cell 77, 713–725. 36. von Knethen A, Lotero A, Brune B (1998). Oncogene 17, 387–394. 37. Surjit M, Liu B, Jameel S, Chow VTK, Lal SK (2004). Biochem J 383, 13–18. 38. Dent P, Yacoub A, Fisher PB, Hagan PMP, Grant S (2003). Oncogene 22, 5885–5896. 39. Kopecky-Bromberg SA, Martinez-Sobrido L, Palese P (2006). J Virol 80, 785–793. 40. Frodin M, Gammeltoft S (1999). Mol Cell Endocrinol 151, 65–77. 41. Moller DE, Xia CH, Tang W, Zhu AX, Jakubowski M (1994). Am J Physiol 266, C351–C359. 42. Zhao Y, Bjorbaek C, Weremowicz S, Morton CC, Moller DE (1995). Mol Cell Biol 15, 4353–4363. 43. New L, Zhao M, Li Y, Bassett WW, Feng Y, Ludwig S, Padova FD, Gram H, Han J (1999). J Biol Chem 274, 1026–1032. 44. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME (1999). Science 286, 1358–1362. 45. Dalby KN, Morrice N, Caudwell FB, Avruch J, Cohen P (1988). J Biol Chem 273, 1496–1505. 46. Buck M, Poli V, Hunter T, Chojkier M (2001). Mol Cell 8, 807–816. 47. Gavin AC, Nebreda AR (1999). Curr Biol 9, 281–284. 48. Smith JA, Poteet-Smith CE, Malarkey K, Sturgill TW (1999). J Biol Chem 274, 2893–2898. 49. Frödin M, Jensen CJ, Merienne K, Gammeltoft S (2000). EMBO J 19, 2924–2934. 50. Jensen CJ, Buch MB, Krag TO, Hemmings BA, Gammeltoft S, Frodin M (1999). J Biol Chem 274, 27168–27176.

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51. Richards SA, Fu J, Romanelli A, Shimamura A, Blenis J (1999). Curr Biol 9, 810–820. 52. Mizutani T, Fukushi S, Saijo M, Kurane I, Morikawa S (2006). FEBS Lett 580, 1417–1424 53. Shuai K, Stark GR, Kerr IM, Darnell JE Jr (1993). Science 261, 1744–1746. 54. Shuai K, Horvath CM, Huang LH, Qureshi SA, Cowburn D, Darnell JE Jr (1994). Cell 76, 821–828. 55. Schindler C, Shuai K, Prezioso VR, Darnell JE Jr (1992). Science 258, 1808–1812. 56. Schindler C, Shuai K, Prezioso VR, Darnell JE Jr (1992). Science 257, 809–813. 57. Rajan P, McKay RD (1998). J Neurosci 18, 3620–3629. 58. Grandis JR, Drenning SD, Zeng Q, Watkins SC, Melhem MF, Endo S, Johnson DE, Huang L, He Y, Kim JD (2000). Proc Natl Acad Sci USA 97, 4227–4232. 59. Mora LB, Buettner R, Seigne J, Diaz J, Ahmad N, Garcia R, Bowman T, Falcone R, Fairclough R, Cantor A, et al. (2002). Cancer Res 62, 6659–6666. 60. Mizutani T, Fukushi S, Murakami M, Hirano T, Saijo M, Kurane I, Morikawa S (2004). FEBS Lett 577, 187–192. 61. Kerr IM, Costa-Pereira AP, Lillemeier BF, Strobl B (2003). FEBS Lett 546, 1–5. 62. Okabayashi T, Kariwa H, Yokota S, Iki S, Indoh T, Yokosawa N, Takashima I, Tsutsumi H, Fujii N (2006). J Med Virol 78, 417–424 63. Leong WF, Tan HC, Ooi EE, Koh DR, Chow VT (2005). Microbes Infect 7, 248–259. 64. Kopecky-Bromberg SA, Martínez-Sobrido L, Frieman M, Baric RA, Palese P (2007). J Virol 81, 548–557. 65. Eblen ST, Catling AD, Assanah MC, Weber MJ (2001). Mol Cell Biol 21, 249–259. 66. Angenstein F, Greenough WT, Weiler IJ (1998). Proc Natl Acad Sci USA 95, 15078–15083. 67. Liu M, Yang Y, Gu C, Yue Y, Wu KK, Wu J, Zhu Y (2007). FASEB J 21, 1586–1596 68. Yan X, Hao Q, Mu Y, Timani KA, Ye L, Zhu Y, Wu J (2006). Int J Biochem Cell Biol 38, 1417–1428. 69. Chang YJ, Liu CY, Chiang BL, Chao YC, Chen CC (2004). J Immunol 173, 7602–7614.

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70. Mizutani T, Fukushi S, Saijo M, Kurane I, Morikawa S (2006). In The Nidoviruses (eds.) Perlman, S, Holmes K. 581, pp. 323–326. 71. Mizutani T, Fukushi S, Saijo M, Kurane I, Morikawa S (2005). Biochim Biophys Acta 1741, 4–10. 72. Mizutani T, Fukushi S, Ishii K, Sasaki Y, Kenri T, Saijo M, Kanaji Y, Shirota K, Kurane I, Morikawa S (2006). Biochem Biophys Res Commun 347, 261–265. 73. Liu M, Yang Y, Gu C, Yue Y, Wu KK, Wu J, Zhu Y (2007). FASEB J 21, 1586–1596. 74. He R, Leeson A, Andonov A, Li Y, Bastien N, Cao J, Osiowy C, Dobie F, Cutts T, Ballantine M, Li X (2003). Biochem Biophys Res Commun 311, 870–876. 75. Kanzawa N, Nishigaki K, Hayashi T, Ishii Y, Furukawa S, Niiro A, Yasui F, Kohara M, Morita K, Matsushima K, et al. (2006). FEBS Lett 580, 6807–6812. 76. Toker A (2000). Mol Pharmacol 57, 652–658. 77. Brazil DP, Hemmings BA (2001). Trends Biochem Sci 26, 657–664. 78. Scheid MP, Woodgett JR (2003). FEBS Lett 546, 108–112. 79. Welch H, Eguinoa A, Stephens LR, Hawkins PT (1998). J Biol Chem 273, 11248–11256. 80. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC (1998). Science 282, 1318–1321. 81. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA (1995). Nature 378, 785–789. 82. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997). Cell 91, 231–241. 83. Mizutani T, Fukushi S, Saijo M, Kurane I, Morikawa S (2004). Virology 327, 169–174. 84. Pap M, Cooper GM (1998). J Biol Chem 273, 19929–19932. 85. Chan CM, Ma CW, Chan WY, Chan HY (2007). Arch Biochem Biophys 459, 197–207. 86. Mizutani T, Fukushi S, Iizuka D, Inanami O, Kuwabara M, Takashima H, Yanagawa H, Saijo M, Kurane I, Morikawa S (2006). FEMS Immunol Med Microbiol 46, 236–243. 87. Konishi H, Shinomura T, Kuroda S, Ono Y, Kikkawa U (1994). Biochem Biophys Res Commun 205, 817–825.

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CHAPTER 15

Impact of Filovirus Infection upon Cellular Signaling Pathways Christopher F. Basler

ABSTRACT The members of the filoviridae family, Ebola virus and Marburg virus, are among the most lethal viral pathogens of humans and nonhuman primates. Filoviral disease is manifested by dysregulated innate and adaptive immune responses and a clinical syndrome similar to disseminated intravascular coagulation. Several experimental vaccines have been produced which effectively protect rodents or nonhuman primates from lethal filovirus disease. However, a licensed vaccine or therapy is not presently available for human use. An understanding of the molecular pathways activated or manipulated by these viruses should provide insights into the severe disease induced by filoviruses and may also provide a key for the identification of new therapeutic targets. Here, we describe what is known about how filoviruses modulate cell signal transduction pathways and discuss how these pathways influence filovirus pathogenesis. Particular focus will be on the impact of the Ebola virus proteins VP35 and VP24 upon the host interferon response and the impact of the Ebola virus glycoprotein (GP) upon MAP kinase signaling and upon the expression of proteins at the cell surface.

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1. INTRODUCTION Filoviruses are filament-shaped, enveloped, viruses with non-segmented negative-sense RNA genomes.1 The filovirus family is composed of two genera, the Ebola viruses (EBOVs) and Marburg viruses (MBGVs). The filoviruses are among the most feared emerging viruses because of their high lethality in humans and because of the dramatic symptoms associated with the hemorrhagic fever caused by these viruses. These viruses continue to cause periodic outbreaks of severe viral hemorrhagic fever in humans and are of concern as potential bioweapons.2,3 Four species of EBOV: Zaire, Sudan, Ivory Coast (Côte d’Ivoire) and Reston, and one species of MBGV have been recognized.1 MBGV, named after the city in which it was identified, was isolated during a 1967 outbreak of hemorrhagic fever among workers in European vaccine production facilities who became ill while preparing monkey kidney cell cultures. This outbreak, which was caused by infected monkeys imported from Africa, was notable for its 23 percent case fatality rate. EBOVs were first identified in the mid-1970s following outbreaks in Zaire (now the Democratic Republic of Congo) and Sudan. Mortality rates in these outbreaks were even higher than that seen in the initial MBGV outbreak, with the Zaire outbreak resulting in an 88 percent mortality rate and the Sudan outbreak having a 53 percent death rate.4 The Reston Ebola virus is notable because although it can cause lethal infections in nonhuman primates, the only known human infections, documented by seroconversion following exposure to infected monkeys imported from the Philippines, did not result in illness.5–8 These data suggest that Reston Ebola virus may be avirulent in humans.

2. FILOVIRUS PATHOGENESIS The best characterized form of filovirus disease is Ebola hemorrhagic fever (for review see Ref. 9). In humans, this illness is highly lethal and is characterized by a febrile syndrome followed by more severe symptoms including hypotension, shock and hemorrhage. A similar syndrome occurs in experimentally infected macaques, and this model has provided the most detailed view of EBOV disease.9 Macaque studies suggest that early

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targets of infection are macrophages and dendritic cells, but the infection disseminates quite rapidly to other target tissues.10 Infection of macrophages and dendritic cells in vitro leads to significant proinflammatory cytokine production,11–13 and significant levels of proinflammatory cytokines are present in the serum of infected animals and humans.2,11,14 In macaques, a syndrome similar to intravascular disseminated coagulation has also been described, and parallels have been drawn between Ebola hemorrhagic fever and severe sepsis.15,16 The activation of coagulation appears to be related to induction of tissue factor production and is also associated with decreased plasma levels of protein C.15–17 Zaire EBOV infections are also characterized by impairment of several components of the adaptive immune system.1,9 For example, apoptotic loss of lymphocytes is seen during infection, despite the fact that these cells are not detectably infected, and experimental data suggests that this loss of lymphocytes is cytokine mediated.18–21 Additionally, infection of dendritic cells, at least in vitro, results in an impaired activation of these cells which may impair mobilization of T cell responses to infection.18,19

3. HOST CELL IFNα /β RESPONSES Several lines of evidence indicate that evasion of the host IFNα /β response is particularly critical for Ebola virus pathogenesis. In general, the IFNα /β response plays a major role in innate immunity response to viral infection and it also modulates adaptive immune responses.22,23 IFNα /β refers to a family of secreted proteins that are encoded, in humans, by a single IFNβ gene and a number of IFNα genes. These bind to a common receptor, the interferon α /β receptor (IFNAR). In most cell types, IFNβ is the predominant form of IFN initially synthesized in response to virus infection. IFNβ transcription is directly stimulated by cellular transcription factors NF-κ B, interferon regulatory factor 3 (IRF-3) and AP-1. In contrast to the case for IFNβ , most IFNα gene promoters are activated specifically by interferon regulatory factor 7 (IRF-7), an interferon-induced protein. Production of IRF-7 thus functions as a positive feedback system which amplifies the IFNα /β response. Specifically, IFNα /β -induced IRF-7 expression results in the transcriptional activation of many IFNα genes that would otherwise remain quiescent. It should also be noted that some

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cell types, such as plasmacytoid dendritic cells (PDCs) rapidly produce IFNα in response to virus infection, in part because they constitutively express IRF-7.24 This allows PDCs to rapidly produce large amounts of IFNα in response to virus infection. IFNα /β production can be induced by a variety of stimuli including viral infection (Figure 2).24 Signaling through selected cellular pattern recognition receptors (PRRs) can lead to induction of IFNα /β gene expression (reviewed in Ref. 24). These PRRs can recognize and respond to a variety of pathogen-associated molecular patterns (PAMPs). Although the role of specific PRRs in host response to filovirus infection has not been defined, those most likely to be relevant to filovirus infection would include Toll-like receptors (TLRs) such as TLR3, which senses double-stranded RNA; TLR7 and TLR8 which can recognize viral singlestranded RNAs; and the intracellular sensors of virus infection, RIG-I and MDA-5.25 The TLRs are type I transmembrane proteins that possess so called Toll/IL-1 (TIR) domains in their cytoplasmic tails.26 Upon recognition of PAMPs, TLRs signal in a manner that requires the TIR-domain of the TLR as well as cellular adapter molecules. As TLR expression is restricted to specific cell types, RIG-I and MDA-5 may play critical roles in the induction of IFN responses in many of the cell types infected by EBOV and MBGV.27 Briefly, RIG-I and MDA5 appear, based on their sequence, to be RNA helicases and possess at their amino-termini protein-protein interaction domains referred to as caspase recruitment domains (CARDs).25 RIG-I can recognize RNAs possessing 5′-triphosphates, such as would be found on genomic RNAs of many RNA viruses; and, experimentally, MDA-5 recognizes the synthetic dsRNA molecule poly (I:C).28,29 These properties appear to confer specificity to RIG-I and MDA-5 in terms of their ability to signal in response to different RNA virus families and RNA molecules with different structures.30 RIG-I was essential for the production of IFN in response to paramyxoviruses, influenza viruses and flaviviruses, and it also signaled in response to transfected in vitro transcribed RNAs with 5′-triphosphates.29 In contrast, MDA5 was essential for production of IFN in response to picornavirus or to transfected polyI:polyC. RIG-I and MDA-5 signaling requires the presence of another CARD-domain containing protein, IPS-1 (also called CARDIF, MAVS or VISA), which localizes to mitochondria.31–34

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Signaling through these IFNα /β -inducing pathways ultimately results in the activation of either of two IRF-3 kinases, IKKε or TBK-1.35,36 These kinases play a critical role in activation of IFNα /β gene expression, because they phosphorylate serine and threonine residues on IRF-3 (and IRF-7 when present), leading to the dimerization and nuclear accumulation of IRF-3.37 Nuclear, activated IRF-3 then participates in IFN gene expression.37 Secreted IFNα /β binds to the IFNAR, activating a JAK/STAT signaling pathway, among other signaling pathways38 (Figure 1). Activation of JAK family tyrosine kinases leads to STAT1 and STAT2 tyrosine phosphorylation which then heterodimerizes via phosphotyrosine-SH2 domain interactions. This complex further associates with interferon-regulatory factor 9 (IRF9) to create the transcription factor complex ISGF-3. ISGF-3 accumulates in the nucleus and activates expression of the genes with promoters containing interferon-stimulated response elements (ISREs). Addition to cells of IFNα /β induces an antiviral state in which virus replication is impaired. The best studied IFN-induced antiviral genes include the dsRNAactivated protein kinase PKR, 2′,5-oligoadenylate synthetase (OAS) and the MxA protein. (MxA is the term used to refer to the human protein, Mx1 refers to its mouse homologue). However, other IFN induced genes also contribute to IFN-induced antiviral effects (e.g., Ref. 39, 40).

4. THE GLOBAL IMPACT OF FILOVIRUS INFECTION UPON THE HOST CELL Filovirus disease is associated with induction of strong proinflammatory responses, potent activation of coagulation cascades and immune suppression.2 To fully understand the molecular basis of filovirus disease and to devise means of therapeutic intervention, it will be important to define how infection of cells induces pathological responses. Therefore, efforts are underway to characterize the impact of EBOV and MBGV infection upon host cell pathways.

4.1 EBOV and IFN Signaling Pathways One major way in which EBOVs have been shown to modulate host cell signaling pathways is by antagonizing the IFN response, inhibiting

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EXTRACELLULAR IFN-β

JAK1

Virus

ATF-2/c-Jun IRF-3 NF-κB

IFN-β IFNAR

VP35

IFN-γ IFNGR

TYK2

STAT1:STAT2 IRF-9 (ISGF-3)

JAK1

VP24

JAK2

STAT1:STAT1

CYTOPLASM NUCLEUS

IFN-β promoter

ISRE promoter

GAS promoter

Figure 1. Interaction of Ebola virus Proteins VP35 and VP24 with the Host IFN system. A simplified schematic diagram of the signaling pathways leading to IFNα /β synthesis following virus infection (left side pathway) and the signaling pathways activated by IFNα /β (center pathway) or by IFNγ (right side pathway). Virus infection can activate cellular transcription factors including the AP-1 transcription factor complex ATF-2/c-Jun, IRF-3 and NF-κ B. These transcription factors cooperate to activate transcription of the IFNβ gene. Expressed IFNβ is secreted and binds to the IFNα /β receptor (IFNAR) triggering the activation of IFNAR-associated Jak family tyrosine kinases Jak1 and Tyk2. These tyrosine phosphorylate STAT1 and STAT2, which form heterodimers and further interact with IRF-9, forming the transcription factor complex ISGF-3. ISGF-3, when in the nucleus, activates transcription of genes with interferon-stimulated response elements (ISREs). IFNγ binds to a distinct receptor, the IFNγ receptor, leading to the activation of Jak1 and Jak2 and the formation of STAT1:STAT1 heterodimers which move to the nucleus and activate promoters with gamma-activated sequence (GAS) elements. EBOV VP35 inhibits the activation of IRF-3, suppressing IFNα /β gene expression. EBOV VP24 prevents the nuclear accumulation of tyrosine-phosphorylated STAT1.

both IFNα /β production as well IFNα /β- and IFNγ-induced signaling. That inhibition of these pathways is relevant to filovirus pathogenesis is supported by data from experimental infections of mice. Specifically, filoviruses that have not been previously adapted to mice do not cause lethal disease in adult or weanling mice, but they can kill newborn mice

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and SCID mice.41 However, by repeated experimental passage in mice, Zaire EBOV evolved a lethal phenotype and killed following mouse intraperitoneal injection.42 However, non-adapted Zaire EBOV was lethal in mice lacking either the IFNα /β receptor or STAT1.43 These data suggest that the IFNα /β response normally controls EBOV infection in mice unless the virus has been adapted to better evade mouse IFN responses. Conversely, induction of innate antiviral responses, including strong IFNα responses, can prevent lethality in mice, even by the mouse-adapted EBOV. For example, 3-deazaneplanocin A, an adenosine analogue, was able to protect mice from an otherwise lethal challenge by mouse-adapted EBOV, and this protection correlated with induction of “massively increased IFNα ” due to the combination of drug and virus.44 Likewise, a subcutaneous inoculation of mice with mouse-adapted EBOV proved to be non-lethal whereas an intraperitoneal inoculation was lethal.45 When the mice were first subcutaneously administered the virus, and then challenged at early time points following the subcutaneous dose, the mice were protected as early as at the 48 hour time point, which correlated with the onset of detectable levels of IFNα in liver, spleen and serum.45 These data again suggest that IFNα can, at least in the mouse system, influence EBOV pathogenesis.45

4.2 EBOV Evades the IFNα /β Response Several studies have demonstrated that EBOV infection blocks cellular IFN responses at more than one level.12,46,47 Zaire EBOV infection of human umbilical vein endothelial cells (HUVECs) inhibited cellular responses to IFNα or IFNγ as manifested by impaired expression of IFN-induced gene expression in infected cells.47 For example, infection reduced the IFNα- or IFNγ-induced expression of IRF-1 and 2′,5′-oligoadenylate synthetase by IFNα or IFNγ.47 This failure of gene expression correlated with the reduced formation of IFNα /β- or IFNγ -induced transcription factor complexes, as assessed by electrophoretic mobility shift assays.47 The inhibition was also specific to the IFN pathways in that infection did not block induction of gene expression by IL-1β nor did it impede formation of functional NF-κ B transcription complexes induced by IL-1β.47

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Zaire EBOV infection also impairs the ability of HUVECs to produce IFNα /β following treatment of the cells with poly I:polyC.46 Uninfected HUVECs treated with dsRNA responded with enhanced expression of a number of IFN-inducible proteins including class I MHC, IRF-1, 2 ′-5′ oligoadenylate synthetase, PKR, ICAM-1 and IL-6. In Zaire EBOVinfected cells, however, poly I:polyC induced IFN responses were impaired.46 Interestingly, the effect on IFNα /β expression did not appear to be due to a general inhibition of dsRNA signaling, because polyI:polyC was able to activate NF-κ B in infected cells.46 These data are consistent with the data of Gupta and colleagues, where human PBMC or macrophages infected with Zaire EBOV did produce proinflammatory chemokines and cytokines but did not produce IFNα or IFNβ until three days postinfection, when only small amounts of IFN were seen.12 Infection also suppressed IFNα /β production induced by poly(I:C).12

4.3 IFNα /β Produced During the Course of EBOV Infection It should be noted that, despite virus-encoded mechanisms to block IFNα /β responses, IFNα /β is produced in EBOV-infected patients and in EBOV-infected nonhuman primates.11,12,48 Sera from EBOV-infected patients had detectable levels of IFNα and IFNβ, in addition to other cytokines, whereas pooled serum from uninfected individuals had undetectable levels of IFNα /β,12 and an IFN-response was clearly detected in microarray analyses of the PBMCs of EBOV-infected nonhuman primates.49 The source of the IFNα /β seen in vivo remains to be defined, but the presence of a systemic IFN response does not exclude a role for EBOV-encoded IFN-antagonists in vivo. Rather, local suppression of IFN responses by the virus only need to be sufficient to permit virus dissemination. The systemic IFN response might simply prove ineffective in terms of suppressing viral spread. It will be of interest to determine whether the systemic IFN is predominantly produced by infected or uninfected cells, whether plasmacytoid dendritic cells might be a major source of this IFN, and why this systemic IFN response fails to arrest disease progression in Zaire EBOV-infected nonhuman primates.

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5. INHIBITION OF IFNα /β PRODUCTION BY THE VP35 PROTEIN 5.1 The Functions of the Ebola VP35 Protein The VP35 protein encoded by EBOV carries out at least three functions: it is an essential component of the viral RNA-dependent RNA polymerase, it plays a structural role, and it inhibits innate immunity, particularly the production of IFNα /β. Each of these functions is likely to be critical for virus replication in vivo. Obviously, if the virus is unable to synthesize RNA, it will be unable to replicate its genome or make mRNA for viral protein expression. That EBOV and MBGV would be unable to synthesize any RNA in the absence of VP35 is supported by experiments which employed a viral “minigenome”. This minigenome, a replica of the viral genome containing only a chloramphenicol acetyl transferase (CAT) reporter gene, expressed CAT only when VP35 was present along with the viral NP, VP30 and L proteins.50,51 The structural role of VP35 is less well characterized. Like all negative-strand RNA viruses, filoviruses must package and deliver to newly infected cells, a functional viral polymerase in order to initiate viral gene expression and replication in the infected cell. Therefore VP35 must be incorporated into viral particles, if the particles were to be infectious. Additionally, EBOV virions contain long filamentous structures which appear to consist predominantly of the viral nucleoprotein (NP) and VP35.52 These filamentous nucleocapsids could be reconstituted by expressing in 293T cells, NP, VP35 and VP24.52 Whether such structures are absolutely required for virus assembly or viral RNA replication is not fully clear; however, VP35 is required for their formation.52

5.2 VP35 Inhibits IFNα /β Production The first evidence that VP35 could inhibit host IFN responses is the observation that VP35 can functionally complement the growth defect of a mutant influenza virus, deltaNS1 virus, a mutant that lacks the influenza virus IFN-antagonist protein NS1 protein.53 The capacity of VP35 to rescue

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delNS1 virus growth correlated with the ability of VP35 to suppress activation of the IFNβ promoter.53 Additional studies reveal that VP35 blocks IFNα /β production by virtue of its ability to prevent activation of IRF-3.54 IRF-3 activation was prevented in Sendai virus-infected, VP35expressing cells, as evidenced by the absence of IRF-3-dependent gene expression, the failure of IRF-3 to accumulate in the nucleus and the absence of IRF-3 hyperphosphorylation.54 This last observation, the absence of IRF-3 hyperphosphorylation, suggests that VP35 acts by inhibiting the virus-induced signaling pathways that activate IRF-3 via serine/threonine phosphorylation.54 As noted above, in most cell types, infection of cells with non-segmented, negative-strand RNA viruses, such as EBOV and MBGV, is thought to induce IFNα /β synthesis through RIG-I-like sensors which signal to activate IRF-3, NF-κ B and AP-1 (Figure 2). Because dsRNA has long

Virus/dsRNA RIG-I -I MDA-5 IPS-1

VP35 IKKε TBK-1

ATF2/cJun

IκB

IRF-3/IRF-7 NF-κB

IFNβ Figure 2. Model of EBOV VP35 IFN-antagonist function. Depicted are the basic components of the pathways that lead from detection of viral replication products by the cellular RNA helicases RIG-I or MDA-5 to the production of IFNβ. RIG-I and MDA-5 signal in an IPS-1-dependent manner and activate the IRF-3 kinases IKKε or TBK-1. These kinases participate in the activation of transcription factors, including IRF-3, required for IFN-β promoter activity. Present data suggests that VP35 inhibits these pathways at or near the level of the IRF-3 kinases.

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been known to induce IFNα /β production, it was of interest to observe that VP35 binds to dsRNA.55 When point mutants were generated that failed to detectably bind to dsRNA, these were found to have some impairment in their ability to inhibit IFNβ production.55 However, these mutants retained the ability to inhibit IFNβ production induced by overexpression of RIG-I, IPS-1 or the IRF-3 kinases, IKKε or TBK-1. These data suggest that VP35 dsRNA binding activity, while potentially contributing to inhibition of IRF-3 activation, is probably not required for this function.55 In this respect, VP35 is like other dsRNA binding proteins, such as the influenza virus NS1 protein, that inhibit IFNα /β production. NS1 can also inhibit IFN production even when its capacity to bind dsRNA is impaired by mutation.56,57 The ability of VP35 to inhibit IFNβ gene expression induced by overexpressed RIGI, IPS-1, IKKε or TBK-1 suggests that VP35 can act at some point proximal to the IRF-3 kinases, although additional effects upstream of the kinases, such as sequestering of dsRNA, cannot be excluded (Figure 2). Defining the precise targets through which VP35 suppresses IRF-3 activation and IFNβ production will be critical to fully understand how VP35 modulates cellular responses to infection.

5.3 Effect of VP35 upon PKR and RNAi PKR is a dsRNA-activated cellular serine/threonine kinase, the expression of which is induced by IFN. PKR has long been known to mediate antiviral responses, and virus-encoded inhibitors of PKR, including the adenovirus VAI RNA, the vaccinia virus E3L protein and the influenza A virus NS1 protein, were among the earliest viral products found to suppress the antiviral effects of IFNs.58–60 PKR primarily suppresses virus replication by inhibiting cellular translation. Upon activation, PKR phosphorylates the α subunit of the translation initiation factor eIF-2, leading to suppression of protein synthesis.61 Demonstration that VP35 can inhibit PKR came from studies in which VP35 was built into a mutant herpes simplex virus 1 (HSV-1) that lacked the γ 134.5 gene. The γ 134.5 protein recruits cellular protein phosphatase 1 to dephosphorylate eIF-2α, and HSV-1 mutants impaired for this function display increased sensitivity to the antiviral effects of IFN. Interestingly, when Vero cells were pretreated with IFNα and then infected

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with either the parental γ 134.5 null virus or the virus expressing VP35, the VP35 virus was relatively resistant to the antiviral effects of IFNα.62 This effect correlated with a relative suppression of PKR activation and eIF-2α phosphorylation in the VP35-expressing cells. Interestingly, an R312A mutation that impairs VP35 dsRNA binding activity55,62 did not abrogate the ability of VP35 to inhibit PKR, despite the fact that PKR is dsRNAactivated.62 These data thus imply that VP35 may counteract the antiviral effects of PKR in EBOV-infected cells. It remains to be determined, however, if IFN mediates an antiviral effect against filoviruses via PKR, although such a role for PKR would seem likely. VP35 also suppresses RNA silencing by a mechanism that requires its dsRNA-binding activity.63 RNA silencing exerts an antiviral effect in plant and insect cells, and viruses that infect plants and insects often encode suppressors of RNA silencing (reviewed in Ref. 64). Several mammalian viruses have also been demonstrated to encode inhibitors of RNA silencing. Examples include the VAI RNA of adenovirus, the NS1 protein of influenza viruses, the E3L protein of vaccinia virus and the Tat protein of human immunodeficiency virus (HIV).65–68 Similarly, expression of VP35 blocked knockdown of a transfected luciferase reporter gene by a cotransfected short hairpin RNA targeting luciferase, and this property was abrogated by mutations previously demonstrated to impair VP35 dsRNA binding activity.63 Additionally, expression of VP35 could functionally substitute for the RNA silencing suppression function of the HIV-1 Tat protein.63 However, whether RNA silencing functions as an antiviral mechanism in mammalian cells remains to be determined.64 Thus, the impact of this function upon EBOV-infected cells, at least for the moment, remains unclear.

5.4 VP35 IFN-Antagonist Function in the Context of Virus Infection Recombinant EBOV with mutagenized VP35s have provided evidence that the ability of VP35 to suppress innate immunity is important for virus replication. Recombinant EBOVs containing either of two VP35 mutations, R305A or R312A, were constructed.69 Based on in vitro studies, these mutations were predicted to impair the ability of VP35 to inhibit IRF-3

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activation.70 When these viruses were compared to wild-type EBOV in Vero cells, U937 cells differentiated into macrophages or the hepatocyte cell line Huh7 and the mutants consistently grew to lower titers.69 The growth attenuation in Vero cells was somewhat surprising given the inability of Vero cells to produce IFNα /β. However, the mutant viruses did induce higher levels of the cytokine RANTES, which is expressed in an IRF-3-responsive manner.69 Thus, the mutant virus presumably exhibits growth defects in Vero cells due to IFN-independent but IRF-3 dependent cellular responses. Consistent with this model, the mutant virus activated IRF-3 nuclear localization more strongly than the wild-type virus did.69 Additionally, microarray analyses comparing Huh7 cellular responses to infection with purified parental wild-type virus and purified R312A virus preparations revealed that the mutant virus induced the potent upregulation of the interferon response at later times in infection while the wild-type virus did not (Hartman and Nichol, personal communication). Finally, a recombinant Zaire EBOV engineered to contain mouse-adaptive mutations in NP and VP24 was further mutated to possess the VP35 R312A mutation. This mouse-adapted VP35 mutant virus was attenuated in mice relative to the parental (wild-type VP35) control virus.71 These data provide compelling evidence that VP35 modulates innate immune responses in the context of EBOV infection. Whether the ability of VP35 to modulate RNA silencing or to inhibit PKR influences cellular responses to virus infection remains to be determined.

6. EBOV VP24 INHIBITS IFN SIGNALING The VP24 protein is the second multifunctional EBOV protein that combats host-IFN responses and influences EBOV pathogenesis.72–74 Early studies demonstrated that VP24 is a virion structural protein and was designated a “minor matrix protein”.1,75 While the major EBOV matrix protein is VP40, and VP40 appears to drive the budding of EBOV particles, recent studies support a possible role for VP24 in viral budding,76 although co-expression of VP24 with VP40 did not appear to enhance budding by VP40.77 In addition, VP24 plays an important role in the assembly of viral nucleocapsids, as EBOV nucleocapsids could be reconstituted by co-expression of the EBOV nucleoprotein (NP), VP35 and

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VP24.52 In these experiments, VP24 could be immunoprecipitated with NP and VP35,52 but VP24 did not co-sediment in density gradients with NP and VP35, suggesting that, while it promotes nucleocapsid formation, VP24 may not be an essential structural component of the nucleocapsid.52 Separately, VP24 was not required for either of the formation or the infectivity of EBOV-like particles carrying an EBOV minigenome.78 More recently, the use of siRNAs targeting MBGV VP24 supports a role for this protein in filovirus assembly and release from infected Vero cells.79 As noted above, EBOV-infected human umbilical vein endothelial cells fail to respond to IFNα or IFNγ .47 However, expression of VP35 was unable to block IFN signaling, suggesting that EBOV encodes different protein(s) to inhibit this arm of the IFN response.54 EBOV proteins were therefore screened for the ability to block IFNβ-induced transcription of a reporter gene, and the VP24 protein repressed this gene expression.73 Additionally, cells expressing VP24 did not become resistant to infection by a GFP-expressing Newcastle disease virus (NDV-GFP) following IFNβ treatment, indicating that VP24 can block the antiviral effects of IFN.73 As VP24 inhibited cellular transcriptional responses not only to IFNα /β but also to IFNγ , it was logical to expect that the cellular target of VP24 would be a factor shared by the IFNα /β and IFNγ signaling pathways. Thus, the impact of VP24 upon STAT1 was assessed. Interestingly, VP24 did not prevent the IFNβ - or IFNγ -induced tyrosine phosphorylation of STAT1, nor did VP24 substantially alter levels of VP24 within cells. Rather, despite the presence of tyrosine-phosphorylated STAT1 (PY-STAT1), STAT1 failed to accumulate in the nucleus. A similar block was seen in EBOV-infected cells. STAT1 became tyrosine phosphorylated in IFN-treated, EBOV-infected Vero cells; however, as was seen in transfection experiments, STAT1 failed to accumulate in the nucleus72 These data thus demonstrates the relevance of this function for viral infection.

6.1 VP24 Interacts with Karyopherin α Proteins An explanation for this block to STAT1 is the ability of VP24 to interact with a subset of human karyopherin α proteins (Figure 3). Nuclear translocation of proteins above a certain size (∼ 50 kDa) usually requires

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A

IFN-induced Gene Transcription

PY STAT1 STAT1 PY Kα1 Impβ

nucleus

cytoplasm

B

PY STAT1 STAT1 STAT1 PY

VP24

IFN-induced Gene Transcription

nucleus

Kα1 Impβ

cytoplasm

Figure 3. Model of EBOV VP24 Inhibition of STAT1 Nuclear Accumulation. Tyrosine phosphorylation of STAT1 by Jak family kinases results in dimerization of STAT1 with itself or other STAT proteins. In the case of STAT1-STAT1 homodimers, as activated by IFNγ (depicted here) or STAT1-STAT2 heterodimers as activated by IFNα /β, STAT1 nuclear accumulation is mediated by karyopherin α 1 (Kα 1). VP24 binds to Kα 1, preventing STAT1 from interacting with Kα 1. This results in the failure of STAT1 to enter the nucleus and activate gene expression.

active transport through the nuclear pore, and this transport is mediated by nuclear localization signals (NLSs).80 Many NLSs mediate binding of cargo proteins to the karyopherin α /β heterodimer (reviewed in Ref. 80). Karyopherin α functions as an adaptor by binding both the NLS and karyopherin β , which in turn mediates docking of the trimeric complex to the nuclear pore. The complex is subsequently translocated into the nucleus.82,83 In humans, there are six karyopherin α s which can be assigned, based on relative sequence similarity, into three subfamilies:84 the Rch1 subfamily (karyopherin α 2),85,86 the Qip1 subfamily (karyopherin α 3 and karyopherin α 4),84,87–89 and the NPI-1 subfamily (karyopherin α1, karyopherin

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α 5 and karyopherin α 6).84,90–93 Within the NPI-1 subfamily, karyopherin α1, α 5 and α 6 share greater than 80% sequence identity.94 Upon IFNα /β or IFNγ signaling, the STAT1:STAT2 heterodimer or the STAT1:STAT1 homodimer has been shown to interact with a specific member of the karyopherin α (also known as importin α ) family of nuclear localization signal receptors, karyopherin α1 (importin α 5).94–96 This interaction with karyopherin α 1 mediates the nuclear accumulation of these STAT1-containing complexes.94–96 The consequence of the activation and nuclear accumulation of these complexes is the specific transcriptional regulation of numerous genes, some of which have antiviral properties.97 Initially, VP24 was found to co-immunoprecipitate with karyopherin α1, but not with karyopherins α 2, α 3, or α 4.73 This observation correlated with earlier reports that STAT1 nuclear import is mediated specifically by karyopherin α 1. In fact, in cells treated with IFNβ , VP24 prevented the association of karyopherin α 1 with PY-STAT1, an observation which seems to explain the ability of VP24 to prevent STAT1 nuclear import.98 Due to the homology among the NPI-1 karyopherin α subfamily members, the ability of both activated STAT1 and VP24 to interact with these karyopherin α s were also assessed.74 By co-immunoprecipitation, PYSTAT1 interacted not only with karyopherin α 1, but also with karyopherins α 5 and α 6. This capacity to interact with all three members of the NPI-1 subfamily of karyopherin α s was mirrored by the interaction of VP24 with the same subset of karyopherin α s.74 As was seen with karyopherin α 1, VP24 was able to prevent interaction of karyopherin α 5 and α 6 with PY-STAT1.74 Mapping of the regions of karyopherin α 1 required for interaction with VP24 led to the conclusion that VP24 and STAT1 bind to overlapping sites on karyopherin α 1, and in vitro binding experiments suggest that VP24 can directly compete with PY-STAT1 for binding to karyopherin α 1.74 However, it remains to be determined whether VP24 blocks IFN signaling simply by acting as a competitive inhibitor of STAT1-karyopherin α 1 interaction, or whether VP24 may also influence the trafficking of the NPI-1 subfamily of karyopherin α proteins. If the latter is true, then the impact of VP24 upon cellular processes would presumably be broader than would be the case if VP24 only affects binding of STAT1 to karyopherin α 1, α 5 and α 6.

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6.2 VP24 as a Determinant of Host-specific Virulence Changes in VP24 have been associated with adaptation of ZEBOV from non-lethal to lethal in mouse and guinea pig models.72,99 At least in the mouse system, these changes appear to influence virus sensitivity to IFNs. When EBOV was adapted to mice, nine nucleotide changes occurred, relative to the parental virus used to initiate the adaptation process.72 Five of these nucleotide changes caused amino acid changes. There were single changes in the NP, VP35 and VP24 proteins, and two in the L protein. Studies employing recombinant EBOVs engineered to contain different combinations of these changes demonstrated that two of the amino acid changes, one in NP and one in VP24, were particularly critical for virulence. Among the different recombinant EBOVs examined, the original mouse-adapted virus exhibited the greatest virulence.72 However, the two changes in the NP and VP24 proteins, combined, were the minimum set of changes required to confer upon the virus a mouse-lethal phenotype.72 Virulence in this system also correlated with the replication capacity of these viruses in the mouse macrophage cell line RAW 264.7, and in the sensitivity of virus replication in these cells to exogenously added IFNα /β. 72 It is tempting to speculate, given these observations, that the mouseadaptive change in VP24 resulted in an altered capacity of the virus to block IFN-induced STAT1 activation in infected cells. Therefore, the ability of the non-adapted and mouse-adapted ZEBOV VP24 to interact with human karyopherin α proteins were compared. In co-immunoprecipitation studies, the non-adapted ZEBOV and mouse-adapted ZEBOV VP24 co-immunoprecipitated with human karyopherin α1, α 5 and α 6 to similar extents.74 Similarly, the non-adapted ZEBOV and mouse-adapted ZEBOV VP24 did not differ in their capacity to co-immunoprecipitate with mouse karyopherin α s (unpublished observation). In reporter gene assays, nonadapted ZEBOV and MA ZEBOV VP24 inhibited IFNβ -induced reporter gene activation to similar extents in two different mouse cell lines and in human cells.74 Therefore, it is presently unclear to what extent mouseadaptation is related to the ability of VP24 to modulate STAT1 function. It should be recognized, however, that VP24 and NP interact, thus it is possible that NP may modulate VP24 function. Alternatively, it may be

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that in EBOV-infected cells, the mouse-adaptive mutations influence VP24 IFN-antagonist function by influencing VP24 expression or VP24 stability. Future studies will be required to address these possibilities.

7. EBOV GLYCOPROTEIN (GP) MODULATES HOST CELL RESPONSES The mature EBOV glycoprotein (GP) is a homotrimer that serves as the viral attachment protein and the viral fusion protein, mediating entry of the virus into host cells.100 When expressed in mammalian cells, GP has several notable effects, including modulation of surface protein levels,101–103 induction of anoikis,104,105 and stimulation of cytokine production from human macrophages103 and dendritic cells.106,107 Further, its vascular cytotoxicity has been proposed to play an important role in virulence,108 and it may possess immune suppressive properties,109 although the relevance of these functions to viral pathogenesis remains to be demonstrated. The cell rounding and detachment phenotype of GP is closely tied to GP cytotoxicity, and has also been tied to changes in cellular MAP kinase signaling. Expression of GP in 293 cells was found to reduce the amount of phosphorylated ERK1 and ERK2 MAP kinases, whereas a GP deleted of the mucin domain, a region of the protein modified with O-linked glycans, (GP∆muc) did not reduce phospho-ERK1 or -ERK2 levels.110 This inhibitory effect was more pronounced for ERK2 and correlated with the ability of GP to induce cell rounding and cell death in a mucin domain-dependent manner.110 As further evidence that inhibition of ERK2 phosphorylation contributes to GP-dependent cytotoxicity, knockdown of ERK2 with siRNAs or expression of a dominant negative form of ERK1/2 enhanced the GP-induced downregulation of α V integrin, whereas expression of a constitutively-activated ERK2 preserved α V integrin expression in GP-expressing cells.110 These data directly link the ability of GP to induce cell detachment and cytotoxicity with its ability to affect MAP kinase signaling. Although it remains to be determined how GP mediates the downregulation of ERK activity, as ERK1 and ERK2 phosphorylate numerous targets, there is expected to be a significant impact of GP-expression on numerous cellular pathways.110

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GP is also required for Ebola virus-like particles to activate NF-κ B and MAP kinase signaling. Macrophages and DCs are early targets of EBOV infection in vivo,10 and they are likely to play a prominent role in EBOV pathogenesis.9 Due to the importance of macrophages and DCs for EBOV infection, it is striking that EBOV virus-like particles (VLPs) trigger the activation of these cell types, inducing production of proinflammatory cytokines and in the case of DCs, triggering the upregulation of co-stimulatory molecules and promoting the stimulation by the DCs of T cell responses.19,103,107,111 EBOV virus-like particles (VLPs) are readily produced by expression of the viral matrix protein VP40 in mammalian cells.112 When GP is co-expressed with VP40, GP becomes incorporated into the VLP membrane. The stimulatory activity of the VLPs toward DCs was shown to involve activation of NF-κ B, and blocking the activity of NF-κ B was sufficient to block VLP-induced cytokine production.113 Interestingly, although expression of GP in cells inhibit ERK2 activation,114 addition of VLPs with wild-type GP to DCs transiently activated ERK1/2 phosphorylation.113 How EBOV VLPs are recognized by macrophages and DCs to trigger their activation remains to be determined. However, the activating property of EBOV VLPs was largely ablated by deletion of the mucin domain (GP∆muc). Despite the fact that VLPs containing GP∆muc associated with DCs to the same extent as VLPs bearing wild-type GP, the GP∆muc VLPs failed to activate the DCs.113 What remains unclear is whether any of the signals induced by VLPs play a significant role during the infection of DCs or macrophages by replication-competent filoviruses.

8. CONCLUSIONS Filoviruses cause profound pathology associated with overwhelming inflammatory responses and immune suppression. Active virus replication is likely required to elicit these host responses. The modulation of cellular IFN pathways likely allow the virus to gain a foothold and establish the level of replication that causes disease. The capacity of GP expression to cause toxicity and modify host responses, combined with excessive host responses, may also contribute to the severity of filovirus disease. However, many of the molecular details as to how these viral proteins

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interact with host signaling pathways remain to be defined. Understanding these interactions should facilitate the development of therapies designed to target these functions.

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78. Watanabe S, Watanabe T, Noda T, Takada A, Feldmann H, Jasenosky LD, Kawaoka Y (2004). J Virol 78, 999–1005. 79. Bamberg S, Kolesnikova L, Moller P, Klenk HD, Becker S (2005). J Virol 79, 13421–13433. 80. Macara IG (2001). Microbiol Mol Biol Rev 65, 570–594, table of contents. 81. Stewart M (2007). Nat Rev Mol Cell Biol 8, 195–208. 82. Gorlich D, Henklein P, Laskey RA, Hartmann E (1996). Embo J 15, 1810–1817. 83. Weis K, Ryder U, Lamond AI (1996). Embo J 15, 1818–1825. 84. Kohler M, Ansieau S, Prehn S, Leutz A, Haller H, Hartmann E (1997). FEBS Lett 417, 104–108. 85. Cuomo CA, Kirch SA, Gyuris J, Brent R, Oettinger MA (1994). Proc Natl Acad Sci USA 91, 6156–6160. 86. Weis K, Mattaj IW, Lamond AI (1995). Science 268, 1049–1053. 87. Miyamoto Y, Imamoto N, Sekimoto T, Tachibana T, Seki T, Tada S, Enomoto T, Yoneda Y (1997). J Biol Chem 272, 26375–26381. 88. Nachury MV, Ryder UW, Lamond AI, Weis K (1998). Proc Natl Acad Sci USA 95, 582–587. 89. Seki T, Tada S, Katada T, Enomoto T (1997). Biochem Biophys Res Commun 234, 48–53. 90. Cortes P, Ye ZS, Baltimore D (1994). Proc Natl Acad Sci USA 91, 7633–7637. 91. Kohler M, Speck C, Christiansen M, Bischoff FR, Prehn S, Haller H, Gorlich D, Hartmann E (1999). Mol Cell Biol 19, 7782–7791. 92. Moroianu J, Blobel G, Radu A (1995). Proc Natl Acad Sci USA 92, 2008–2011. 93. O’Neill RE, Palese P (1995). Virology 206, 116–125. 94. Melen K, Fagerlund R, Franke J, Kohler M, Kinnunen L, Julkunen I (2003). J Biol Chem 278, 28193–28200. 95. Sekimoto T, Imamoto N, Nakajima K, Hirano T, Yoneda Y (1997). Embo J 16, 7067–7077. 96. McBride KM, Banninger G, McDonald C, Reich NC (2002). Embo J 21, 1754–1763. 97. Levy DE, Garcia-Sastre A (2001). Cytokine Growth Factor Rev 12, 143–156. 98. MMWR Morb Mortal Wkly Rep 54 (2005) 308–309.

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99. Volchkov VE, Chepurnov AA, Volchkova VA, Ternovoj VA, Klenk HD (2000). Virology 277, 147–155. 100. Feldmann H, Volchkov VE, Volchkova VA, Stroher U, Klenk HD (2001). J Gen Virol 82, 2839–2848. 101. Takada A, Watanabe S, Ito H, Okazaki K, Kida H, Kawaoka Y (2000). Virology 278, 20–26. 102. Simmons G, Wool-Lewis RJ, Baribaud F, Netter RC, Bates P (2002). J Virol 76, 2518–2528. 103. Wahl-Jensen V, Kurz SK, Hazelton PR, Schnittler HJ, Stroher U, Burton DR, Feldmann H (2005). J Virol 79, 2413–2419. 104. Alazard-Dany N, Ottmann Terrangle M, Volchkov V (2006). Med Sci (Paris) 22, 405–410. 105. Ray RB, Basu A, Steele R, Beyene A, McHowat J, Meyer K, Ghosh AK, Ray R (2004). Virology 321, 181–188. 106. Bosio CM, Moore BD, Warfield KL, Ruthel G, Mohamadzadeh M, Aman MJ, Bavari S (2004). Virology 326, 280–287. 107. Ye L, Lin J, Sun Y, Bennouna S, Lo M, Wu Q, Bu Z, Pulendran B, Compans RW, Yang C (2006). Virology 351, 260–270. 108. Yang ZY, Duckers HJ, Sullivan NJ, Sanchez A, Nabel EG, Nabel GJ (2000). Nat Med 6, 886–889. 109. Yaddanapudi K, Palacios G, Towner JS, Chen I, Sariol CA, Nichol ST, Lipkin WI (2006). Faseb J 20, 2519–2530. 110. Zampieri CA, Fortin JF, Nolan GP, Nabel GJ (2007). J Virol 81, 1230–1240. 111. Warfield KL, Bosio CM, Welcher BC, Deal EM, Mohamadzadeh M, Schmaljohn A, Aman MJ, Bavari S (2003). Proc Natl Acad Sci USA 100, 15889–15894. 112. Noda T, Sagara H, Suzuki E, Takada A, Kida H, Kawaoka Y (2002). J Virol 76, 4855–4865. 113. Martinez O, Valmas C, Basler CF (2007). Virology 364, 342–354. 114. Sullivan NJ, Peterson M, Yang ZY, Kong WP, Duckers H, Nabel E, Nabel GJ (2005). J Virol 79, 547–553.

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CHAPTER 16

Responses to Ebola and Marburg Virus Infections Thomas W. Geisbert, Friederike Feldmann, Joan Geisbert & Heinz Feldmann

ABSTRACT Marburg and Ebola viruses, from the family Filoviridae, are prototype viral hemorrhagic fever pathogens that cause a fulminant hemorrhagic disease in humans and nonhuman primates. Following unspecific symptoms, patients display increased paraendothelial permeability, hypotension, coagulation disorders, hemorrhages and immune suppression. Disturbances of the blood tissue barrier, primarily controlled by endothelial cells, and immune suppression seem to be the key pathogenic factors of the disease. The endothelium is affected in two ways: directly by virus infection leading to activation and perhaps lytic replication, and indirectly by a mediator-induced inflammatory response. Those mediators originate from virus-activated cells of the mononuclear phagocytic system which are the primary target cells. Immune suppression may result from lytic infection of circulating and sessile cells of the mononuclear phagocytic system, inactivation of neutrophils, impairment of antigen-presenting cells, and lymphoid depletion. Despite being clearly immunosuppressive, there is evidence of protective immunity during filovirus hemorrhagic fever. In contrast to survivors and asymptomatic cases that show humoral responses to viral antigens, fatal infections usually end with high viremia and little evidence of a humoral immune response. The transmembrane glycoprotein can be used to 371

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provoke a protective immune response in animal models including nonhuman primates.

1. INTRODUCTION Filoviruses were first brought to our attention with the discovery of Marburg virus (MARV) in 1967.1 Ebola virus (EBOV), the other member of the Filoviridae family, was discovered nine years later in 1976.2,3 The initial discoveries of these agents were followed by long periods of sporadic episodes of filovirus disease in humans. This ended dramatically when the news media provided extensive coverage of the 1989–1990 outbreak of Reston ebolavirus in quarantined nonhuman primates in the United States,4 and the reemergence of EBOV in Kikwit, Democratic Republic of the Congo (formerly Zaire).5 Since then, several other larger outbreaks have occurred in Central Africa6 including the emergence of MARV in Angola7 and the emergency of a putative new EBOV species in Bundibunyo District of Uganda.8 Today, MARV and EBOV are known worldwide as highly pathogenic agents that can now pose a biothreat to public health.9,10 Approximately 500 cases of Marburg hemorrhagic fever (MHF) and more than 1800 cases of Ebola hemorrhagic fever (EHF) have been reported (Figure 1A). Almost all of these cases are associated with outbreaks in the tropical African ecosystem located between the latitudes of 10° north and 10° south (Figure 1B). So far, the vast majority of outbreaks have been caused by different strains of the species Lake Victoria marburgvirus (genus Marburgvirus), Zaire ebolavirus and Sudan ebolavirus (genus Ebolavirus) with case fatality rates ranging from 70–90%, 60–90% and 50–60%, respectively.6,9,11 The epidemiology of human infections in nature is unknown. However, the time between the occurrence of index cases, the recognition of the subsequent large outbreaks and the addition of possible asymptomatic infections all suggest that sporadic cases of filovirus infections can pass unnoticed. Transmission of the disease generally results from close contact with blood, secretions or tissues from patients or infected animals (e.g., gorillas, chimpanzees). Virus has been detected in biological fluids of convalescent patients until three months after the onset of symptoms.

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Figure 1. Reported viral hemorrhagic fever outbreaks caused by Marburg virus (MARV) and Ebola virus (EBOV), species Sudan ebolavirus, (SEBOV), Zaire ebolavirus (ZEBOV) and Cote d’Ivoire ebolavirus (CIEBOV). (a) Case numbers and fatality rates. The number of cases in each outbreak is indicated above the bars. The colour code indicates the fatality rate. (b) Geographic distribution of outbreaks/cases. Reported outbreaks of hemorrhagic fever caused by MARV (blue dots) and EBOV (red dots) are indicated with the corresponding year. Countries affected by the outbreaks are specifically named and the borders are highlighted in bold. The natural vegetation zones from tropical rain forest (dark green) to semi desert (brown opaque) are presented in different underlying colours. Note the monkeys that initiated the original 1967 MARV outbreak in Europe were shipped from Entebbe at Lake Victoria. [altered from Ref. 13]

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(Continued) Figure 1.

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Nosocomial infections are reported from some of the outbreaks and are mainly the result of low hygiene standards in local hospitals with frequent reuse of contaminated needles and syringes. Transmission through mucosal exposure or via a person-to-person airborne route may occur in single cases but does not appear to be a major contributing mechanism for outbreaks since all epidemics to date have been successfully controlled using isolation techniques without specific airborne precautions.6,9 The natural reservoir(s) of filoviruses have remained elusive since their initial discovery.12,13 However, a recent survey of small vertebrate animals collected during EHF outbreaks in 2001 and 2003 in Gabon and the Republic of Congo suggests that asymptomatic infection may exist in three species of fruit bats.14,15 This supports earlier experimental data demonstrating replication of EBOV in bats.16 Similarly to EBOV, some African bat species have recently been found to be positive for MARV, suggesting bats could play a role as a natural reservoir for both viruses.17 Further laboratory and ecological investigations will be required to determine the relevance of these findings.18

2. HOST RESPONSE TO INFECTION 2.1 Clinical Presentation In general, filovirus infections are considered the most severe of the viral hemorrhagic fevers (VHFs) (Figure 2). After an incubation period of 2–21 days, there is an abrupt onset averaging 4–10 days that is characterized by flu-like symptoms (fever, chills, malaise, and myalgia). The subsequent signs and symptoms indicate multisystem involvement and include systemic (prostration), gastrointestinal (nausea, vomiting, abdominal pain, diarrhea), respiratory (chest pain, shortness of breath, cough), vascular (conjunctival injection, postural hypotension, edema), and neurologic (headache, confusion, coma) manifestations. Hemorrhagic manifestations may develop during the peak of the illness and include petechiae, ecchymoses, uncontrolled bleeding from venipuncture sites, mucosal hemorrhages, and postmortem evidence of visceral hemorrhagic effusions. There is often a macropapular rash associated with varying degrees of erythema and desquamation. In later stages of the disease,

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DIAGNOSTIC WINDOW

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Figure 2. Appearance of clinical symptoms and diagnostic window. The bottom part of the graph shows the average time frame for the appearance of clinical symptoms. The upper part shows the preferred time frame for specimen collection. The uninterrupted lines indicate the period in which clinical samples are most likely to be tested positive. The interrupted lines indicate periods in which diagnostic test might become positive but are not reliable if negative. The photo shows the classical rash occurring in infected nonhuman primates (Cynomolgus macaque infected with Zaire ebolavirus).

shock, convulsions, severe metabolic disturbances, and, in more than half of the cases, diffuse coagulopathy supervene (Figure 2) occurs.6,9,12,19,20 Fatal cases develop clinical signs early during infection and demise typically occurs between days 7–16 mainly due to the consequences of a hypovolemic shock. Non-fatal cases have fever for about 5–9 days and improvement typically occurs around days 7–11, about the time the humoral antibody response is noted (Figure 2). Convalescence is prolonged and sometimes associated with myelitis, recurrent hepatitis, psychosis or uveitis. There is an increased risk of abortion for pregnant women, and clinical observations indicate a high death rate for children of infected mothers.6,9,12,19,20

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2.2 Pathogenesis The main entry mechanism for filoviruses seems to be via small skin lesions and mucus membranes.21 In vivo, virus was shown to spread from initial infection sites by monocytes and dendritic cells to regional lymph nodes, most probably via lymphatics, to the liver and spleen through the blood and subsequently, to EBOV-infected resident macrophages, dendritic cells and fibroblastic reticular cells. Mononuclear phagocytic cells located in multiple organs including the liver (Kupffer cells), spleen, lymph nodes, lung (alveolar macrophages), serous cavities (pleural and peritoneal macrophages) and nervous system (microglia) were infected. However, the lymph nodes, liver and spleen seem to be the organs consistently affected the most by virus replication (Figure 3).22,23

2.2.1 Impairment of immune system A central role for the innate immune system in filovirus infections has been demonstrated by many findings (Figure 3). In vivo, inflammatory responses, accompanied by substantial cytokine production, can be detected as a result of infection. Monocytes/macrophages and dendritic cells (DCs) have been shown to be early and sustained targets of filoviruses in vivo and their infection might contribute to immune suppression.22,24–26 For EHF, studies have correlated increased levels of IL-10, neopterin and IL-1 receptor A (IL-1RA) with fatal outcome, whilst the presence of IL-1β and elevated concentrations of IL-6 in the plasma during the symptomatic phase have been indicated as markers of non-fatal infections (Figure 3).27–30 In vitro, EBOV has been shown to selectively suppress the production of interferon (IFN)-α and IFN-γ as well as the production of IFN-α in response to double-stranded RNA.31–35 Two EBOV proteins have been described to interfere with the IFN-response. Virion protein (VP) 35 blocks phosphorylation of the IFN regulatory factor (IRF)-3, which acts as transcription factor for IFN production,36,37 while VP24 has been suggested to block IFN signalling by binding to karyopherin alpha 1 and blocking STAT1 nuclear accumulation.38,39 The role of IFN-antagonism in vivo has not yet been extensively analyzed.

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Figure 3. Pathogenesis model. Primary target cells for MARV and EBOV are monocytes/macrophages (M∅) and monocyte-derived dendritic cells (DC) cells. Infected M∅ become activated independently of virus replication, produce virus progeny, and spread the virus through extravasation into tissues. The production of cytokines by M∅ either promotes or inhibits the immune response. Proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the activation of endothelial cells (EC) and increase vascular leakage. In contrast to M∅, infected DC are rather impaired in their function but they propagate the virus. Both infected M∅ and DC trigger apoptosis of cytotoxic T cells (Tc). Key: DIC: disseminated intravascular coagulopathy; IL: interleukin; NO: nitric oxide; RA: receptor antagonist; dashed line: fatal infection; solid line: non-fatal infection [altered from Ref. 63].

However, treatment of nonhuman primates with high doses of IFN had only very minor benefits.40 It has been noted earlier that monocytes, macrophages and DCs are the most important early target cells during infection with EBOV (Figure 3). However, the extent of the infection of monocytes and macrophages that impairs the function of these cells has not been extensively studied.

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In contrast, for DCs, which play a crucial role for both innate and adaptive immunity, it has been clearly shown in vitro that after infection with filoviruses, they fail to produce proinflammatory cytokines or express co-stimulatory molecules such as CD80 or CD86 and are impaired in their ability to support T cell proliferation and undergo anomalous maturation.41,42 Since non-infectious virus-like particles (VLPs) are able to elicit these responses, it appears that infectious virus actively interferes with the function of DCs.43 Further, EBOV is able to induce the proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DCs both in vitro and in vivo. However, there is no evidence of apoptosis in EBOVinfected DCs in vivo.42,44 One other class of innate immune cells affected by EBOV infection are natural killer (NK) cells. Although they do not seem to be infected, their numbers drop dramatically during in vivo infection, most likely due to apoptosis.27,45–47 The role of adaptive immunity in filovirus infection is more difficult to assess. Animal models are less helpful because of their relatively fast fatal disease progression with virtually no adaptive immune response. To date, the limited data available from human infections show profound differences in the adaptive immune responses of fatal and non-fatal cases.27,47 Survivors show specific IgM antibodies as early as two days and IgG antibodies six to eight days after the onset of symptoms (Figure 2). In contrast, in fatal cases even low levels of specific IgM are detected in only 30% of patients whereas specific IgG is never detected.27,48 One common observation is the decrease in T cell numbers in fatal cases prior to death.27,47 In accordance with the data obtained in human patients, a strong depletion of both CD4+ and CD8+ lymphocytes, as well as plasma cells, can be found in nonhuman primates. It is interesting to note that, similar to NK cells, lymphocytes are not infected, but undergo “bystander apoptosis” (Figure 3).45,46 Currently it is believed that an early inflammatory response as well as differences in the adaptive immune response are factors that determine disease outcome.

2.2.2 Impairment of the vascular system During filovirus infection the endothelium appears to be affected in two ways: directly by virus replication, leading to activation and eventual cytopathogenic replication, and indirectly by a mediator-induced inflammatory

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response (Figure 3).9 Endothelial dysfunction can lead to a multitude of vascular effects that may cause disturbances in vascular permeability, coagulation disorders, dysregulation of vascular tone and/or hemorrhage. In vitro, data has shown that virus-induced cytokine release leads to the activation of the endothelium that can be defined as an increased expression and/or release of adhesion molecules on endothelial cells including intravascular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E- and P-selectin as well as a breakdown of the endothelial barrier function.49 Recent in vivo experiments confirmed that EBOV infection primarily affects the function rather than the structure of endothelial cells, and that EBOV-induced coagulopathy results primarily from vascular disruption induced by factors secreted from infected monocytes/macrophages and dendritic cells including cytokines and tissue factor (Figure 3).23 While the molecular mechanisms for the breakdown of endothelial barrier function are not completely understood, there is evidence of changes in the protein organization of the endothelial cell junctions, particularly the VE-cadherin/catenin complex.49 Activation and subsequent endothelial cell gap formation will promote leukocyte recruitment and transmigration, including primary target cells and thus, might support virus spread in the infected host (Figure 3). Surprisingly, in vivo data obtained from EBOV-infected animals described a lack of leukocyte infiltration in areas of focal necrosis, thereby suggesting a deficient immunoreaction of unknown etiology.25 The phenomenon might be explained by recent data showing that the soluble glycoprotein sGP, the primary product of the EBOV glycoprotein gene, might have a protective effect on the endothelium and counteract the initial activation of the endothelium.50 During filovirus infections, disseminated intravascular coagulation (DIC) can be regularly observed in nonhuman primates and has also been described in humans.51,52 Recent in vivo data indicated that the coagulation abnormalities might be triggered by tissue factor release from infected mononuclear phagocytic cells rather than the destruction of the endothelium itself (Figure 3).23,53 Treatment of nonhuman primates with recombinant nematode anticoagulant protein c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood coagulation, resulted in 30% protection of lethally infected nonhuman primates.54

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3. MEDICAL RESPONSE TO INFECTION Despite all the achievements in laboratory diagnostics in the past decades, it should be kept in mind that the diagnosis of filovirus infections initially has to be based on clinical assessment (Figure 2). For this purpose, contingency plans should be developed that are still missing in many, particularly developing countries. As clinical microbiology and public health laboratories are not generally equipped for diagnosis of VHF, particularly MHF and EHF, it is necessary that samples are sent to national and/or international reference laboratories capable of performing the required assays. Outbreaks often occur in remote sites where sophisticated medical support systems are limited and timely diagnostic services are extremely difficult to provide. In the past, isolation of patients and use of strict barrier nursing procedures, including protective clothing and respirators, have been instrumental and sufficient to rapidly interrupt transmission in hospital settings. For community members, residual risks are present from deceased people, which should be handled and buried accordingly. Under specific circumstances, the use of high-efficiency particulate air (HEPA) filter respirators for protection against aerosols might be indicated.55 One of the important elements is the provision of sterile equipment for injections and personal protection equipment for medical personnel in local hospitals and outpatient clinics, which are remarkably and tragically lacking in Central Africa today. Provision of a fieldable laboratory to provide basic diagnostics for filoviruses, and other agents that may be confounding to the diagnosis, could aid in the management of patients specifically and the outbreak in general.56

3.1 Diagnostics Rapid and reliable laboratory diagnosis becomes a key element in the response. Laboratory diagnosis of filovirus infections is primarily achieved through the detection of viral particle components in infected individuals. Today, reverse transcriptase-polymerase chain reaction (RTPCR) and antigen detection “Enzyme-linked Immunosorbent Assay” (ELISA) are the primary assays of choice to diagnose an acute infection (Figure 2). Due to relatively high viremia levels in infected individuals, electron microscopy has been useful in the diagnosis of filovirus infections.

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Less frequently, immunohistochemistry (IHC) on formalin-fixed material and paraffin-embedded tissues, as well as immunofluorescence (IF) on impression smears of tissues are used.6,57,58 An alternative approach is by measuring the host-specific humoral immune response (Figure 2). The most commonly used assays for antibody detection are direct IgG/IgM ELISAs and IgM capture ELISA. Confirmatory tests include western blot and the indirect immunofluorescence assay (IFA). Furthermore, virus isolation in tissue culture and susceptible animals from serum or other clinical material should be attempted if a biocontainment level 4 (BSL4) facility is available.6,57,58 Noteworthy, there are no commercially available diagnostic kits for the detection of MARV and EBOV infections and diagnosis seems only reliable if performed by qualified personnel. International quality assurance tests would be advisable to validate the method currently used in the different centers.

3.2 Treatment Currently, supportive therapy is the only therapeutic option and should be directed towards maintenance of effective blood volume and electrolyte balance. Shock, cerebral edema, renal failure, coagulation disorders, and secondary bacterial infection have to be managed as these treatment may be life-saving. Given its benefit for some arenavirus and bunyavirus infections which can cause a similar VHF, ribavirin may initially be indicated but should be discontinued after a confirmed filovirus diagnosis, due to a lack of proven efficacy against MARV and EBOV.59,60 Research over the past decade has identified new targets for potential therapeutic intervention. However, no single treatment is likely to be sufficiently potent to offset the severe and rapid progression of filoviral disease. However, slowing disease progression may provide enough time for the adaptive immune response to develop enough momentum to overcome the infection. In the following we discuss the most promising options. For a more detailed review the readers are referred to previously published review articles.61–63 Although no definite therapeutic conclusion can be drawn from the current studies,64–69 data indicate a principle value of passively acquired

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antibodies in reducing the viral burden during infection, and if used in combination with other pharmaceutical agents, they might be beneficial. Coagulation abnormalities are a hallmark of filovirus infections and considered a key factor in pathogenesis. Recently a breakthrough was achieved in the treatment of EBOV-infected nonhuman primates using a nematodederived anticoagulation protein (rNAPc2) which primarily targets signaling through the extrinsic blood coagulation pathway.54 Additional benefits might be gained by using inhibitors of Factor X, targeting the common pathway thereby blocking signaling through both the extrinsic and intrinsic blood coagulation pathways. It might also be beneficial to counteract the rapid decrease of plasma protein C during EBOV infection53 which might be a critical component to the observed coagulation dysfunction in filovirus VHF. Efforts are on the way to investigate the effect of interfering with host responses during infection. Increased plasma levels of TNF-α have been shown in EBOV-infected humans and nonhuman primates,27,28 indicating a potential benefit from anti-TNF therapy. Neutralizing antibodies to TNF-α have been partially successful in rodent models of MHF,60,70 but have not been evaluated in nonhuman primates. Attempts to inhibit apoptosis of lymphocytes during infection and to modulate the dysregulated cytokine/chemokine response are currently being investigated. As mentioned earlier, filoviruses seem to be resistant to the antiviral effects of type I interferon;36–39 but despite varying success in rodent models, treatment with exogenous IFN-α did not show much effect in nonhuman primates.40,70 Similar results were found with treatment of a S-adenosylhomocysteine hydrolase inhibitor which has partially been associated with a mechanistically unexplained strong increase of IFN-α production.61

3.3 Prophylaxis In the past there have been discussions about the usefulness of vaccine development for filoviruses given the rare occurrence of outbreaks in restricted rural areas of Central Africa (Figure 1B) and thus, the lack of a market for a commercialized vaccine. Nevertheless, vaccine efforts started early and have been enforced by the biothreat potential of EBOV and MARV. The efforts resulted in several promising experimental vaccines,

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which are discussed below, that show cross-protection within but not across filovirus species. Today, protective filovirus vaccines would not only be favored for at-risk medical personnel, first responders, military personnel and researchers, but for targeted vaccination in effected populations as well. Inactivated whole virus preparations, as well as to be generated attenuated MARV and EBOV strains are unlikely to be approved as vaccines due to the uncertainty of complete inactivation and potential occurrence of revertants.9 Thus, more recent efforts concentrate on the use of subunit vaccines based on single or a combination of viral structural proteins to induce protective immunity using naked DNA, adenovirus, vaccinia virus, Venezuelan equine encephalitis virus (VEEV) replicons and vesicular stomatitis viruses (VSV) as delivery mechanisms. Most of these vaccines showed protective efficacy in rodent models (mouse and guinea pig) but failed to protect nonhuman primates.71 The first successful strategy to protect nonhuman primates from lethal filovirus challenge used a VEEV replicon system expressing the MARV glycoprotein,72 an approach that was unsuccessful for EBOV.9,71 Later, a DNA prime (glycoprotein and nucleoprotein) adenovirus boost (glycoprotein) approach was successfully used to protect cynomolgus macaques against lethal EBOV challenge.73 This approach could be accelerated by a single immunization with only the recombinant adenovirus expressing the EBOV glycoprotein.74 From these studies, it seems that antibody and T-helper cell memory are essential for protection, and that cell-mediated immunity whilst possibly important, is not an absolute requirement. Subsequently, VSV vectors expressing the EBOV and MARV glycoproteins are developed as live attenuated vaccine candidates. These vectors seem to be able to more potently stimulate the innate and adaptive (humoral and cellular) immune responses and completely protected nonhuman primates against lethal MARV and EBOV challenge.75 A unique and remarkable ability of the VSV-based vaccine is the rapidity by which immunization induces protection. Nonhuman primates can survive a lethal challenge when vaccinated 30 minutes after exposure to EBOV76 and MARV.77 Cross-protection was also demonstrated with the VSV-MARV vaccine in nonhuman primates against a closely related strain of MARV. However, cross-protection against EBOV was not achieved following immunization with the VSV-MARV vaccine.78 Despite these promising

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results, the safety of the VSV live attenuated vaccine vectors needs further investigation. The vectors have been safe in four animal species: mice, guinea pigs, goats and nonhuman primates (own unpublished data). More recently, another recombinant replication-competent vaccine based on human parainfluenza virus 3 was able to induce mucosal and systemic immunity, and protect guinea pigs and nonhuman primates from lethal EBOV challenge.79,80 This approach seems promising but as with human adenovirus-based vectors, it might have the limitation of pre-existing immunity against the vector in the human population.81 Currently, researchers investigate virus-like particles (VLPs), which are generated by the transfection of the viral glycoprotein and nucleoprotein, as vaccine candidates.82 VLPs may provide an interesting alternate delivery system for a protective cellular and humoral host immune response that may also better address the safety limitations associated with the live attenuated vaccine vectors.

4. CONCLUSION Basic research on filovirus biology and pathogenesis has advanced over the last decade, and has led to the development of potential treatment strategies and vaccine candidates. However, no specific therapy or prophylaxis is currently licensed for EHF or MHF. Regulatory authorities need to consider the progress in experimental treatment options to define if treatment schemes could be offered in the future. Nevertheless, with the reservoir still unknown, a lack of clinical information on human infections, and an incomplete understanding of the immunologic response to infection, there is still much room for improvement. A major drawback is the biocontainment needed for in vitro and in vivo work with the infectious agents. Building new biocontainment level 4 (BSL4) facilities, as are currently being done, is one way to respond and political support is also mostly guaranteed in crisis situations. But maintaining facilities, longterm funding and most importantly, establishment of a comfort level of welltrained personnel are critical issues that have to be addressed in the future.

ACKNOWLEDGMENTS Work on filoviruses in Winnipeg was funded by Health Canada, Public Health Agency of Canada, Canadian Institutes of Health Research (MOP-49321),

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Chemical Radiological-Nuclear, and Explosives (CBRNE) Research and Technology Initiative (CRTI) and Deutsche Forschungsgemeinschaft (DFG). Studies on filovirus at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) was supported by the Medical Chemical/Biological Defense Research Program and Military Infectious Diseases Research Program, US Army Medical Research and Material Command (project numbers 02-4-4J-081, 03-4-7J-020, and 04-4-7J-012). Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army.

REFERENCES 1. Siegert R, Shu HL, Slenczka W, Peters D, Mueller G (1967). Dtsch Med Wochenschr 92, 2343–2370. 2. World Health Organization (1978). Bull WHO 56, 247–270. 3. World Health Organization (1978). Bull WHO 56, 271–290. 4. Jahrling PB, Geisbert TW, Dalgard DW, Johnson ED, Ksiazek TG, Hall WC, Peters CJ (1990). Lancet 335, 502–505. 5. World Health Organization (1995). Wkly Epidemiol Rec 70, 241–242. 6. Sanchez A, Geisbert TW, Feldmann H (2007). In Fields Virology (eds.) Knipe DM, Howley PM, Griffin DE, Lamb A, Martin MA, Roizman B, Strauss SE. Lippincott, Williams and Wilkins, Philadelphia, pp. 1409–1448. 7. Jeffs B, Roddy P, Weatherill D, de la Rosa O, Dorion C, Iscla M,Grovas I, Pablo Palma P, Villa L, Bernal O, et al. (2007) J Infect Dis 196(Suppl 2), S154–S161. 8. Alsop Z (2008). Lancet 370, 2085. 9. Feldmann H, Jones S, Klenk HD, Schnittler HJ (2003). Nat Rev Immunol 3, 677–685. 10. Groseth A, Jones S, Artsob H, Feldmann H (2005). In Bioterrorism and Infectious Agents: A New Dilemma for the 21st Century (eds.) Fong I & Alibek K. Plenum Press, New York, pp. 169–187. 11. Feldmann H, Geisbert TW, Jahrling PB, Klenk HD, Netesov SV, Peters CJ, Sanchez A, Swanepoel R, Volchkov VE (2005). In Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses (eds.) Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Elsevire/Academic Press, London, UK, pp. 645–653.

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12. Peters CJ, Leduc JW (1999). J Infect Dis 179(Suppl 1), ix–xvi. 13. Feldmann H, Wahl-Jensen V, Jones SM, Stroeher U (2004). Trends Microbiol 12, 433–437. 14. Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba P, Délicat A, Paweska JT, Gonzalez JP, Swanepoel R (2005). Nature 438, 575–576. 15. Pourrut X, Delicat A, Rollin PE, Ksiazek TG, Gonzalez JP, Leroy EM (2007). J Infect Dis 196 (Suppl 2), S176–S183. 16. Swanepoel R, Leman PA, Burt FJ, Zachariades NA, Braack LE, Ksiazek TG, Rollin PE, Zaki SR, Peters CJ (1996). Emerg Infect Dis 2, 321–325. 17. Towner JS, Pourrut X, Albariño CG, Nkogue CN, Bird BH, Grard G, Ksiazek TG, Gonzalez JP, Nichol ST, Leroy EM (2007). PloS One 2, e764. 18. Groseth A, Feldmann H, Strong JE (2007). Trends Microbiol 15, 408–416. 19. Martini GA, Siegert R (1971). In Marburg virus disease (eds.) Martini GA & Siegert R. Springer, New York, pp. 1–230. 20. Pattyn SR (1978). In Ebola virus haemorrhagic fever (ed.) Pattyn SR. Elsevier/North-Holland Biomedical Press. 21. Schnittler HJ, Feldmann H (1999). Curr Top Microbiol Immunol 235, 175–204. 22. Geisbert TW, Hensley LE, Larsen T, Young HA, Reed DS, Geisbert JB, Scott DP, Kagan E, Jahrling PB, Davis KJ (2003). Am J Pathol 163, 2347–2370. 23. Geisbert TW, Young HA, Jahrling PB, et al. (2003). Am J Pathol 163, 2371–2382. 24. Geisbert TW, Jahrling PB, Hanes MA, Zack PM (1992). J Comp Pathol 106, 137–152. 25. Ryabchikova E, Kolesnikova LV, Luchko SV (1999). J Infect Dis 179 (Suppl. 1), S199–S202. 26. Zaki SR, Peters CJ (1997). In Diagnostic Pathology of Infectious Diseases (eds.) Connor DH, Schwartz DA. Appleton and Lange, Stamford, CT, pp. 347–364. 27. Baize S, Leroy EM, Georges-Courbot MC, Capron M, Lansoud-Soukate J, Debré P, Fisher-Hoch SP, McCormick JB, Georges AJ (1999). Nat Med 5, 423–426. 28. Villinger F, Rollin PE, Brar SS, Chikkala NF, Winter J, Sundstrom JB, Zaki SR, Swanepoel R, Ansari AA, Peters CJ (1999). J Infect Dis 179 (Suppl 1), S188–S191. 29. Leroy EM, Baize S, Volchkov VE, Fisher-Hoch SP, Georges-Courbot MC, Lansoud-Soukate J, Capron M, Debré P, McCormick JB, Georges AJ (2000). Lancet 355, 2210–2215.

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30. Baize S, Leroy EM, Georges AJ, Georges-Courbot MC, Capron M, Bedjabaga I, Lansoud-Soukate J, Mavoungou E (2002). Clin Exp Immunol 128, 163–168. 31. Harcourt BH, Sanchez A, Offermann MK (1998). Virology 252, 179–188. 32. Harcourt BH, Sanchez A, Offermann M (1999). J Virol 73, 3491–3496. 33. Gupta M, Mahanty S, Ahmed R, Rollin PE (2001). Virology 284, 20–25. 34. Cárdenas WB, Loo YM, Gale M Jr., Hartman AL, Kimberlin CR, MartínezSobrido L, Saphire EO, Basler CF (2006). J Virol 80, 5168–5178. 35. Feng Z, Cerveny M, Yan Z, He B (2007). J Virol 81, 182–192. 36. Basler CF, Wang X, Mühlberger E, Volchkov V, Paragas J, Klenk HD, García-Sastre A, Palese P (2000). Proc Natl Acad Sci USA 97, 12289–12294. 37. Basler CF, Mikulasova A, Martinez-Sobrido L, Paragas J, Mühlberger E, Bray M, Klenk HD, Palese P, García-Sastre A (2003). J Virol 77, 7945–7956. 38. Reid SP, Leung LW, Hartman AL, Martinez O, Shaw ML, Carbonnelle C, Volchkov VE, Nichol ST, Basler CF (2006). J Virol 80, 5156–5167. 39. Reid SP, Valmas C, Martinez O, Sanchez FM, Basler CF (2007). J Virol 81, 13469–13477. 40. Jahrling PB, Geisbert TW, Geisbert JB, Swearengen JR, Bray M, Jaax NK, Huggins JW, LeDuc JW, Peters CJ (1999). J Infect Dis 179(Suppl 1), S224–S234. 41. Bosio CM, Aman MJ, Grogan C, Hogan R, Ruthel G, Negley D, Mohamadzadeh M, Bavari S, Schmaljohn A (2003). J Infect Dis 188, 1630–1638. 42. Mahanty S, Hutchinson K, Agarwal S, McRae M, Rollin PE, Pulendran B (2003). J Immunol 170, 2797–2801. 43. Bosio CM, Moore BD, Warfield KL, Ruthel G, Mohamadzadeh M, Aman MJ, Bavari S (2004). Virology 326, 280–287. 44. Geisbert TW, Hensley LE, Larsen T, Young HA, Reed DS, Geisbert JB, Scott DP, Kagan E, Jahrling PB, Davis KJ (2003). Am J Pathol 163, 2347–2370. 45. Geisbert TW, Hensley LE, Gibb TR, Steele KE, Jaax NK, Jahrling PB (2000). Lab Invest 80, 171–186. 46. Reed DS, Hensley LE, Geisbert JB, Jahrling PB, Geisbert TW (2004). Viral Immunol 17, 390–400 47. Sanchez A, Lukwiya M, Bausch D, Mahanty S, Sanchez AJ, Wagoner KD, Rollin PE (2004). J Virol 78, 10370–10377.

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48. Ksiazek TG, Rollin PE, Williams AJ, Bressler DS, Martin ML, Swanepoel R, Burt FJ, Leman PA, Khan AS, Rowe AK, et al. (1999). J Infect Dis 179 (Suppl 1), S177–S187. 49. Schnittler HJ, Feldmann H (1999). Curr Top Microbiol Immunol 235, 175–204. 50. Wahl-Jensen VM, Afanasieva TA, Seebach J, Ströher U, Feldmann H, Schnittler HJ (2005). J Virol 79, 10442–10450. 51. Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J (1998). J Infect Dis 178, 651–661. 52. Fisher-Hoch SP, Platt GS, Neild GH, Southee T, Baskerville A, Raymond RT, Lloyd G, Simpson DI (1985). J Infect Dis 152, 887–894. 53. Geisbert TW, Young HA, Jahrling PB, Davis KJ, Kagan E, Hensley LE (2003). J Infect Dis 188, 1618–1629. 54. Geisbert TW, Hensley LE, Jahrling PB, Larsen T, Geisbert JB, Paragas J, Young HA, Fredeking TM, Rote WE, Vlasuk GP (2003). Lancet 362, 1953–1958. 55. Dowell SF, Mukunu R, Ksiazek TG, Khan AS, Rollin PE, Peters CJ (1999). J Infec Dis 179 (Suppl 1), S87–S91. 56. Grolla A, Lucht A, Dick D, Strong J, Feldmann H (2005). Bull Soc Pathol Exot 98, 205–209. 57. Ksiazek TG (1991). Lab Animal 20, 34–46. 58. Strong J, Grolla A, Jahrling PB, Feldmann H (2006). In Manual of Clinical Laboratory Immunology (eds.) Detrick B, Hamilton RG, Folds JD. ASM Press, Washington DC, pp. 774–790. 59. Huggins JW (1989). Rev Infect Dis (Suppl 4), S750–S761. 60. Ignatyev G, Steinkasserer A, Streltsova M, Atrasheuskaya A, Agafonov A, Lubitz W (2000). J Biotechnol 83, 67–76. 61. Bray M, Paragas J (2002). Antiviral Res 54, 1–17. 62. Feldmann H, Jones SM, Schnittler HJ, Geisbert T (2005). Curr Opin Investig Drugs 6, 823–830. 63. Stroher U, Feldmann H (2006). Expert Opin Investig Drugs 15, 1523–1535. 64. Mupapa KD, Massamba M, Kibadi K (1999). J Infect Dis 179(Suppl 1), S18–S23. 65. Maruyama T, Parren PW, Sanchez A, Rensink I, Rodriguez LL, Khan AS, Peters CJ, Burton DR (1999). J Infect Dis 179(Suppl 1), S235–S239. 66. Wilson JA, Hevey M, Bakken R, Guest S, Bray M, Schmaljohn AL, Hart MK (2000). Science 287, 1664–1666.

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67. Parren PW, Geisbert TW, Maruyama T, Jahrling PB, Burton DR (2002). J Virol 76, 6408–6412. 68. Mikhailov VV, Borisevich IV, Chernikova NK, Potryvaeva NV, Krasnyanskii VP (1994). Vopr Virusol 39, 82–84. 69. Jahrling PB, Geisbert J, Swearengen JR, Jaax GP, Lewis T, Huggins JW, Schmidt JJ, LeDuc JW, Peters CJ (1996). Arch Virol 11 (Suppl 1), 135–140. 70. Ignatyev GM, Streltsova MA, Agafonov AP, Kashentseva EA, Prozorovskii NS (1996). Vopr Virusol 41, 206–209. 71. Geisbert TW, Pushko P, Anderson K, Smith J, Davis KJ, Jahrling PB (2002). Emerg Infect Dis 8, 503–507. 72. Hevey M, Negley D, Pushko P, Smith J, Schmaljohn A (1998). Virology 251, 28–37. 73. Sullivan NJ, Sanchez A, Rollin PE, Yang ZY, Nabel GJ (2000). Nature 408, 605–609. 74. Sullivan NJ, Geisbert TW, Geisbert JB, Xu L, Yang ZY, Roederer M, Koup RA, Jahrling PB, Nabel GJ (2003). Nature 424, 681–684. 75. Jones SM, Feldmann H, Ströher U, Geisbert JB, Fernando L, Grolla A, Klenk HD, Sullivan NJ, Volchkov VE, Fritz EA, et al. (2005). Nat Med 11, 786–790. 76. Feldmann H, Jones SM, Daddario-DiCaprio, KM, Geisbert JB, Stroeher U, Grolla A, Bray M, Fritz EA, Fernando L, Feldmann F, et al. (2007). PLoS Pathog 3, e2. 77. Daddario-DiCaprio KM, Geisbert TW, Stroeher U, Geisbert JB, Grolla A, Fritz EA, Fernando L, Kagan E, Jahrling PB, Hensley LE, et al. (2006). Lancet 367, 1399–1404. 78. Daddario-DiCaprio KM, Geisbert TW, Geisbert JB, Stroeher U, Hensley LE, Grolla A, Fritz EA, Feldmann F, Feldmann H, Jones SM (2006). J Virol 80, 9659–9666. 79. Bukreyev A, Yang L, Zaki SR, Shieh WJ, Rollin PE, Murphy BR, Collins PL, Sanchez A (2006). J Virol 80, 2267–2279. 80. Bukreyev A, Rollin PE, Tate MK, Yang L, Zaki SR, Shieh WJ, Murphy BR, Collins PL, Sanchez A (2007). J Virol 81, 6379–6388. 81. Kobinger GP, Feldmann H, Zhi Y, Schumer G, Gao G, Feldmann F, Jones S, Wilson JM (2005). Virology 346, 394–401. 82. Warfield KL, Swenson DL, Demmin G, Bavari S (2005). Expert Rev Vaccines 4, 429–440.

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CHAPTER 17

Host Gene Expression Triggered by West Nile Virus Infection Janusz T. Pawe ska

ABSTRACT A small percentage of individuals infected with West Nile virus (WNV) exhibit a life-threatening meningoencephalitis syndrome. The reasons why WNV causes severe illness in only a subset of cases have not yet been elucidated, but host-dependent and/or virus-dependent genetic factors might be important. Identification of the host genes that determine the outcome of virus-host interaction is imperative for understanding the molecular pathways that govern WNV pathogenicity, including neuroinvasiveness, neurovirulence and immunopathology. These genes are numerous and many of them interact with each other and have additive effects. Recent studies involving mice deficient in vital components of the immune defenses and DNA microarray technology have contributed significantly to revealing the genetic basis of host susceptibility to flavivirus infection and disease.

1. INTRODUCTION West Nile virus (WNV) is closely related to other important human pathogens within the Japanese encephalitis (JE) sero-group of the family Flaviviridae, including yellow fever virus (YFV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus (MVEV). The virus is maintained in an enzootic cycle 391

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between mosquitoes and birds but can also infect and cause disease in humans and other vertebrates. In the last decade, there was a marked increase in the frequency and severity of WNV outbreaks in humans worldwide, characterized by concurrent high mortality in birds.1 The majority of WNV infections in humans are asymptomatic, but approximately 20% to 30% of infected individuals develop febrile illness with a subset of cases progressing to meningitis, encephalitis, or an acute flaccid paralysis. Rare human cases of acute fatal hepatitis and pancreatitis have also been reported. Host and/or viral factors that allow virus entry from the blood into the central nervous system and development of fatal encephalitis are not well understood. The elderly and immunocompromised individuals are at greatest risk of developing severe neurological disease.2 Studies in animal models established that various components of the innate immunity, humoral immunity, and T-cell mediated immunity are all crucial to effectively control WNV infection.3,4 Most recently, DNA microarray technology has been applied to assess changes in host cell gene expression following flavivirus infection in both in vitro and in vivo models.5–10 This chapter focuses on aspects of host gene expression during infection with WNV. Viral life cycle, dissemination, pathogenesis and immune defenses, as well as host genetic determinants of WNV infection are discussed briefly in order to provide important background information to the primary topic of this chapter.

2. MOLECULAR BIOLOGY 2.1 Genome Structure and Protein Function WNV is an enveloped virus with a single-stranded, positive sense, RNA genome of approximately 11.3 kb. Translation of the single long open reading frame produces a large polyprotein that is cleaved by host and viral proteases into three structural and seven non-structural proteins. The structural proteins include a capsid protein (C), a pre-membrane (prM) protein that blocks premature viral fusion and may chaperone protein folding; and an envelope (E) protein that mediates virus-host cell binding, membrane fusion, viral assembly and elicits most of the virus-neutralizing

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antibodies, thus determining the serological specificity of the virus.11,12 Replication functions are associated with the non-structural proteins, which may also modulate responses to viral infection. NS1 has cofactor activity for the viral replicase, NS2A inhibits interferon responses and may participate in virus assembly, while NS3 has protease, NTPase, and helicase activities. NS3 alone triggers the apoptotic cell death by activation of caspase-based pathways. NS2B is a cofactor required for NS3 proteolytic activity, NS4A and NS4B modulate interferon signaling, and NS5 encodes the RNA-dependent RNA polymerase.13–17

2.2 Life Cycle Several cell surface molecules have been postulated to be involved in the attachment of flaviviruses to cells in vitro, including CD209 (DC-SIGN), CD290L (DC-SIGN-R), and the integrin α vβ 3. Virions enter cells via receptor-mediated endocytosis that may involve clathrin-coated pits. However, attachment receptors in vivo for target cell types, such as neurons, remain unknown. A pH-dependent conformational change in the E protein is required for fusion of the viral and endosomal membranes and subsequent release of the viral nucleocapsid into the cytoplasm.18,19 Flaviviruses replicate in the membranes of rough endoplasmic reticulum (ER) to generate a negative-strand intermediate RNA that serves as a template for nascent positive-strand RNA synthesis. Positive-strand RNA is either packaged within progeny virions or serves as a template for translation of additional viral proteins. Virus particles are thought to assemble by budding into the ER to form immature particles containing the prM protein. During transport of nascent virions through the trans-Golgi network, furin-mediated cleavage of prM protein to M protein results in generation of mature, infectious virions that are released by exocytosis.20,21

2.3 Molecular Basis of Virulence Genome sequences and phylogenetic analyses have defined two phylogenetic lineages of WNV, lineages 1 and 2; with lineage 1 including isolates from North Africa, Europe, Asia, North America, as well as Kunjin virus from Australia, while lineage 2 consists solely of isolates from Southern

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Africa and Madagascar.22 Two unique strains, Rabensburg virus and strain LEI-Krnd88-190, have recently been isolated from Culex pipiens mosquitoes and Dermacentor marginatus ticks in eastern Europe that form either two new lineages (lineages 3 and 4) or two distinct members of the Japanese encephalitis virus (JEV) group based on genetic distance analysis.23 Indian isolates of WNV appear to constitute an additional lineage (lineage 5).24 Initial genome sequences, phylogenetic studies, and reports on severe human infections support the perception that the emergence of increased virulence is associated with lineage 1 strains. However, neuroinvasive lineage 2 isolates have also been identified.6,22,25 Identification of specific sequence determinants of WNV virulence is currently a subject of intensive molecular studies. Mutations of the E-protein glycosylation sites lead to attenuation of viral replication and pathogenesis. E-protein glycosylation modulates WNV virulence by altering virion stability, viral replication and maturation processes.12,26 Mutations of the E-protein residues alter its affinity for glycosaminoglycans (GAG) and result in attenuation of virulence in a number of flaviviruses, including WNV.27 The C-type lectins DC-SIGN and DC-SIGN-R have recently generated considerable interest as cellular targets for flaviviruses.19 These membrane proteins tie glycosylated virions to the cell surface, thereby facilitating infection of lectin-bearing cells or transfer of bound virions to permissive target cells. A polymorphism in the DC-SIGN promoter region, associated with altered transcriptional activity in vitro, was recently linked to a decreased risk of severe dengue fever.28 It has been demonstrated that flavivirus non-structural proteins inhibit the activation of many known IFN-inducible genes by delaying IFN regulatory factor 3 (IRF-3) activation and IFN-β gene transcription, and preventing the phosphorylation and activation of the Janus tyrosine kinases JAK1 and Tyk2.13,17–21 Evidence is growing that the replication fitness and virulence of lineage 1 and 2 strains are determined by their ability to inhibit type I (α /β IFN) IFN responses.29 A single amino acid substitution in the non-structural protein NS2A disabled WNVs’ ability to inhibit α /β IFN induction and resulted in virulence attenuation in mice.14 Due to the low-fidelity of RNA-dependent RNA polymerase, flaviviruses have the propensity to generate quasispecies.30 The resulting antigenic

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variants may escape antibody-mediated neutralization, especially when newly emerged strains would have mutations at the dominant neutralizing epitope in domain III of the E protein.31

3. REPLICATION AND PATHOGENESIS IN SUSCEPTIBLE VERTEBRATE HOSTS Current knowledge of tissue tropism, dynamics of WNV replication and dissemination, mechanisms of pathogenesis and protection against WNV infection in vertebrates is mostly based on studies carried out in wild-type mouse strains and in mice that are deficient in vital functions of the immune responses to viral infection. In recent years it has been clearly demonstrated that both innate and adaptive murine immune responses determine protection against infection with WNV.32 Type I and type II IFNs,33,34 complement,35 γδ cells36 and early humoral responses37–39 limit viremia and dissemination into the CNS. B cells, macrophages, and dendritic cells38,40,41 play a crucial role in T cell activation and proliferation during WNV infection. Cellular immunity plays a vital role in the recovery from WNV encephalitis.42–44 After peripheral inoculation or bite by competent mosquito vectors, initial WNV replication is believed to occur in the skin’s Langerhans dendritic cells, which migrate to draining lymph nodes, where viral amplification occurs. Subsequently, WNV enters the bloodstream and spreads to other peripheral organs. Within few days of infection, WNV disseminates to the brain and spinal cord, and in many animals, WNV infects a variety of neurons in the hippocampus, cerebellum, brain stem, cerebral cortex, and anterior horn of the spinal cord.25,32 By the end of the first week post infection (p.i.), WNV is virtually not detectable in the serum and peripheral organs but infection of the central nervous system (CNS) is observed in a subset of immunocompetent animals. Pathological changes in the CNS of animals that succumb to infection are similar to those observed in human WNV cases, including infection and injury of brain stem, hippocampal, and spinal cord neurons. But interestingly, infection of nonneuronal cell populations within the CNS is difficult to demonstrate or is insignificant.25,45–47 In surviving wild-type mice, WNV is cleared from all tissues within two to three weeks p.i. but persistent viral infection

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has been shown in the brains of mice that lacked CD8+ T-cells or classical class Ia major histocompatibility complex (MHC) molecules.43 It was also shown in perforin-deficient mice34,48 and in the brains and kidneys of infected hamsters.25,49 Prolonged viremia has been demonstrated in a WNV-infected immunocompromised patient.50 Once WNV reaches the CNS, CD8+ T-cells seem to play an essential role in controlling viral replication and clearing WNV from infected neurons in the mouse model. Results of experiments in the C57BL/6J strain of inbred wild-type, congenic CD8 α-chain−/−, and congenic MHC class Ia-deficient mice suggested that at least part of WNV virus clearance occurs through a cytolytic mechanism. This is supported by the fact that both newly expanded and memory populations of purified CD8+ T-cells kill syngeneic targets that express WNV antigens in a class I MHC-restricted manner43; and it has been demonstrated that cytolytic lymphocytes proliferate, kill, and release inflammatory cytokines after incubation with WNV-infected cells.51 CD8+ effector T cells were shown to be critical in clearing WNV infection from tissues and preventing viral persistence;43,44 however, the development of CD8 T cell memory response to VNV is not fully understood. Results of very recent studies highlighted the importance of caspase 3-dependent apoptosis in the pathogenesis of lethal WNV encephalitis.52

4. GENETIC DETERMINANTS OF SUSCEPTIBILITY TO WNV INFECTION 4.1 Genetic Resistance in Mice Studies on susceptibility to flavivirus infection indicated that a single locus on chromosome (Chr) 5, designated flavivirus resistance (Flv), is responsible for phenotype differences in laboratory inbred mice, and is basically controlled by two alleles: dominant Flvr inducing resistance and recessive Flvs associated with susceptibility.53 A mutation of the Oas1b/L1 gene encoding the 2′-5′-oligoadenylate synthetase (OAS) was found to be associated with WNV susceptibility in the mouse model.54,55 The importance of this finding is related to that fact that 2′-5′- OAS family, latent endoribonuclease (RNase L), and other interferon (IFN)inducible proteins play important roles in the innate antiviral immune

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responses.56 While most laboratory mouse strains are susceptible to experimental flavivirus infection, most wild mice are resistant. An intraperitonal inoculation of adult mice of the classical laboratory strains, BALB/c and C57BL/6, caused encephalitis and 100% mortality for all of these animals, whereas mice from unrelated inbred strains derived from wild ancestors (Mus musculus domesticus — strain WMP/Pas; Mus m. musculus — strains MAI/Pas, MBT/Pas, PWK/Pas; Mus spretus — SEG/Pas, STF/Pas) or from laboratory strain PL/J, were resistant under similar experimental conditions. Back-crosses led to the identification of a WNV resistance/susceptible locus within an interval of 0.4 cM on Chr5, designated Wnv,54 which corresponds to the region where Flv had been mapped previously.57 Among the genes identified within the interval defined by D5Mit408 and D5Mit242 that includes the Wnv/Flv locus, is the gene cluster encoding the IFN-inducible OAS family of proteins. Although the Oas1b/L1 gene exhibits several single nucleotide polymorphisms (SNPs) among the different strains or species of mice, all susceptible mice tested so far have a T to C transition in the fourth exon sequence. This substitution results in a codon change, from CGA (arg) in resistant strains to UGA (stop) in susceptible strains. The correlation between susceptibility to viral infection and the occurrence of a stop codon supported the hypothesis that a truncated, and presumably inactive form of Oas L1 is indeed the cause of the innate susceptibility to flavivirus infection.58 In stable mouse neuroblastoma cell clones, overexpressing either the mutant or wild type Oas1b, replication of the WNV was less efficient in cells that produce the normal copy of Oas1b than those expressing the mutant form of the protein.59 Results of this study seem to further confirm the critical role of the Oas1b gene in the endogenous antiviral pathway, and therefore, its essential role in determining WNV pathogenesis in mice. Nevertheless, available data52,57 indicated that the phenotype of resistance/susceptibility is not specific to WNV infection, but also include other flaviviruses.58 Knock-in of the Oas1b resistance allele into a susceptible mouse strain produced mice with YLV-resistance phenotype.60 With extensive development of mouse genetics and the generation of new mutant alleles, it is likely that more genes with an impact on flavivirus resistance will be identified.

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4.2 Genetic Resistance in Humans A study in a limited number of WNV-infected patients suggested that SNPs in human genes are associated with susceptibility to WNV disease. The most prominent association was observed with the transition from T to C in SNP rs 3213545, which is located in exon 2 of the OasL gene. It was predicted that the nucleotide change T210C results in a new exon splice enhancer sequence that could eliminate activation of RNase L and, consequently, reduce degradation of viral RNA. Since 48% of the control subjects in the study were homozygous at the incriminated allele, additional factors are likely to be involved in virulence, and consequently, other loci in the 2′-5′ OAS family should also be investigated.61 The first strong evidence for a genetic factor correlated with increased susceptibility to WNV infection has been published by Glass et al.62 These authors established that homozygous CCR5∆ 32 is a significant genetic risk factor for symptomatic, laboratory-confirmed WNV infection. It is worth mentioning here that the homozygous CCR5∆ 32 genotype has been strongly associated with genetic resistance to HIV, implying that chemokine receptor CCR5 plays opposing roles in susceptibility to HIV and WNV infection. Although CCR5 might be a logical target for new HIV/AIDS drug development, the benefits of blocking CCR5 carries the risk of an increased clinical WNV disease in co-infected patients.63 Previous results in C57BL/6 mice infected subcutaneously (s.c.) with WNV-NY99 have shown that CCR5 is a critical antiviral and survival determinant in WNV infection. WNV-NY99 infection in CCR5−/− mice was rapidly and uniformly fatal.64

5. GENE EXPRESSION IN VIVO 5.1 Mouse Model Venter et al.6 used recent DNA microarray technology to measure mRNA transcription levels in 3- to 4-week old male NIH Swiss mice inoculated s.c. with 104 TCID50 of seven WNV strains, representing genetic lineage 1 and 2. Two lineage 1 strains were associated with fatal bird (NY385/99) and human (NY2001Hu) encephalitis in the USA, and the

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other two represented an Ethiopian strain isolated from a healthy bird (AN4766) and an Australian strain (Kunjin virus) recovered from mosquitoes (MRM16). Two of the lineage 2 isolates were associated with benign febrile disease (H442, AR381/00) and a third with fatal hepatitis (SPU116/89) in humans in South Africa. Based on the LD50 titration results in mice, these strains were classified as either highly neuroinvasive (strains SPU116/89, H442, NY385/99, NY2001Hu) or less neuroinvasive (strains AN4766, AR381/00, MRM16). Mice were sacrificed on day 5 p.i. to measure mRNA levels in brain, liver and spleen. In comparison to mean gene expression in mock inoculated mice, in WNV-infected mice, at least 127 differentially expressed genes were detected in the brain, of which 84 had increased and 43 decreased expression; 350 genes in the liver, of which 230 had upregulated and 120 downregulated expression; and 353 genes in spleen, of which 94 had increased and 259 decreased expression, in response to infection with at least one of the WNV strains. Using additional statistical criteria for selection, genes with greater expression change in response to infection with the highly neuroinvasive relative to the less neuroinvasive strains, 47 genes in the brain, 111 genes in the liver, and 70 genes in the spleen were selected for final analysis. The four highly neuroinvasive strains induced increased expression of genes involved in IFN-pathwayrelated proteins such as transcription factors and antiviral proteins, protein degradation, T-cell recruitment, MHC class I and II antigen presentation, apoptosis, acute proteins, CNS-specific proteins, and proteins associated with T-cell hepatitis. Genes with the highest average fold change induced by the highly neuroinvasive WNV strains vs. less neuroinvasive WNV strains in the brains of mice included: IRF1, STAT1, ubiquitin-specific protease 18 (Usp 18), proteosome subunit beta type 8 (Psmb8), guanylate nucleotide binding protein 2 and 3 (Gbp2, Gbp3), IFN-inducible protein 1 (Ifi1), IFN-α inducible protein (G1p2), Oas1g, Oasl 2, MX1, IFN-induced transmembrane protein 3 (Ifitm3), chemokine (C-X-C motif) ligand 11 (CXCL11), programmed cell death 1 ligand 1 (Pdcd1lg1), apolipoprotein D (Apod), and peptidylprolyl isomerase C-associated protein (Ppicap). A number of genes with as yet unknown functions were also identified, including IFN-induced protein with tetratricopeptide repeats 2, (Ifit2) which had the greatest mRNA expression level (14-fold) in the brains of

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mice. Ifit2 was also significantly overexpressed in human embryonic kidney cells infected with tick-borne encephalitis virus.65 Genes with the highest average fold change induced by the highly neuroinvasive WNV strains vs. less neuroinvasive WNV strains in the liver of mice included: tripartite motif protein 30 (Trim30), IFN-regulatory factor 7 (Irf7), Sat-1, solute carrier family 16 (Slc16a14), Usp 18, proteosome subunit beta type 9 (Psmb9), Z-DNA binding protein (Zbp1), macrophage expressed gene 1 (Mpeg1), serum amyloid A2 (Saa2), T-cell specific GTPase (Tgtp), G1p2, viral hemorrhagic septicemia induced gene 1 (Mm.24045), 28 kDa IFN-α responsive protein (Ifrg28), Oasl1, Oasl2, Oas1g, IFN-γ induced GTPase (Igtp), 3 genes of lymphocyte antigen 6 complex (Ly6c, Ly6f, Ly6a), insulin-like growth factor binding protein 1 (Igfb1), Ifi1, scotin gene (Scotin), and placenta-specific 8 (Plac8). Genes with the highest average fold change induced by the highly neuroinvasive WNV strains vs. less neuroinvasive WNV strains in the spleen of mice included: Trim30, Irf7, potassium voltage-gated channel of subfamily Q member 1 (Ly6F), USP18, Tgtp, Ly6c, Ly6a, Oas1a, Oasl1, Oasl2, and granzyme A and B (Gzma, Gzmb). IFN-pathway-related genes constituted one of the major groups with increased expression. STAT1 was postulated to play a role in protection against IFN-α mediated injury to the CNS, and is upregulated by other neurotropic viruses.15 The enhanced expression of CXCL11 indicates T cell involvement in response to neuroinvasive strains of WNV. Infection with WNV induces production of CCL5 from glial cells, resulting in recruitment of CCR5-expressing T cells and macrophages to the CNS where they have protective effect both in mice and humans.62,64 Members of the Oas family found to have increased expression in the brains of mice infected with highly neuroinvasive WNV strains, are implicated in conferring resistance to flavivirus infections.54,59 Proapoptotic genes upregulated in mice infected with highly neuroinvasive WNV strains could help clear virus infection but also mediate neuronal and hepatocyte damage. CNS-specific proteins implicated in the pathogenesis of neurological diseases which had enhanced expression in the study by Venter et al.6 included Apod and Gfap. Apod has been associated with brain injury9,66 and Gfap with gliosis resulting in the inability of neurons to regenerate axons.67 Despite much lower levels of WNV RNA in the liver and spleen

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than in the brain, the majority of genes with increased expression tend to fall into the same functional categories, but do not necessarily have the same levels of increased expression. These differences might be due to different levels of viral replication, differences in viral tissue tropism, cell damage and stage of defense responses in various tissues at the one time point (day 5 p.i.) when gene expression was measured in that study.

5.2 Knockout Mouse Models Knockout mice have been used extensively to research virulence mechanisms, pathology, and immune responses associated with WNV infection. Congenic mice genetically deficient in B cells and antibody (strain µ MT) inoculated s.c. with very low doses of the 2000 New York isolate of WNV (3000.0259) were very susceptible to lethal infection and developed high viral loads in serum and the CNS. Passive transfer of serum from infected and immune animals protected the µ MT mice against severe illness, thus confirming a critical role of antibodies and B cells in the defense against WNV infection.38 Samuel and Diamond33 evaluated the role of IFN-α /β in controlling WNV replication, spread, and tropism in IFN-α /β receptordeficient (IFN-α /β R−/−) mice. Compared to congenic wild-type 129v/Ev mice, the IFN-α /β R−/− mice had uncontrolled viral replication, rapid dissemination to the CNS, enhanced mortality as well as altered cellular tropism, and increased infection of macrophages, B cells, and T cells in the spleen. The role of IFN-α /β in protecting against lethal WNV infection was further confirmed by these authors in primary neuronal cultures from superior cervical ganglia (SCG), which were treated with IFN-β , IFN-α or IFN-γ before inoculation with the 3000.0259 isolate of WNV. Pretreatment of SCG neurons with a combination of IFN-β and IFN-γ resulted in the greatest reduction of viral replication. Subsequent studies in mice deficient in either IFN-γ or IFN-γ receptor (IFN-γ R) infected s.c with the 3000.0259 isolate of WNV demonstrated that the absence of IFN-γ function in mice results in higher viremia and greater viral replication in peripheral lymphoid tissues, and consequently leads to rapid spread to the CNS and early death.34 These studies provided an important insight as to the crucial antiviral role of types I and II IFN in limiting early dissemination of WNV in a susceptible host. Comparison of the genetic, pathogenic,

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and IFN-α /β -regulatory properties of WNV strains representing lineage 1 and 2 has shown that viral control of INF action and JAK-STAT signaling is critical for high replication fitness and virulence.68 Sitati and Diamond42 investigated the function of CD4+ T cells in controlling WNV infection in mice with genetic and acquired deficiencies of these cells. Their studies demonstrated that CD4+ T cells contribute to protection against WNV infection by sustaining antibody production and CD8+ T cells that have an important role in clearing infection from tissues and preventing viral persistence.43 Wang et al.69 demonstrated that γδ T cells produced IFN-γ within the first few days after WNV infection, and contributed in reduction of viral dissemination and partial protection of mice from death. Follow-up studies in the γδ T cells-deficient (TCRδ −/−) mice have shown that γδ T cells are also involved in the development of adaptive immunity to WNV infection but the mechanisms through which these cells regulate CD8+ T cell memory responses are not fully understood.36 Mice deficient in both the interferon-induced PKR and the endoribonuclease of the 2′-5′-OAS-RNase L system (PKR−/− × RL−/−) were highly susceptible to s.c. WNV infection and had increased viral loads in draining lymph nodes, serum, peripheral organs, and neuronal tissues compared to congenic wild-type mice. Mice deficient in RNase L showed a higher viral replication in the CNS and enhanced death rate but were less susceptible than the PKR−/− × RL−/− animals, thus indicating an antiviral role for PKR in controlling WNV infection. Although PKR and RNase deficiencies decreased the ability of type I IFN to inhibit WNV replication in primary macrophages and cortical neurons, this was not the case in the peripheral neurons of the superior ganglia of PKR−/− × RL−/− mice, thus suggesting the existence of cell-specific requirements for utilizing PKR and RNase L in inhibiting viral infection.70 PKR gene knockout, posttranscriptional gene silencing of PKR mRNA, and chemical inhibition of PKR function all interfered with IFN synthesis in mouse embryo fibroblasts following infection with WNV-derived virus-like particles, indicating that PKR serves as pathogen recognition receptor and that it is involved in WNV-induced INF synthesis.71 Klein et al.72 demonstrated that neurons infected with WNV secrete the chemokine CXCL10, which recruits CD8+ T cells via the chemokine receptor CXCR3, and by controlling viral

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infection in the CNS, it enhances survival rate in mice. Neutralization or a genetic deficiency of CXCL10 decreases CD8+ T cells trafficking into the CNS, resulting in increased viral burden in the brain and severe encephalitis in mice.

6. GENE EXPRESSION IN VITRO 6.1 CNS Cells Different mechanisms of WNV entry into the CNS were postulated, including entry via leukocytes, direct entry across the blood brain barrier (BBB) or entry by retrograde axonal transport via the peripheral nervous system.73 The Toll-like receptor 3 (TLR3) activated by WNV infection, indirectly enhances the permeability of the BBB via tumor necrosis factor (TNF).74 Activation of TLR3, associated with increased cellular adhesion molecule expression by immune cytokines, might allow increased leukocyte migration into the CNS,75 which could further be facilitated by increased vascular permeability due to CNS endothelial damage.76 Results in a murine footpad inoculation model indicate that virus-infected peripheral neurons can introduce WNV into the CNS by a retrograde transport mechanism.77 Various groups have hypothesized that the neuronal injury mechanisms are either due to the viral infection leading to apoptotic-mediated cell death78 or due to CTL responses following infection with WNV.79 Two important cell types that respond to infection of the CNS include microglia and astrocytes. These cells express TLRs which are important in pathogen recognition, processing and presentation of antigen to T cells.80 However, these cells can also produce large quantities of TNF, nitric oxide (NO) and other soluble mediators of potentially damaging effects to neurons, including production of chemokines in response to TNF secretion during the initial phase of infection.81 Reduced TNF output from microglia in TLR3-deficient animals decreased neuronal damage and promoted BBB stability.74 Thus, although TLR3 is important for viral recognition, it contributes to the role of the innate immune response in immunopathology in WNV infection. Studies in primary murine microglia demonstrated that these cells recognize double-stranded RNA,

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which is produced during WNV replication, through TLR3.82 In mice infected with MVEV the TNF stimulation of microglia and astrocytes seems to precipitate the neuropathological process. TNF triggers the production and secretion of neutrophil-attracting chemokine N51/KC resulting in a large influx of neutrophils into the CNS, whose activation leads to production of damaging levels of NO.83 On the other hand some chemokines and chemokine receptors appear to be important in viral clearance. For example, CCR5-deficient mice have enhanced brain viral burdens, with reduced T cell, macrophage and NK cell infiltration into the CNS, resulting in enhanced mortality.64 Infection of human brain glioblastoma (A172) cells with the Sarafend strain of WNV yielded high virus titer with advanced cytopathic effect within 24 h p.i. Activated A172 cells have macrophagic activity; therefore their response to virus infection allows investigation of immune-mediated neuropathologic processes that might reflect those in the natural CNS host cells. Using this system, a total of 173 cellular genes were identified to be differentially expressed and of these, 23 were selected as having potential roles in the pathogenesis of WNV-infected A172 cells. These included genes related to immune responses, apoptosis and the energy synthesis pathways.73 Upregulated genes related to immune response included IFNinduced protein with tetratricopeptide repeats 1 and 2, IFN α -inducible protein 27, IFN-induced transmembrane protein 1 and 2, IFN α -inducible protein, the HLA-C gene coding for MHC class I antigens, indoleaminepyrolle 2,3-dioxygenase (INDO), and the pentraxin-related gene (PTX3).71 INDO is expressed in antigenic-presenting cells84 (macrophages and dendritic cells), and there is now increasing evidence of its role in inflammatory responses including an inhibitory effect on proliferation of T lymphocytes.85 It has also been demonstrated recently that INDO may be responsible for the inhibition of T-cell reactivity against human tumors. T lymphocytes undergo proliferation arrest when exposed to tryptophan shortage, which can be provoked by INDO when its production is induced by several inflammatory mediators including IFN-γ .86 The PTX3 is induced most notably in dendritic cells and macrophages, in response to TLR engagement and inflammatory cytokines (interleukin-1β and TNF-α ) and has been implicated in the regulation of innate resistance to pathogens, inflammatory response, chemotaxis, and clearance of apoptotic cells.87

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Genes involved in apoptosis that are upregulated in WNV-infected A 172 cells, included tumor necrosis factor superfamily (TNFSF14), nuclear factor of κ light-chain gene (NFKBIA), TNF receptor-associated factor (TRAF1), and spermidine/spermine N1-acetyltransferase (SAT)73 which are responsible for cellular self-destruction, thus limiting the spread of WNV.88 Although analysis of the influence of WNV infection on the gene expression of A172 cells using microarrays indicated upregulation of apoptosis-related genes, a distinct mechanism involved in A172 cell death was not identified in that study. Gene delivery of WNV capsid protein into Balb/c mice induced apoptosis through a mitochondrial based caspase-9 pathway,78 and the WNV NS3 structural protein was shown to trigger apoptotic cell death by induction of caspase-8 and -3 in different cell lines.16 In the mouse neuroblastoma cell line Neuro-2a, WNV infection upregulated expression of the BAX gene, which triggers mitochondria outer membrane permeabilisation, followed by the release of cytochrome c from mitochondria into the cytoplasm.89 WNV infection induced caspase-3 activation and apoptosis in the brains of wild-type C57BL/6 mice and in primary CNS-derived mouse neurons. Accordingly, treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased WNV-induced neuronal cell death.90 These observations were recently further extended by results obtained in the brain-derived tumor cell line T98G.91 It was demonstrated that WNV replication in T98G cells decreased cell viability and induced apoptosis as indicated by the activation of the effector caspase-3, the initiator caspases-8 and -9, poly (ADP-ribose) polymerase cleavage and release of cytochrome c from the mitochondria. Western blot analysis demonstrated the truncation of BID (a proapoptotic BCL-2 family member), indicating that both intrinsic and extrinsic apoptotic pathways play a role in WNV-induced death of T98G cells. Numerous genes relating to the mitochondria, ribosomes, and protein biosynthesis were downregulated in WNV-infected A 172 cells, including succinate dehydrogenase (SDHC), cytochrome c oxidase (COX5B/COX6B), various genes of the ATP synthase complex, and two antioxidant enzymes of the peroxiredoxin family (PRDX5 and PRDX3). Inefficient energy production and increase in oxidative stress may lead to neurodegeneration.92

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6.2 Human Retinal Pigment Epithelial Cells Infection with WNV is associated with self-limiting chorioretinitis and vitritis but the mechanism of ocular disease caused by this virus is not well understood. Primary human retinal pigment epithelial cells (RPE) were recently used as in vitro models of ophthalmic WNV infection and to investigate interaction of the virus with IFN signal-transduction.93 Infection of RPE cells with the NY385-99 strain of WNV yielded maximum titres 24 h p.i., and significantly increased mRNA levels of IFN-β were observed as early as 12 h p.i. (6-fold) with maximum peak on day 4 p.i. (265-fold). IFN-α mRNA expression was not induced before 24 hours, and on day 4 p.i. its expression level was about 70 times less compared to that of IFN-β. Infection of RPE cells with WNV resulted in upregulated expression of IFN-stimulated genes (ISGs), including transducer and activators of transcription 1 and 2 (STAT1/2), IFN regulatory 1 and 9 (IRF1 and IRF9), and myxovirus (influenza virus) resistance 1 and 2 (MX1 and MX2). Addition of recombinant IFN-β before and after virus inoculation completely suppressed the expression of WNV NS1 protein in RPE cells. Furthermore, it has been demonstrated that superinfection of WNVinfected RPE cells with human cytomegalovirus strain Hi91 completely inactivated replication of the latter. This indicates that WNV induces an unrestricted general antiviral state in RPE cells. In contrast to these findings, WNV inhibited IFN signaling through blockage of STAT1 and STAT2 activation by WNV non-structural proteins in different humantransformed cell lines.94,95 It has been suggested that, because the retinal pigment epithelium forms the outer blood-retina barrier, and as such, constitutes a front-line defense against invading organisms, RPE cells may utilize mechanisms which allows more efficient activation of innate immune responses when compared to other cell types which do not have an immune-privileged status.93

6.3 Human Skin Fibroblast Cells The anti-NS1WNV specific monoclonal antibody together with RT-PCR, was used by Arnold et al.96 for analysis of cell surface human leukocyte antigen (HLA) expression and class I-associated molecules on human

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skin fibroblast (HFF) cells infected with WNV. This study demonstrated altered surface HLA expression on both infected HFF and neighboring uninfected HFF cells. Increased expression of HLA was due to virusinduced interferon IFNα /β as indicated by inhibition of HLA expression by IFNα /β -neutralizing antibodies. Although infection with WNV induced increased levels of mRNAs for HLA-A, -B, and -C genes, and HLA-associated low molecular weight polypeptide-2 (LMP-2) and transporter associated with antigen presentation-1 (TAP-1), they were not diminished in HFF cells cultured with IFNα /β -neutralizing antibodies. Large amounts of tumor necrosis factor-α (TNFα ) and IFNβ mRNAs were found in infected HFF cells 24 h p.i. These findings indicate that infection of HHF cells with WNV results in increased surface HLA expression via IFNα /β and TNFα -dependent and -independent mechanisms, suggesting that skin fibroblasts are important in supporting initial host immune responses to control WNV infection.

6.4 Human Embryonic Kidney Cells Human embryonic kidney cells (293) were used by Fredericksen et al.10 for the analysis of WNV-host interactions and especially to define the molecular mechanisms by which the pathogenic WNV-NY strain evades the host cell’s innate antiviral response. 293 cells infected with WNV-NY at multiplicity of infection 5 yielded peak infectious virus production at 24 h p.i. Under these experimental conditions, 8.3% of the cellular transcripts were differentially regulated by at least two-fold in WNV-infected cells compared to mock-infected cells at 24 h p.i. Biochemical and microarray analyses demonstrated that WNV-NY induced the expression of IFNβ and several IFN-stimulated genes late in the infection of 293 cells, notably ISG6-16, as well as IFN regulatory factor 3 (IRF-3), and STAT1α. The late expression of these antiviral genes was due to the delayed activation of the IRF-3, a transcription factor which is critical to the initiation of the antiviral response. Subsequent studies confirmed that delayed activation of IRF-3 is essential for WNV-NY to achieve maximum virus production, and that WNV-NY evades activation of IRF-3 through the retinoic acid-inducible gene I (RIG-I) pathway.97

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6.5 Mouse Embryo Fibroblasts It has been shown that even though infection of primary mouse embryo fibroblast (MEFs) with WNV induced IFN-β production as early as 12 h p.i. did not inhibit JAK-STAT signaling pathway, viral replication was efficient in these cells.98 Consequently, to gain insights into possible viral countermeasures used by this virus to suppress the host response in the early stages of infection, the kinetics of activation/expression of IFN regulatory factors (IRFs) and the expression of IFN-stimulated genes (ISGs) were examined in primary MEFs beginning at 2 h p.i.99 The majority of the upregulated genes in WNV-infected primary MEFs included those involved in IFN pathways. Comparison of ISG expression levels in mock-infected, IFN-treated, and virus-infected primary MEFs demonstrated IRF-3 activation/upregulation, and IRF-7 expression before 12 h p.i., while the induction of a number of ISGs was delayed until 24 h p.i. Virus-induced gene suppression was sufficient to overcome the effect of exogenous IFN pretreatment for 1 h but not for 4 h prior to infection. Whether suppressed expression of the subset of ISGs identified by Scherbik et al.99 was mediated directly or indirectly by the virus, the reported data indicate that WNV virus can selectively counteract the host response soon after infection. Results obtained by Fredericksen and Gale97 in RIG-I null MEFs suggest that activation of the host response by RIG-I early in infection is also important for controlling replication of WNV. Studies in resistant and susceptible MEFs demonstrated that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response, but, RNase L activation might not be a major component of Oas1b-mediated flavivirus resistance phenotype.98

7. CONCLUSION In the last few years, there have been considerable advances in the understanding of the intricate kinetics of flaviviral disease, principally through the availability of genetic mutant mice models and recent advances in DNA microarray technology. However, interpretation of results in the murine model and various in vitro models using these new research tools

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should be viewed with caution since even closely related strains of WNV stimulate different host responses. Attempts to strengthen our approaches in understanding the diversity and complexity of pathogenesis of WNV infection in different species of susceptible hosts should be expanded by monitoring changes in the kinetics of gene expression and their products during different stages of acute infection as well as during the disease recovery process.

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57. Shellam GR, Sangster MY, Urosevic N (1998). Rev Sci Tech Off Int Epizoot 17, 231–248. 58. Guénet J-L (2005). Brif Funct Genomic Proteomic 4, 225–240. 59. Lucas M, Mashimo T, Frenkiel MP, Simon-Chazottes D, Montagutelli X, Ceccaldi PE, Guenet JL, Despres P (2003). Immunol Cell Biol 81, 230–236. 60. Scherbik SV, Kluetzman K, Perelygin AA, Brinto MA (2007). Virology 368, 232–237. 61. Yakub IK, Lillibridge KM, Moran A, Gonzales OY, Belmont J, Gibbs RA, Tweardy DJ (2005). J Infect Dis 192, 1741–1748. 62. Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, Frank WA, Pape J, Cheshier RC, Murphy PM (2006). J Exp Med 203, 35–40. 63. Lim JK, Glass WG, McDermott DH, Murphy PA (2006). Trends Immunol 27, 308–312. 64. Glass WG, Lim JK, Cholera R, Pletnev AG, Gao J-L, Murphy PM (2005). J Exp Med 202, 1087–1098. 65. Gavirlov BG, Monastyrskaia GS, Velikodvorskaia TV, Filiukova OB, Konovalova SN, Kachko AA, Protopopova EV, Nikolaev LG, Loktev VB, Sverdlov ED (2003). Bioorg Khim 29, 175–180. 66. Johnston JB, Silva C, Hiebert T, Buist R, Dawood MR, Peeling J, Power C (2002). J Neuro Virol 8, 420–431. 67. Menet V, Prieto M, Privat A, Gimenez Y, Ribotta M (2003). Proc Natl Acad Sci USA 100, 8999–9004. 68. Keller BC, Fredericksen BL, Samuel MA, Mock RE, Mason PW, Diamond MS, Jr MG (2006). J Virol 80, 924–9434. 69. Wang T, Scully E, Yin Z, Kim JH, Wang S, Yan J, Mamula M, Anderson JF, Craft J, Fikrig E (2003). J Immunol 171, 2524–2531. 70. Samuel MA, Whitby K, Keller BC, Marri A, Barchet W, Williams BRG, Silvermann RH, Jr MG, Diamond MS (2006). J Virol 80, 7009–7019. 71. Gilfoy FD, Mason PW (2007). J Virol 81, 11148–11158. 72. Klein RS, Lin E, Zhang B, Luster AD, Tollet J, Samuel MA, et al. (2005). J Virol 79, 11457–11466. 73. Koh W-L, Ng M-L (2005). Emerg Infect Dis 11, 629–632. 74. Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA (2004). Nat Med 10, 1366–1373. 75. Shen J, Scrieber L, King NJ (1997). J Virol 71, 9323–9332. 76. Dropulic B, Masters CL (1990). J Infect Dis 161, 685–691.

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77. Hunsperger EA, Roehring JT (2006). J Neurovirol 12, 129–139. 78. Yang J-S, Ramanathan MP, Muthumani K, Choo AY, Jin S-H, Yu Q-C, Hwang DS, Choo DK, Lee MD, Dang K (2002). Emerg Infect Dis 8, 1379–1384. 79. Kesson AM, King NJ (2001). J Infect Dis 184, 947–954. 80. So EY, Kang MH, Kim BS (2006). Glia 53, 858–867. 81. Chen CJ, Chen JH, Chen SY, Liao SL, Raung SL (2004). J Virol 78, 12107–12119. 82. Town T, Jeng D, Alexopoulou L, Tan J, Flavell RA (2006). J Immunol 176, 3804–3812. 83. Andrews DM, Matthews VB, Sammels LM, Carrello AC, McMinn PC (1999). J Virol 73, 8781–8790. 84. Mellor AL, Munn DH (2003). J Immunol 170, 58–5813. 85. Mellor AL, Munn DH (2004). Nat Rev Immunol 4, 762–774. 86. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, Boon T, Van den Eynde BJ (2003). Nat Med 9, 1269–1274. 87. Garlanda C, Bottazzi B, Bastone A, Mantovani A (2005). Annu Rev Immunol 23, 337–366. 88. Chu JJH, Ng ML (2003). J Gen Virol 84, 3305–3314. 89. Parquet MC, Kumatori A, Hasebe F, Morita K, Igarashi A (2001). FEBS Lett 500, 17–24. 90. Samuel MA, Morrey JD, Diamond MS (2007). J Virol 81, 214–2623. 91. Kleinschmidt MC, Michaelis M, Ogbomo H, Doerr H-W, Cintal Jr J (2007). BMC Microbiol 7:49 doi: 10.1186/1471-2180-7-49. 92. Binienda Z, Przybyla-Zawislak B, Virmani A, Schmmued L (2005). Annu NY Acad Sci 1053, 174–182. 93. Cintal J Jr Michaelis M, Fleckenstein C, Bauer G, Kabic ková H, Scolz M, Rabenau HF, Doerr HW (2006). Invest Ophthalmol Vis Sci 47, 645–651. 94. Guo JT, Hayashi J, Seeger C (2005). J Virol 79, 1343–1350. 95. Liu WJ, Wang XJ, Mokhonov VV, Shi P-Y, Randall R, Khromykh AA (2005). J Virol 79, 1934–1942. 96. Arnold S, Osvath SR, Hall RA, King NJC, Sedger LM (2004). Virology 324, 286–296. 97. Fredericksen B, Gale Jr M (2006). J Virol 80, 2913–2923. 98. Scherbik SV, Paranjape JM, Stockman BM, Silverman RH, Brinton MA (2006). J Virol 80, 2987–2999. 99. Scherbik SV, Stockman BM, Brinton, MA (2006). J Virol 81, 12005–12018.

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CHAPTER 18

West Nile Virus-Host Interaction: An Immunological Perspective Raghavan Bhuvanakantham & Mah-Lee Ng

ABSTRACT West Nile virus (WNV) is a positive-stranded RNA virus that produces double-stranded RNA (dsRNA) during replication. Engagement of Tolllike receptor 3 with dsRNA initiates tumor necrosis factor-alpha receptor 1 signaling in peripheral lymphoid tissues. This could disrupt the integrity of the blood-brain barrier, thereby facilitating WNV entry into central nervous system (CNS). In this way, WNV infection could induce fatal encephalitis. Prompt cellular responses are thus vital to fight against WNV infection. These are regulated by more complicated networks whereby specific signaling pathways such as interferon, complement, innate and adaptive immune responses are induced for the successful removal of the invading pathogen. Interferons and γδ T cells play a vital role in limiting WNV replication in peripheral tissues and its dissemination into CNS. Complement activation primes the adaptive immune responses by triggering the protective humoral antibody responses, which helps to control WNV infection. Inflammatory chemokines like CXCL10 modulate the recruitment of CD8+ T leukocytes into the infected tissues in CNS via the chemokine receptors CXCR3 and CCR5. CD8+ T cells are the foremost population involved in both protection and immunopathology of WNV while CD4+ T cells help to sustain virus-specific CD8+ T cell responses. Fas-mediated cytolytic mechanisms of CD8+ T cells are one of the most prominent 415

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effector functions in protection against WNV. An increase in cell surface expression of MHC class I induced by NF-κ B activation could be the host antiviral strategy associated with immunopathology. This review will also discuss how WNV evades host immune responses for better replication.

1. INTRODUCTION West Nile virus (WNV) is an arbovirus originally isolated from the blood of a febrile woman in the West Nile province of Uganda in 1937. WNV was subsequently found endemic in Africa, Asia, Egypt, Europe and Israel. In 1999, the virus emerged as the common cause of encephalitis in the United States. Following its introduction in New York city, WNV rapidly broadened its territory across North America, Mexico, South America, Carribean and Central America [reviewed in Refs. 1 and 2]. WNV is a single-stranded RNA virus which belongs to the Japanese encephalitis serocomplex of the genus Flavivirus within the family Flaviviridae. It is an enveloped, spherical virus of about 50 nm in diameter and its RNA genome encodes a single large polyprotein, which is processed by viral and host proteases into three structural proteins: the capsid (C), membrane (M) and envelope (E), as well as seven non-structural (NS) proteins.3,4 This review will briefly summarize the modes of transmission, phylogenetic analysis, pathology and clinical manifestations associated with WNV infection. The mechanisms by which the host immune system limits the dissemination of the virus and how the virus evades the immune system to establish successful infection will also be highlighted.

2. TRANSMISSION WNV is transmitted by Culex mosquitoes primarily between birds, the amplifying hosts of the virus. They also function as bridge vectors for transmission to humans, equines and other mammals.5 Humans are considered dead-end hosts because they usually develop viremia at an insignificant level to facilitate further transmission of the virus. WNV transmission was also reported during organ transplantation,6–10 blood

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transfusion,11–14 pregnancy15–18 and lactation.19 Occupational WNV infections in laboratory workers have also been documented.20,21

3. PHYLOGENETIC ANALYSIS Nucleotide sequencing and phylogenetic analysis of WNV performed based on the entire or partial viral gene sequences distinguished WNV into two lineages: lineage I and lineage II.22,23 Lineage I viruses have been further classified into three distinct clades, namely clades Ia, Ib and Ic. Clade Ia includes the pathogenic strains from Africa, Europe and North America. Kunjin virus and Indian isolates have been grouped into clades Ib and Ic, respectively. Lineage II comprises viruses isolated exclusively in Africa and Madagascar. Although the more pathogenic strains have been classified under lineage I, it was shown that lineage II viruses could also cause major outbreaks.24 Recently, Rabensburg and Russian isolates were suggested to form third and fourth lineages of WNV, respectively.25,26 Bondre and colleagues27 recently proposed that WNV isolates from India form the fifth lineage of WNV.

4. PATHOGENESIS West Nile virus is thought to replicate in dendritic cells after being bitten by an infected mosquito or at the site of inoculation.28 These cells then migrate to lymph nodes and cause primary viremia and subsequent infection of peripheral organs. Viremia peaks between two to four days after infection and later the virus load is considerably reduced in the blood and peripheral tissues. WNV is cleared from all tissues in two to three weeks postinfection although persistent viral infection was reported in CD8+ T-cell or perforin-deficient mice.29–31 Persistent infection has also been observed in immunosuppressed patients.32 The mechanism by which WNV establishes prolonged infection remains unclear. The exact mechanism by which WNV breaches the blood-brain barrier (BBB) and enters the central nervous system (CNS) is not clear. WNV could enter the CNS through three different means, namely direct entry across the BBB, entry by means of leukocytes or entry by bidirectional spread (retrograde and anterograde directions) along peripheral neurons.33–38

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WNV directly infects neurons especially in the nuclei and grey matter of the brain, brainstem, hippocampus and spinal cord.31,39–45 In addition to injury to the neurons, collateral damage to the bystander neurons and immune-mediated tissue damage might also contribute to the pathological changes.37,46,47

5. CLINICAL MANIFESTATIONS While the majority of WNV infections are asymptomatic, it can cause debilitating disease in humans and animals, with symptoms ranging from febrile illness to fatal encephalitis. About 20% of infected patients display a range of symptoms including fever, headache, malaise, back pain, myalgias, eye pain, pharyngitis, nausea, vomiting, diarrhea, and abdominal pain. Incubation period typically lasts up to 14 days.48 Out of that 20%, maculopapular rash appears in approximately half, a subset of which would acquire a form of neuroinvasive disease.49,50 More serious manifestations of WNV are categorized as: encephalitis, meningitis, and flaccid paralysis with the former two being more common.51 Muscle weakness and flaccid paralysis is particularly suggestive of WNV infection.52 Asymmetric acute flaccid paralysis syndrome may also occur independent of encephalitis and has been noted to be a sign of impending respiratory failure.53 West Nile encephalitis is commonly reported in patients above the age of 55 and is higher among organ transplant recipients.54,55 West Nile poliomyelitis, West Nile choreoretinitis, hepatitis, pancreatitis, cardiac dysrhythmia and myocarditis have also been documented [reviewed in Ref. 56]. Following a European outbreak in 1998, WNV came into the spotlight in 1999 when it was identified for the first time in North America.57 The number of cases peaked in 2003 following the spread of WNV to the whole of Northern America, México, and Canada. Table 1 summarizes the case numbers from 2000 to 2007. WNV, thus, present a growing threat to public health in the U.S. and the rest of the world. There is now a strong demand for the development of vaccines and/or antiviral lead compounds. In order to achieve this, better understanding of virus morphogenesis and the associated immunological responses is indispensable.

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Table 1. Confirmed WNV case numbers from 2000 to 2007. Year

Case Numbers

Reference

21 66 4156 9862 2744 4261 1395

58 58 59 60 61 62 63

2000 2001 2002 2003 2005 2006 2007 (1 Jan–11 Sept)

6. IMMUNE RESPONSES TO WNV INFECTION Prompt and regulated cellular response is vital to immunity. Viral infection of susceptible cells initiates a series of intracellular events within the infected cell and in neighboring cells. By orchestrating the activation of several genes, antiviral and innate immune responses will be established to eliminate the virus. Interferons play an important role in host defense not only as important mediators of innate antiviral immunity, but also as links between the innate and adaptive immune systems. There is considerable research being undertaken to elucidate the immunological responses to flaviviruses including WNV. It is clear that both the innate and adaptive arms of the immune system are important in reducing viremia and in viral clearance.

6.1 Innate Immune Responses 6.1.1 Interferons Interferons (IFNs) are widely expressed cytokines that have potent antiviral and growth-inhibitory effects. These cytokines form the first line of defense against viral infections. The IFN family includes two main classes of related cytokines: type I IFNs and type II IFN. There are many type I IFNs but the most important forms in humans are IFN-α and IFN-β .64 In contrast, there is only one type II IFN, IFN-γ. 65 Type I and II IFNs have been well studied in the context of WNV infection.

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Type I IFN is secreted by many cell types including lymphocytes, macrophages, fibroblasts, endothelial cells and osteoblasts. They stimulate both macrophages and NK cells to elicit an antiviral response. The importance of type I IFN signaling in controlling the outcome of WNV infection by restricting tropism and viral burden has been clearly demonstrated.66,67 Recent studies further highlighted the significance of type I IFNs68–71 in restricting WNV spread and thereby reducing its virulence. Another crucial component of cytokine-mediated immune responses that eliminate viruses from the CNS is IFN-γ , the only type II IFN. IFN-γ is secreted mainly by NK cells, γδ T cells, ThI CD4+ and CD8+ cells. IFN-γ could restrict viral replication directly by inducing an antiviral state or indirectly by modulating the adaptive immune response through activation of macrophages/microglia, and upregulating cell surface expression of major histocompatibility complex (MHC) class I molecules [reviewed from Ref. 67]. IFN-γ limits early viral dissemination and its invasion into CNS.72 Getts and colleagues73 also showed that IFN-γ plays an important role in the immunopathogenecity of WNV. IFN-γ produced by γδ T cells also show protection against WNV-induced lethal encephalitis.74 During WNV infection, γδ T cells displayed the characteristic features of innate immune cells by their ability to produce IFN-γ , thus enabling the host to clear the virus. Although the role of (γδ T cells-produced) IFN-γ in controlling WNV infection is innate and antiviral, the ability of γδ T cells to induce CD8+ T cell response makes these cells the connecting bridge between innate and adaptive immunity during WNV infection.

6.1.2 Interferon-regulatory factors Interferon-regulatory factors (IRFs) are a family of transcription factors mainly involved in the regulation of IFNs and other genes that have an essential role in CNS antiviral defense. Currently, there are nine members of the mammalian IRF family (IRFs 1 to 9). Each IRF contains a conserved DNA-binding domain at its amino terminus that recognizes Interferon-stimulated response element (ISRE). The impact of WNV infection upon global host cell gene expression over the course of an infection was examined by microarray analysis (Refs. 75, 76; unpublished data, Ng). Detailed analysis indicated that

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WNV infection triggers events that lead to the activation of IRF-1, IRF-2, IRF-3, IRF-7, IRF-target gene expression and IFN production, which helps to combat WNV. The protective role of IRF-3 in limiting infectious particle production during WNV infection was further enlightened by pattern recognition receptors studies.77,78 However, activation of IRF-3 was delayed up to 12 to 16 h postinfection. This could be a unique mechanism applied by WNV to circumvent stimulation of host antiviral response. These observations lead to the conclusion that although IRF-3 stimulated gene controls the rampant spread of WNV in the host, it fails to block WNV infection. Similarly WNV failed to completely block host antiviral response but succeeded in delaying the induction of IRF-3 pathways during early infection.

6.1.3 Pattern recognition receptors Pattern recognition receptors (PRR) recognize pathogen-associated molecular patterns, such as lipopolysaccharide and viral nucleic acids. Engagement of PRR activates various signaling pathways in host cells depending on the type and the location of the invading pathogen to fight against the specific pathogen. Two classes of PRR have been defined, namely the transmembrane PRR and the cytosolic PRR. Transmembrane PRR includes Toll-like receptors (TLR) while cytosolic PRR includes the retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), IFN-inducible double-stranded RNA (dsRNA)-dependent protein kinase (PKR), and nucleotide-binding oligomerization domain (NOD) proteins. Both classes of PRR are shown to be involved in WNV infection.37,77–81 The involvement of Toll-like receptor-3 (TLR3) in mediating WNV entry into the CNS was initially demonstrated by Wang and colleagues37 using TLR3-deficient [TLR3(−/−)] mice. Although enhanced viral load was observed in the peripheral tissues, reduced viral load, inflammatory responses and neuropathology were detected in the brain of TLR3(−/−) mice compared to wild-type mice. TLR3-deficient mice had also impaired cytokine production (IFN-β , IL-6, and TNF-α ) and were more resistant to lethal WNV infection. It was also shown that tumor necrosis factor-alpha receptor 1 signaling was initiated in peripheral lymphoid tissue by TLR3,

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playing a vital role in compromising the BBB. As a result, WNV can cross the BBB into the CNS and cause neuronal injury. Town and colleagues81 further demonstrated that microglia recognized dsRNA through TLR3 signaling pathways. Microglia are glial cells that act as the immune cells of the CNS and express TLRs on the surface. Microglial cells play numerous important roles in protecting the CNS against invading dsRNA-producing viruses. It was shown that microglia elicited innate immune response against WNV infection through the activation of MAPKs, production of proinflammatory cytokines and increased surface activation markers. Mice lacking both the interferon-induced, dsRNA-activated protein kinase (PKR) and the endoribonuclease of the 2′, 5′-oligoadenylate synthetase (OAS)-RNase L system [PKR(−/−) × RL(−/−)] showed increased viral loads in the CNS during early infection. These results suggested that PKR and RNase L contributed to IFN-mediated protection to recognize and control WNV infection.79,80 In addition, these studies implied that PKR played a more vital antiviral role in the control of WNV infection compared to RNase L. The participation of RIG-1 and MDA-5 were also recently studied in the context of cellular recognition of WNV.77,78 Interferon-inducible proteins such as OAS aid in eliciting the endogenous antiviral pathway. The genetic determinant of WNV susceptibility in mice has been delineated to a mutation in the Oas1b gene. This mutation produced a truncated 2′, 5′-OAS isoform that lacks 30% of the C-terminal sequence of the resistance counterpart.82,83 Replication of WNV was less efficient in cells that produce the normal copy of Oas1b as compared to those expressing the truncated isoform.84 In humans, although there is no insertion, deletion or nonsense mutation in OAS gene, a synonymous single nucleotide polymorphism (SNP) in OAS was observed. This SNP resulted in dominant-negative OASL isozyme similar to the nonsense/truncation mutant form of OAS1b in mice.85 These results indicated that Oas genes are critical in determining the susceptibility of the host to WNV infection. Kajaste-Rudnitski and colleagues86 reported that OAS1b exerts its inhibitory effect on WNV replication by preventing viral RNA accumulation inside infected cells. Although RNase L plays a role in the cellular antiviral response to flaviviruses by cleaving WNV genomic RNA, RNase L activation is not a major component of the OAS1b-mediated flavivirus resistance phenotype.87

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In light of such complexities, the detailed underlying mechanism needs to be elucidated. In brief, PRRs function as important mediators of intracellular antiviral responses by preventing virus replication and cell-to-cell spread. Table 2 summarizes the role of IFNs and PRRs in eliciting immune responses against WNV infection. Figures 1 and 2 illustrate the brief outline of IFNs and IRF activation by PRR during WNV infection and the mechanism of immune evasion by WNV. The dsRNA generated during WNV genome replication (replication intermediate) interacts with the RNA-helicase domain of PRRs and activates IRF-3 and IRF-7 via phosphorylation. Subsequent translocation from the cytoplasm to the nucleus affects the transcription of ISRE-containing genes including IFN-α, IFN-β , CXCL10 and RANTES. A protein complex termed the enhanceosome, which includes NF-κ B, ATF2/c-Jun, and IRF-3, in general, regulates transcription of type I IFN. Activation of IRF-3 and NF-κ B is well documented during WNV infection,36,75–78,88,89 Table 2. Role of IFNs and PRRs in eliciting immunoprotection against WNV. Mouse Strain/ Cell Line

WNV Strain

IFN-α /β R(−/−)

NY

IFN-γ (−/−) or IFN-γ R(−/−) INF γ (−/−)

NY Sarafend, KUN

RIG-I null MEFs PKR(−/−) × RL(−/−)

NY NY

RL(−/−)

NY

C57BL/6 RNase L(−/−) cells TLR3(−/−)

Strain Eg101

TLR3(−/−) Microglia

NY

NY

Characteristics High virulence, increased mortality. Increased viremia, early entry into CNS, high mortality. INF-γ does not play a significant role in WNV infection. Delayed onset of the host response. Increased viremia and viral burden, early entry into CNS, high mortality. Increased viremia and higher CNS viral burden. High viremia. Impaired cytokine production, increased lethality. Impaired cytokine production, increased lethality.

Refs 71 72 128, 134 75 79, 80

79, 80 87 37 81

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Figure 1. Host immune responses against WNV infection and immune evasion by WNV. WNV enters the cell by clathrin-mediated endocytosis using α Vβ 3 integrin as the

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which could positively regulate the transcription of IFNs, other proinflammatory cytokines and chemokines. Type I IFNs binds to the specific type I IFN receptor and activates the transcription of IRF-7 genes and expression of RIG-1 and MDA-5. This positive feedback generates large amounts of IFN-α and IFN-β, thus amplifying type I IFN signaling to elicit the host antiviral response against WNV infection. The binding of secreted type I IFNs to the IFN receptor triggers the activation of the Janus kinase and signal transducers and activators of the transcription (JAK/STAT) signal transduction pathway (Figure 2). This leads to the induction of expression of a wide variety of IFN-stimulated genes (ISGs) that function to constrain WNV infection and limit cell-tocell virus spread. In this way, the eukaryotic immune system fights against invading WNV. Despite these series of host responses, WNV is still able to replicate efficiently, suggesting that WNV possesses the capability to attenuate the host antiviral response through several mechanisms. Studies by Guo and group90 showed that WNV block the IFN signal transduction pathway by inhibiting the phosphorylation and activation of the IFN receptor-associated kinases, Janus kinase 1 (JAK1) and Tyrosine kinase 2 (Tyk2). This in turn block the phosphorylation of STAT proteins and activation of IFN-induced genes. Keller and colleagues69 demonstrated that lineage I (strain TX02) and lineage II (strain MAD78) WNV differentially regulate JAK/STAT signaling and IFN action. Lineage I virus prevent IFNinduced tyrosine phosphorylation of Tyk2 but did not have any impact on JAK1 activation. Blockade of Tyk2 in turn, prevented the phosphorylation Figure 1. (Continued ) receptor. Double-stranded RNA is generated as the replication intermediate in the cytosol of infected cells during WNV replication. Binding of dsRNA with the RNA-helicase domain of PRRs activates IRF-3 and IRF-7 via phosphorylation and forms homo- and hetero-dimers. These dimers are translocated to the nucleus and affect the transcription of ISRE-containing genes including IFN-α, IFN-β , CXCL10 and RANTES. Double-stranded RNA binding to PRRs also activates NF-κ B. Activated NF-κ B enters the nucleus and induces the transcription of proinflammatory cytokines such as IL-6. These cytokines, chemokines and IFNs function to protect the infected cells. In this way, innate immune responses are triggered against WNV infection. By suppressing the activation of IRF-3 and IRF-7 and by activating NF-κ B-repressing factor during early infection, WNV escape the host-mediated antiviral mechanisms.

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Figure 2. Activation of ISGs and WNV strategy to triumph over host’s response. Binding of IFN to type I receptor on target cells activates the receptor-associated kinases JAK1 and Tyk2. Activated kinases in turn phosphorylate and activate the downstream effectors, STAT1 and STAT2 which eventually forms dimer. These STAT1/STAT2 heterodimers interact with IRF-9. This heterotrimeric complex induces the transcription of interferon-inducible genes (ISGs) through ISRE in the nucleus. These ISGs execute the antiviral functions against WNV infection. By blocking JAK/STAT signaling pathway, WNV prevents the induction of ISGs and its action.

of STAT proteins. Lineage II virus was attenuated in this function and showed reduced replication fitness and inability to cause the blockade of JAK/STAT signaling cascade. This suggested that JAK/STAT signaling could be one of the major determinants of virus fitness and virulence.

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All the non-structural proteins except NS1 and NS5 were reported to have a role in this inhibition although NS2A and NS4B of WNV were sufficient to inhibit the IFN response.91–94 It was also suggested that structural genes through yet unknown interactions could result in the inhibition of the IFN signaling pathway during WNV infection. By blocking the JAK/STAT pathway and allowing the activation of IRF-3 pathway only during late infection, WNV thus evades host immune responses. This creates a micro-environment virtually unchallenged by the host cell at early times postinfection for better replication.

6.1.4 Chemokines Chemokines are a family of structurally related glycoproteins with potent leukocyte activation and/or chemotactic activity. The major role of chemokines is to guide the migration of cells such as macrophages and microglia. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine. Following WNV infection, Cheeran and colleagues95 detected the expression of RANTES and IP-10 in an in vitro model. Using in vivo animal model, the upregulation of several chemokines such as CCL5 (RANTES), CCL3 (macrophage inflammatory protein [MIP]-1α), CCL4 (MIP-1β), and CXCL10 (IP-10) were reported in the brain of infected mice.96 In addition to these chemokines, Garcia-Tapia and colleagues97 have observed the upregulated expression of MCP-5 (CCL12) and MIG (CXCL9). They suggested that MCP-5, MIG and CXCL10 are the important mediators of neuroinflammation. Other chemokines like CCL12, CCL6, CXCL9, CCL3, CCL4 and CCL7 also played an important role in inducing a potent cellular helper-T cell (Th)-1 type immune responses against WNV in the CNS. Klein and group98 demonstrated that the chemokine CXCL10 secreted by neurons recruited CD8+ T cells via the chemokine receptor CXCR3, controlling WNV infection in the CNS and increasing survival. The roles of the chemokine receptor CCR5 in recruiting leukocytes into the brain and in reducing the risk of developing encephalitis in WNV infection illustrate the importance of CCR5 during WNV infection.99–101 Table 3 summarizes the role of chemokines in controlling WNV infection.

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Table 3. Chemokines and immunoprotection during WNV infection. Mouse Strain/ Cell Line

WNV Strain

Astrocytes, Microglial cells BALB/c

NY1999

BALB/c

Eg101 strain

C57BI/6

WNV isolate

CXCL10(−/−)

NY2000

CCR5(−/−)

NY1999

CCR5(−/−)

NY1999

NY1999

Characteristics

Ref.

Secreted chemokines such as CXCL10 and CCL5 and CCL2 could induce neuroinflammation. Upregulated of CXCL10, CCL3, CCL4, CCl5 caused high viremia in peripheral tissues and CNS, increased mortality. Low viremia and reduced or no CNS invasion. Secreted chemokines such as MCP-5, IP-10, MIG triggers neuroinflammation. Decreased T-cell infiltration in brain, increased viral load in CNS, increased mortality. Decreased T-cell infiltration in brain, increased viral load in CNS, increased mortality. Decreased NK cells, macrophages and T-cell infiltration in brain, increased viral load in CNS, increased mortality.

95

96

96 97

98

99

101

6.1.5 Complement The complement system consists of a number of small proteins found in the blood, which work together to kill target cells by direct opsonization and/or cytolysis, chemotaxis, immune clearance, and modulation of Band T-cell functions. The complement system is not adaptable and does not change much over the course of an individual’s lifetime; as such it belongs to the innate immune system. However, it can be recruited and brought into action by the adaptive immune system. Complement activation can occur through the classical, lectin, and alternative pathways. Recent studies have shown the involvement of complement system in limiting WNV infection.102,103 Complement system directly neutralizes WNV

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predominantly by antibody-dependent and to a lesser extent, by antibodyindependent mechanisms.102 It was also shown that C3 component is essential for controlling the early spread of WNV into the CNS. Histological studies showed prominent staining in the cerebral cortex, hippocampus and brain base in C3-deficient mice, suggesting severe injury to neurons. Experiments performed using C3- and CR1/2-deficient mice showed blunted WNV-specific antibody responses and increased susceptibility to infection. This indicated that the complement activation is necessary for the development of a protective humoral response during primary infection since the attenuated antibody response in C3- and CR1/2-deficient mice directly resulted in increased viral infection and mortality.102,103 Addition of neutralizing antibodies (MAb E16) or inactivated immune serum could block WNV-induced lethality in C3-deficient mice.102 Complement activation through classical, lectin and alternative pathways are thus required for normal humoral and T cell responses to protect individuals from WNV infection.103 Table 4 summarizes the involvement of complement system in clearing WNV infection. On the other front, WNV NS1 protein decreased complement activation by binding to and recruiting the complement regulatory protein factor H.104 This immunomodulatory function of NS1 protein could be the immune evasion strategy of WNV to establish successful infection. Table 4. Complement activation during WNV infection. Mouse Strain

WNV Strain

Characteristics

Ref.

Clq(−/−)

NY2000

103

C4(−/−)

NY2000

fB(−/−)

NY2000

C3(−/−)

NY2000

CR1/CR2(−/−)

NY2000

Increased viral burden in CNS, delayed IgG production, increased lethality. Increased viral burden in CNS, reduced humoral and T-cell responses, increased lethality. Increased viral burden in CNS, reduced humoral and T-cell responses, increased lethality. Increased viral load in CNS, decreased humoral response, increased mortality. Increased viral load in CNS, decreased humoral response, increased mortality.

103

103

102 102

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All these observations suggest that complement is absolutely required to generate a rapid and effective antibody response and for priming the adaptive immune responses against WNV infection. WNV in turn, minimizes the effect of host complement system to establish infection.

6.1.6 Immune recognition molecules Flavivirus infection of mammalian cells upregulates cell surface expression of major histocompatibility complex class I (MHC class I) molecules. Cheng and colleagues88 reported that two different pathways were involved in WNV-induced upregulation of MHC class I namely a NF-κ Bdependent, IFN-independent pathway and an NF-κ B-independent, IFNdependent pathway. WNV-mediated upregulation of MHC class I is not a predominant consequence of virus-induced IFN responses since WNV infection of trophoblast, a cell line refractory to IFN-mediated induction of MHC class I, induced MHC class I expression.105 The upregulation of MHC I could be a by-product of flavivirus replication.106 NK cells also have the potential to control WNV infection through recognition and elimination of virus-infected cells. However, WNV could evade natural killing by increasing surface expression of MHC molecules.107 WNV infection induces NF-κ B activation, which in turn modulates the expression of several immune recognition molecules such as MHC class II, ESelectin, ICAM-1 and VCAM-1.88,89,108,109 It was also shown that upregulation of MHC molecules and ICAM-1 was abrogated when NF-κ B nuclear translocation was reduced.89 Microarray analysis (unpublished data, Ng) showed the upregulation of NF-κ B repressing factor during early WNV infection. This suggested that the repressing factor could modulate/mask the function of NF-κ B during early infection, aiding the efficient replication of the virus without host interference.

6.2 Adaptive Immune Responses 6.2.1 Humoral responses Humoral response is important in controlling enhanced virus infection and dissemination. Humoral immunity is an essential aspect of immune

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system-mediated protection from WNV.31,40,110,111 Diamond and group40 illustrated that B cells and IgG play critical early roles in host defense against WNV infection. B-cell-deficient mice uniformly died after WNV infection. Moreover, passive transfer of heat-inactivated serum from infected and immune wild-type mice protected B-cell deficient mice against morbidity and mortality. Induction of a specific, neutralizing IgM response is essential to limit viremia and its dissemination into the CNS as sIgM−/− mice developed high viremia and showed high lethality.31 The importance of CD40-CD40L interactions for efficient production of virus specific antibodies by B cells during WNV infection was recently documented.112 Eliciting neutralizing antibodies that can recognize specific epitopes located on viral proteins is a common host strategy against viral infection. Envelope (E) and Non-structural protein 1 (NS1) were the more commonly reported viral proteins recognized by neutralizing/protective antibodies in WNV albeit E protein is a major key player. The E protein monomer folds into three structural domains, namely domain I, domain II and domain III. Studies have shown that E-specific neutralizing antibodies map to all three domains, although the most potent inhibitory antibodies recognize a dominant neutralizing epitope present on DIII.111,113–117 Human single-chain variable region antibody fragments against domains I and II of WNV E protein were protective.118 Oliphant and colleagues116 also reported that 25 residues on DI and DII of E protein formed the neutralization epitopes. These studies portrayed the presence of neutralizing epitopes on DI and DII of WNV E protein. Oliphant and colleagues111 tested several monoclonal antibodies against DIII region and identified strong neutralizing ability of those antibodies. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. In post-exposure therapeutic trials in mice, a single dose of humanized E16 antibody protected mice against WNV-induced mortality, and may therefore be a viable treatment option for WNV infection in humans. There are increasing evidences which suggest that DIII-specific antibodies are potent inhibitors because they block at a post-attachment stage, possibly by inhibiting viral fusion.119,120 Structural analysis of DIII in complex with E16 antibody demonstrated that E16 antibody engaged 16 residues positioned on four loops of DIII,

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a consensus neutralizing epitope sequence conserved in WNV and distinct in other flaviviruses. Additional crystallographic modeling and cryoelectron microscopy studies have shown that only 120 of the available 180 E-protein epitopes were occupied by E16 antibody, suggesting that epitope saturation was not required for neutralization. The E16 antibody neutralized WNV by blocking the initial rearrangement of the E glycoprotein before fusion with cellular membrane.114 NS1 is a cofactor in replication and is detected in the serum of infected animals during the acute phase of WNV disease.3,121 The NS1 proteins of WNV are capable of eliciting significant neutralizing antibodies in mice and offers protection against WNV infection.122,123 Studies have shown that neutralizing antibodies against WNV NS1 protein were localized to three discrete regions of the protein. Antibodies against NS1 offered protection against lethal WNV infection via Fc-gamma receptordependent pathway.104,124

6.2.2 T-cell responses CD8+ cytotoxic lymphocytes play an important role in the immune response against viruses by two mechanisms: one being cytotoxicity and the other via the release of cytokines. Cytotoxic lymphocytes execute the target cell killing through NK cells and Tc cells. Several experiments have demonstrated that T lymphocytes are an essential component of protection against WNV infection.29,30,125–127 The prevalence of CD8+ T lymphocytes in the CNS of WNV-infected mice indicated that T cells participate in recovery and immunopathology of the infected cells.127 It is known that CD8+ effector T cells are critical in clearing WNV infection from tissues and preventing viral persistence.29,127 A small subset of T cells that possess a distinct T cell receptor (TCR) on their surface are γδ T cells. γδ T cells induce a protective CD8+ T cell response against WNV.128 γδ T cells are peculiar in that they do not seem to require antigen processing and MHC presentation of peptide epitopes although some recognize MHC class IB molecules.129 These characteristics of γδ T cells could also contribute to a protective role in inhibiting WNV-induced lethal encephalitis observed by Wang and colleagues.74

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West Nile virus infection can be controlled by CD8+ T cells either by producing antiviral cytokines (e.g., IFN-γ or TNF-α) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. As mentioned earlier, WNV infection upregulates MHC class I and class II cell surface expression. Cytotoxic T lymphocytes proliferate and could recognize infected cells expressing MHC class I molecules and release proinflammatory cytokines and lyse the infected cells. Mice deficient in CD8+ T cells or class I MHC molecules showed normal humoral responses but increased viral burdens in the spleen and CNS, and increased mortality.29,127 This suggest that virus-specific antibodies are responsible for terminating viremia and CD8+ T cells have an important function in clearing infection from tissues and preventing viral persistence. Studies performed using mice deficient in granule exocytosis- and/or Fas-mediated cytolytic effector function of CD8+ T cells show that defects in either exocytosis itself (perforin and granzymes) or perforinFas-mediated cytolytic mechanisms, but not perforin alone, increase the severity of WNV encephalitis.130 In contrast to the phenotype observed with the WNV strain Sarafend-infected perforin knockout mouse, Shrestra and colleagues30 reported that perforin-deficient mice showed increased viral load and mortality, suggesting a perforin-dependent mechanism to clear WNV from infected neurons. Using genetically deficient mice, it was shown that CD40-CD40L interaction was required to facilitate T-cell migration across the BBB to control WNV infection.112 CCR5-deficient mice presented reduced T cell infiltration into the CNS, which ultimately caused increased mortality.99 The potential risk factors for developing encephalitis from WNV infection were associated with impaired T-cell function.131,132 All these studies clearly portray the function of T-cell-mediated immunity in controlling WNV infection and its disease severity. CD4+ T helper cells contribute to the clearance of acute viral infections through several mechanisms, including activation and priming of B cells and CD8+ T cells, production of inflammatory and antiviral cytokines, as well as direct cytotoxicity on infected cells. B and CD8+ T cells require co-stimulatory signals from CD4+ T cells to elicit both humoral and cellular responses. CD4+ T cells proliferate after exposure to WNV, recognize peptides from structural and non-structural proteins, and lyse infected

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Table 5. B-cell and T-cell responses during WNV infection. Mouse Strain

WNV Strain

µ MT IgM(−/−)

NY2000 NY2000

Perforin(−/−)

NY2000

Perforin(−/−) Granzyme A/B(−/−) Perforin(−/−) × Granzyme A/B(−/−) TCRδ(−/−)

Sarafend Sarafend

Isolate2741

CD4(−/−)

NY2000

Sarafend

Characteristics High viremia, increased mortality. High viremia, reduced IgG responses, increased mortality. High viremia, reduced T-cell infiltration in brain, increased mortality. No difference. High viremia in CNS and increased mortality. High viremia in CNS and moderate mortality. Early dissemination to CNS, reduced CD8 memory cells. Persistent virus load in CNS, reduced antibody response, reduced CD8+ cells recruitment, high mortality.

Ref. 40 31 30 130 130 130

128 133

targets or peptide-pulsed cells (reviewed in Ref. 67). Experiments using CD4+ T cell-deficient mice indicated that the absence of CD4+ T-cell function result in persistent WNV levels in the CNS and increased lethality. Blunted antibody response and less efficient recruitment of CD8+ cells were observed in T-cell-deficient mice, which suggest that CD4+-dependent antibody responses and active recruitment of CD8+ T cells provide protection against infection.133 Table 5 summarizes the role of adaptive immune responses during WNV infection.

7. CONCLUSION Extensive studies performed using animal models have advanced our understanding of the host immune responses elicited against WNV infection. Host immune system incite protection against WNV infection by using different arms of the immune system. The coordinated involvement of innate immunity, humoral immunity, and T-cell-mediated immunity control viral replication and its dissemination into CNS. Disruption of one or

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more of these orchestrated responses could result in severe disease. WNV have also evolved several mechanisms through which they can alter the host immune response and actively spread in the presence of an active immune system. Establishment of the disease is thus a balance between effectiveness of the host immune response and the efficacy of immune evasion by the virus. Figure 3 summarizes the innate, humoral, T-cell mediated responses against WNV infection and strategies evolved by WNV to evade host immune response. Lack of IFN-γ seems to have little impact on WNV infection in the mouse model when strains of Sarafend or KUN were used.128,134 This contrasts with the other studies reporting that IFN-γ is important to control viral infection where the more virulent New York strain was used.72 Similarly, Granzymes seem to play an important role in controlling WNV infection with the lineage II isolate Sarafend, with perforin having a more limited role in modulating infection.130 In contrast, CD8+ T cells require perforin to control lineage I WNV infection.30 Similarly, lineage I and II viruses responded differently in blocking JAK/STAT signaling pathway.69 One of the possible explanations for these discrepancies could be the use of WNV strains of different virulence. These discrepancies illustrate that virus-host interaction and dependency on immune responses cannot be generalized even between different strains of the same type of virus, WNV in this case. PKR and RNase L mediate the antiviral effects of IFN in cortical neurons after WNV infection, but these molecules were dispensable for protection in peripheral neurons.80 The nutshell of these divergences is that different strains of the same virus and different cell types could stimulate the type and the extent of immune responses differentially. All these factors must be considered carefully while testing antiviral drugs or vaccines against WNV. A number of important issues remain to be addressed at this moment: (1) How does the virus remain undetected by the host immune system during early infection while it utilizes host transcription and translation machinery for active replication? (2) What kind of immune responses result in symptomatic and asymptomatic disease pattern? (3) What kind of immune responses decide the severity of symptomatic disease? Cell death or neuronal injury could be a consequence of two factors: the immune response initiated to eradicate virus becomes pathological, causing

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Figure 3. Activation of different arms of host immune responses and the immune evasion by WNV. Host cell triggers various signaling pathways such as interferons, interferon-regulatory factors, NF-κ B activation, complement, chemokines, proinflammatory cytokines, B-cell and T-cell responses following WNV infection. These innate and adaptive immune responses act together to eliminate the virus. Long dashed lines represent the connecting bridge between innate and adaptive immunity during WNV infection. Explosions in red represent the strategy of WNV to evade the host responses. WNV attenuates the host immune responses through several mechanisms. WNV achieved this immune evasion by suppressing the activation of IRFs: (1), by activating NF-κ Brepressing factor; (2), by blocking JAK/STAT pathway; (3), during early infection and by decreasing complement activation (4).

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immune-mediated damage to the infected neurons or the inability of the immune responses to control the viral infection. To reiterate, such factors must be considered carefully while designing vaccines against WNV. The desired vaccine should elicit the protective antiviral immune response and at the same time reduce the degree of immune-mediated destruction.

ACKNOWLEDGMENT We apologize to the authors of many papers that are significant to the subject, but not cited in this review due to the space limitation.

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92. Liu WJ, Wang XJ, Clark DC, Lobigs M, Hall RA, Khromykh AA (2006). J Virol 80, 2396–2404. 93. Evans JD, Seeger C (2007). J Virol 81, 11809–11816. 94. Munoz-Jordan JL, Laurent-Rolle M, Ashour J, Martinez-Sobrido L, Ashok M, Lipkin WI, Garcia-Sastre A (2005). J Virol 79, 8004–8013. 95. Cheeran MC, Hu S, Sheng WS, Rashid A, Peterson PK, Lokensgard JR (2005). J Neurovirol 11, 512–524. 96. Shirato K, Kimura T, Mizutani T, Kariwa H, Takashima I (2004). J Med Virol 74, 507–513. 97. Garcia-Tapia D, Hassett DE, Mitchell WJ Jr, Johnson GC, Kleiboeker SB (2007). J Neurovirol 13, 130–138. 98. Klein RS, Lin E, Zhang B, Luster AD, Tollett J, Samuel MA, Engle M, Diamond MS (2005). J Virol 79, 11457–11466. 99. Glass WG, Lim JK, Cholera R, Pletnev AG, Gao JL, Murphy PM (2005). J Exp Med 202, 1087–1098. 100. Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, Frank WA, Pape J, Cheshier RC, Murphy PM (2006). J Exp Med 203, 35–40. 101. Lim JK, Glass WG, McDermott DH, Murphy PM (2006). Trends Immunol 27, 308–312. 102. Mehlhop E, Whitby K, Oliphant T, Marri A, Engle M, Diamond MS (2005). J Virol 79, 7466–7477. 103. Mehlhop E, Diamond MS (2006). J Exp Med 203, 1371–1381. 104. Chung KM, Liszewski MK, Nybakken G, Davis AE, Townsend RR, Fremont DH, Atkinson JP, Diamond MS (2006). Proc Natl Acad Sci USA 103, 19111–19116. 105. King NJ, Maxwell LE, Kesson AM (1989). Proc Natl Acad Sci USA 86, 911–915. 106. Lobigs M, Mullbacher A, Lee E (2004). Immunol Cell Biol 82, 184–188. 107. Momburg F, Müllbacher A, Lobigs M (2001). J Virol 75, 5663–5671. 108. Cheng Y, King NJ, Kesson AM (2004). Virology 329, 361–370. 109. Shen J, Devery JM, King NJ (1995). Immunology 84, 254–264. 110. Ben-Nathan D, Lustig S, Tam G, Robinzon S, Segal S, Rager-Zisman B (2003). J Infect Dis 188, 5–12. 111. Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, Gorlatov S, Mehlhop E, Marri A, Chung KM et al. (2005). Nat Med 11, 522–530.

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112. Sitati E, McCandless EE, Klein RS, Diamond MS (2007). J Virol 81, 9801–9811. 113. Chu JH, Chiang CC, Ng ML (2007). J Immunol 178, 2699–2705. 114. Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, Fremont DH, Kuhn RJ, Rossmann MG (2006). Proc Natl Acad Sci USA 103, 12400–12404. 115. Li L, Barrett AD, Beasley DW (2005). Virology 335, 99–105. 116. Oliphant T, Nybakken GE, Engle M, Xu Q, Nelson CA, Sukupolvi-Petty S, Marri A, Lachmi BE, Olshevsky U, Fremont DH, et al. (2006). J Virol 80, 12149–12159. 117. Oliphant T, Nybakken GE, Austin SK, Xu Q, Bramson J, Loeb M, Throsby M, Fremont DH, Pierson TC, Diamond MS (2007). J Virol 81, 11828–11839. 118. Gould LH, Sui J, Foellmer H, Oliphant T, Wang T, Ledizet M, Murakami A, Noonan K, Lambeth C, Kar K, et al. (2005). J Virol 79, 14606–14613. 119. Gollins SW, Porterfield JS (1986). Nature 321, 244–246. 120. Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH (2005). Nature 437, 764–769. 121. Macdonald J, Tonry J, Hall RA, Williams B, Palacios G, Ashok MS, Jabado O, Clark D, Tesh RB, Briese T, et al. (2005). J Virol 79, 13924–13933. 122. Chung KM, Nybakken GE, Thompson BS, Engle MJ, Marri A, Fremont DH, Diamond MS (2006). J Virol 80, 1340–1351. 123. Lieberman MM, Clements DE, Ogata S, Wang G, Corpuz G, Wong T, Martyak T, Gilson L, Coller BA, Leung J, et al. (2007). Vaccine 25, 414–423. 124. Chung KM, Thompson BS, Fremont DH, Diamond MS (2007). J Virol 81, 9551–9555. 125. Kesson AM, Blanden RV, Mullbacher A (1987). J Gen Virol 68 (Pt 7), 2001–2006. 126. Liu Y, Blanden RV, Mullbacher A (1989). J Gen Virol 70 (Pt 3), 565–573. 127. Wang Y, Lobigs M, Lee E, Mullbacher A (2003). J Virol 77, 13323–13334. 128. Wang T, Gao Y, Scully E, Davis CT, Anderson JF, Welte T, Ledizet M, Koski R, Madri JA, Barrett A, et al. (2006). J Immunol 177, 1825–1832. 129. Holtmeier W, Kabelitz D (2005). Chem Immunol Allergy 86, 151–183. 130. Wang Y, Lobigs M, Lee E, Mullbacher A (2004). Immunol Cell Biol 82, 170–173.

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131. Murray K, Baraniuk S, Resnick M, Arafat R, Kilborn C, Cain K, Shallenberger R, York TL, Martinez D, Hellums JS, et al. (2006). Epidemiol Infect 134, 1325–1332. 132. Pruitt AA (2004). Semin Neurol 24, 435–452. 133. Sitati EM, Diamond MS (2006). J Virol 80, 12060–12069. 134. Wang Y, Lobigs M, Lee E, Koskinen A, Mullbacher A (2006). J Gen Virol 87, 3599–3609.

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CHAPTER 19

Hepatitis C Virus (Flaviviridae): Host Immune Responses to Hepatitis C Virus Christoph Neumann-Haefelin & Robert Thimme

ABSTRACT Hepatitis C virus (HCV) causes chronic infection in approximately two third of cases, leading to liver disease, liver failure, and hepatocellular carcinoma in a substantial proportion of those 170 million people infected worldwide. The virus has developed multiple mechanisms to evade innate, humoral, and cellular immune responses which will be discussed in this chapter. Although HCV rapidly induces a strong innate immune response, viral replication is not sufficiently suppressed in the early phase of infection. HCV interrupts recognition of viral double-stranded RNA and also interferes with the interferon (IFN) response. Several lines of evidence suggest that the cellular immune response plays the most important role in determining the outcome of HCV infection. Firstly, vigorous, multispecific and sustained CD4+ and CD8+ T cell responses have been associated with viral clearance. Secondly, depletion studies in chimpanzees, the only animal which can be infected with HCV, have shown that both, CD4+ and CD8+ T cells are required for the elimination of HCV. Thirdly, the host’s HLA alleles which restrict the repertoire of CD4+ and CD8+ T cell responses influence the outcome of infection. However, multiple mechanisms may be involved in the failure of the HCV-specific T cell response in the majority of patients who progress to viral persistence. Note that protective immunity has been 445

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demonstrated in population-based studies as well as experimentally infected chimpanzees. Thus, it seems reasonable that the precise understanding of the mechanisms contributing to the failure of the antiviral immune response may allow the development of successful prophylactic and therapeutic vaccination strategies.

1. INTRODUCTION Infection with hepatitis C virus (HCV) results in chronic infection in approximately two thirds of individuals, causing chronic liver inflammation, fibrosis and finally cirrhosis with a high risk of hepatocellular carcinoma in a subset of patients. With an estimated 170 million people infected worldwide, HCV represents a major global healthcare issue. Current therapy of chronic HCV infection consists of pegylated interferon-α and ribavirin, which is associated with a wide range of side effects, and leads to viral clearance in only 50 to 80% of patients, depending on viral genotype. Thus, the development of more efficient and better tolerable treatment regimes and the design of prophylactic or therapeutic vaccine strategies are great challenges. Clearly, a better understanding of the mechanisms of HCV immunity is necessary for the establishment of these new therapies. In the last few years, tremendous work was performed in order to better characterize the host immune response to HCV and reveal the multiple mechanisms by which HCV evades from this immune response in the majority of cases. In this chapter we will describe the innate, humoral, CD4+ and CD8+ T cell responses to HCV infection and discuss the mechanisms of viral immune evasion.

2. INNATE IMMUNE RESPONSE Innate immunity includes a wide range of different mechanisms, such as physical and chemical barriers, innate immune cells (e.g., macrophages, dendritic cells, natural killer cells), and cytokines such as type I interferons. Indeed, type I interferons, including IFN-α and IFN-β , are of special interest in HCV infection, since (pegylated) IFN-α in combination with ribavirin is the standard treatment regime in chronic HCV infection, leading to viral clearance in 50–80% of patients (depending on viral genotype).

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In the natural course of infection, type I interferons are thought to play an important role in the initial control of viral spread, until an adaptive immune response has been established. We will briefly introduce interferon induction, cellular interferon signaling, interferon response, and summarize some evasion mechanisms by which HCV evades from the type I interferon system.1,2 There are two different pathways of interferon induction by viral infections. Firstly, viral RNA can be sensed by two intracellular RNA helicases, RIG-I and MDA5, starting a signal cascade which finally results in IFN-β mRNA synthesis (for details, see Figure 1A). Secondly, the signal cascade can be induced after sensing of virus by toll-like receptor (TLR) 3 and/or 7 (Figure 1A). After a first wave production of IFN-β , positive feedback enhances IFN-β production and leads to the additional synthesis of IFN-α subtypes as a second wave. IFN-α and β bind to a common type I interferon receptor, activating the JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway that leads to the induction of interferon-stimulated genes (ISGs) (for details, see Figure 1B). More than 300 ISGs with antiviral, antiproliferative, and immunomodulatory functions have been identified. In experimentally infected chimpanzees, it has been demonstrated that HCV infection rapidly induces a strong type I interferon response,3–5 and type I interferons have been shown to suppress viral replication in the replicon model.6 In addition, several ISGs have been shown to inhibit HCV, including protein kinase R (PKR),7 the RNA-specific adenosine deaminase 1 (ADAR 1),8 the 2′-5′ oligoadenylate synthetase (2–5 OAS)/ RNaseL system,9 and P56.10 However, the induction of ISGs was demonstrated irrespective of outcome of acute infection.3–5 Several mechanisms have been identified by which HCV disturbs the type I interferon response at different levels, e.g., interferon induction, interferon signaling, and function of ISGs. With respect to interferon induction, the NS3/4A protease specifically cleaves Cardif as well as TRIF, thus blocking both pathways of interferon induction (compare Figure 1).11,12 In addition, NS3 can directly interact with TBK1 and inhibit its activation.13 With respect to interferon signaling, the core protein suppresses JAK-STAT signaling by inducing different inhibitors of this pathway, such as SOCS-3 and PIAS-1.14,15

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Figure 1. Type I Interferon System. (A) Type I interferon induction. Double-strand RNA (dsRNA) is sensed by the RNA helicases RIG-I and MDA-5. The adaptor protein Cardif (CARD adaptor inducing IFN-β ), also called IPS-1 (IFN-β promotor stimulator 1), VISA (virus-induced signaling adaptor), or MAVS (mitochondrial antiviral signaling), activates the kinases TBK-1 and IKKε which phosphorylate IRF-3 (interferon regulatory factor). Upon dimerization and translocation to the nucleus, IRF-3 initiates IFN-β mRNA synthesis. (B) Intracellular Type I interferon signaling. Binding of IFN-α or -β to the type I interferon receptor (IFNAR) leads to the heterodimerization of the receptor subunits, leading to the activation of the Janus kinases JAK-1 and TYK-2 which in turn phosphorylate STAT-1 and -2. STAT-1, -2, and IRF-9 bild a heterotrimer (called ISGF-3, interferon stimulated gene factor 3) which translocates to the nucleus and initiates mRNA synthesis of interferon stimulated genes (ISGs), e.g., protein kinase R (PKR), RNA-specific adenosine deaminase 1 (ADAR-1), 2′–5′ oligoadenylate synthetase (OAS), and P56.

HCV proteins can also interfere with different ISG products. For example, NS5A binds to the dimerization domain of PKR and thus interrupts kinase dimerization. As a result, PKR-mediated phosphorylation of the eukaryotic translation initiation factor 2 is abolished, leading to unbridled translation of cellular and viral mRNA.16 In sum, HCV has developed

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mechanisms to evade the strong type I interferon response at different levels, inhibiting definitive viral control by type I interferons. NK cells are also an important component of the innate immune system, secreting antiviral cytokines and lysing infected cells at an early stage of viral infections. They are regulated by different activating and inhibitory signals, including the interaction between activating or inhibitory KIRs and HLA class I molecules on target cells. Of note, different KIRs have been shown to be associated with different outcomes of HCV infection, e.g., KIR2DL1, 2 and 3.17 KIR2DL1 is present in almost all individuals and strongly binds HLA-C alleles with a lysine at position 80 (HLA-C2), leading to a strong inhibitory signal on NK cells. KIR2DL2 and especially 3, in contrast, bind only weakly to HLA-C alleles with an asparigine at position 80 (HLA-C1), and thus only result in weak inhibition of NK cells. Probably caused by this reduced threshold of NK cell activation, individuals homozygous for KIR2DL3 and HLA-C1 alleles have been reported to have a greater chance of eliminating HCV; the frequency of the homozygous genotype was ∼20% in individuals with resolved HCV infection, but only ∼10% in individuals with persistent infection after needle stick infection.17 This protective effect was not found in individuals who obtained inocula with high viral titres (e.g., contaminated blood transfusions). This might be due to the inhibition of NK cell function by viral factors such as the HCV E2 glycoprotein.18,19

3. HUMORAL IMMUNE RESPONSE During acute infection, the vast majority of individuals produce antibodies against many epitopes within the structural as well as non-structural proteins. While these antibodies serve as a screening parameter for the diagnosis of acute, chronic, or resolved HCV infection, most of them have no relevant antiviral activity. Only a small fraction of antibodies is able to inhibit virus binding, entry, or uncoating. These antibodies can potentially abolish HCV infectivity and are called “neutralizing antibodies”. The first evidence for the existence of neutralizing antibodies in HCV infection came from studies in experimentally HCV-infected chimpanzees.20 The association of antibodies targeting similar epitopes with viral clearance in patients suggested that antibodies with neutralizing effect also exist in

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man. However, the further characterization of neutralizing antibodies has been hampered by the lack of suitable model systems for many years. Recently, growing knowledge about neutralizing antibodies has been forthcoming, stemming from the use of recombinant HCV envelope glycoproteins, HCV-like particles which are produced by self-assembly of HCV structural proteins in insect cells, and by HCV pseudotype particles consisting of HCV envelope glycoproteins assembled on retroviral or lentiviral core particles. Using these different in vitro models, multiple linear as well as conformational epitopes targeted by neutralizing antibodies have been identified in the envelope glycoproteins E1 and E2, with a “hot-spot” in and adjacent to the hypervariable region 1 (HVR-1). These epitope regions have important functions in cell binding and entry, including CD81 binding and membrane fusion. In chronic infection, HCV-specific neutralizing antibodies can be detected in most patients, and multiple mechanisms for failure of the humoral immune response have been suggested. For example, evolution of viral quasispecies within targeted epitopes may lead to continuous escape from neutralizing antibodies;21 interactions of HCV glycoproteins with high-density lipoprotein (HDL) and the HCV receptor scavenger receptor B1 (SR-B1) may accelerate viral entry and protect from neutralizing antibodies22; specific glycans on envelope protein E2 also modulate cell entry and confer protection from neutralizing antibodies23; and conformational changes or binding of non-neutralizing antibodies may prevent binding of neutralizing antibodies.24 The role of neutralizing antibodies in acute infection, and most importantly, in viral clearance has been poorly defined so far. Many studies have suggested that the majority of patients with acute-resolving HCV infection lack neutralizing antibodies and that patients with a chronic course of infection develop neutralizing antibodies only after persistence has been established.25,26 A recent study, however, detected neutralizing antibodies in the early phase of infection in the majority of patients with resolving HCV infection, while patients with a chronic course of infection showed a delayed induction of neutralizing antibodies.27 This study was performed in a well-characterized and homogenous group of young women infected by an anti-D immunoglobulin charge contaminated with the

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same viral inoculum. Thus, in contrast to previous studies, heterogeneity between the autologous infecting virus and the viral strain used when testing for neutralizing antibodies could be avoided. Although this study showed a strong association between an early neutralizing antibody response and HCV clearance, it remains elusive whether the neutralizing antibody response indeed mediates viral clearance. Alternatively, they may be the result of efficient CD4+ help that has been shown to be associated with viral clearance.

4. T CELL RESPONSE 4.1 CD4+ and CD8+ T Cell Response in Acute HCV Infection The important roles of CD4+ and CD8+ T cells in HCV clearance have been demonstrated by depletion studies in chimpanzees: following the antibody-mediated depletion of CD8+ T cells, experimental infection of a chimpanzee led to the persistence of HCV viremia until the CD8+ T cells recovered and an HCV-specific CD8+ T cell response emerged.28 After depletion of CD4+ T cells, however, viremia also persisted and CD8+ escape variants emerged, finally resulting in chronic HCV infection.29 Studies of acutely HCV-infected patients revealed that a strong, multispecific and sustained HCV-specific CD4+ T cell response (Figure 2A) is associated with a self-limited course of infection.30–32 Recently, a comprehensive analysis of the HCV specific CD4+ T-cell response was performed using overlapping peptides covering the NS3/NS4 proteins33 and the complete HCV protein, respectively.34 These studies identified ten and six peptides, respectively, which were each recognized in >30% of patients with acute resolving HCV infection demonstrating the existence of immunodominant CD4+ T cell epitopes. With respect to CD8+ T cells, vigorous virus-specific CD8+ T cell responses that target multiple epitopes (Figure 2A) can be detected approximately four to eight weeks after infection with an acute-resolving course, and their emergence is temporally associated with the onset of liver disease.32,35,36 However, the virus-specific CD8+ T cells are not able to secrete antiviral cytokines such as IFN-γ in this early phase of infection, a status referred to as “stunned phenotype”.32,35 In a later phase of infection, virus-specific CD8+

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C Figure 2. T Cell Response to HCV Infection. (A) Virus-specific T cell responses are strong, multi-specific, and sustained in acute resolving HCV infection. (B) Virus-specific T cell responses may be initially similiarly strong and multi-specific, but wane in the course of actue persisting HIV infection. (C) Virus-specific T cell responses are weak and narrowly focused or even absent in chronic HCV infection.

T cells regain their ability to secrete antiviral cytokines, and this is temporally associated with a rapid decline of viremia and finally viral clearance. Knowledge about the intrahepatic virus-specific CD4+ and CD8+ T cell response during acute HCV infection was obtained from experimentally infected chimpanzees. These studies revealed that virus-specific T cell responses accumulate in the liver 8–14 weeks after infection and coincide with liver disease as well as viral clearance.5,37 After resolution of infection, virus-specific CD4+ and CD8+ T cell responses persist for decades and can even outlast humoral responses.38 Virus-specific T cells also play a role in mediating protective immunity. Indeed, evidence for protective immunity came from both, epidemiological studies,39 as well as experimental studies. For example, chimpanzees re-challenged by HCV

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showed a shorter period and lower level of viremia than naïve animals.40 Sterilizing immunity against HCV, however, may not exist, since multiple episodes of heterologous or homologous re-infection have been observed in both humans and chimpanzees. In contrast to acute resolving HCV infection, T cell responses are thought to be weaker and only monospecific or even absent in acute persisting HCV infection. Indeed, patients without any evidence of control of HCV replication often lack any significant CD4+ T cell response.2 A larger subset of patients who transiently control viral replication but then progress to chronic infection, initially displays HCVspecific CD4+ T cell responses, but these responses are not sustained, and disappearance of CD4+ T cell responses is followed by a viral rebound (Figure 2B). Complete physical disappearance of virus-specific CD4+ T cells is proceeded by functional alterations, including reduced proliferation and IFN-γ production.41 Regarding the role of CD8+ T cells during acute persistent HCV infection, the picture is currently less clear. Previous reports comparing the CD8+ T cell response in acute resolving versus acute persisting HCV infection in chimpanzees5,37 and man32,42 found significantly weaker and more narrowly focused virus-specific CD8+ T cell responses in those subjects developing persistent infection. More recent studies, however, could not confirm this finding.36,43,44 For example, Cox et al. performed a prospective longitudinal study in young i.v. drug users and analyzed the T cell response in four individuals with resolution of acute HCV infection and 15 individuals who progressed to chronic infection. Although all four individuals with resolving infection mounted virus-specific CD8+ T cell responses and those four individuals who lacked CD8+ T cell responses developed chronic infection, the CD8+ T cell response did not differ significantly between resolvers and persistenly infected individuals.36 Urbani et al. studied six patients with acute resolving and 11 patients with acute persisting HCV infection and found an association between strong and multispecific CD4+, but not CD8+ T cells with viral clearance. However, patients developing chronic infection displayed prolonged CD8+ T cell dysfunctions and maturational defects.43 This discordant role of CD4+ and CD8+ T cells was confirmed by Kaplan et al. albeit their analysis was limited to two HLA-A2 restricted CD8+ T cell epitopes.44

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The important role of virus-specific T cell responses in the outcome of HCV infection is further supported by the finding that class I and II alleles are associated with the natural course of infection. Indeed, since CD8+ T cells recognize antigens presented by human leukocyte antigen (HLA) class I molecules it has been suggested that different HLA class I alleles are associated with differential outcome of HCV infection, e.g., viral clearance versus persistence.45 A cohort of Irish women accidentally infected with HCV (genotype 1b) from a single source more than 20 years ago, represents a homogeneous group in which the role of HLA alleles in the outcome of HCV infection could be studied.46 Importantly, the HLA class I alleles A3, B27 and Cw*01 were significantly associated with viral clearance, while B8 was associated with viral persistence. Interestingly, the strongest protective effect was observed for HLA-B27: 80% (12/15) of B27 positive women were able to clear the infection spontaneously, while only a minority developed chronic infection. We recently identified an immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope, which was targeted in the majority (5/6) of B27 positive Irish women who had cleared the infection.47 Of note, such a clear dominance of a single epitope-specific CD8+ T cell response has not been described for any other HLA allele in HCV infection. Thus, a single immunodominant HLA-B27 restricted CD8+ T cell epitopes might mediate clearance of HCV infection in the majority of B27 positive individuals, further indicating a dominant role of virus-specific CD8+ T cell responses in HCV infection. While HLA class I molecules present antigens to CD8+ T cells, HLA class II molecules restrict CD4+ T cell responses. Similar to the findings described for HLA class I, certain HLA class II alleles have been correlated with different outcomes of HCV infection. In heterogenous study cohorts, the HLA class II alleles most reproducibly associated with viral clearance are DRB1*1101 and DQB1*0301, which are genetically closely linked, a phenomenon referred to as linkage disequilibrium.48 The results from the well-defined Irish cohort contrasted somewhat, since in this cohort, DRB1*01, DRB1*0401, and DRB1*15 were protective alleles.46 It is somewhat intriguing that most of the CD4+ T cell epitopes described so far are restricted by HLA alleles for which a protective effect has been shown; however, it is important to point out that CD4+ T cell epitopes are

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highly promiscuous and can often be restricted by multiple HLA class II molecules.

4.2 CD8+ T Cell Response in Chronic HCV Infection In contrast to acute resolving infection, CD4+ T cell responses are barely detectable in chronic HCV infection (Figure 2C).30,41,49 In addition, HCVspecific CD4+ cells circulating at a very low frequency in chronically infected patients, have a strongly impaired proliferative capacity41,50 and show an altered cytokine production pattern, with very low IL-2 production, but rather enhanced IFN-γ production.51 CD8+ T cell responses are usually weak or even absent in chronic HCV infection, targeting only few epitopes52–56 (Figure 2C). In this context, it is important to point out that in at least some chronically infected patients, the CD8+ T cell response targets several epitopes.53,56 Importantly, however, these HCV-specific CD8+ T cells display functional impairments, including reduced cytotoxicity, reduced secretion of antiviral cytokines such as IFN-γ , and a reduced proliferative capacity.57–59 In addition, many CD8+ T cell responses do not target the present antigen, but rather a historical antigen due to viral escape (see below). Several different mechanisms might be involved in the failure of the HCV-specific CD4+ and CD8+ T cell response in viral clearance in the majority of infected patients (Figure 3). Currently, most experimental data is available for CD8+ T cell failure, thus, we will focus on this T cell subset in the following.

4.3 Mechanisms of T Cell Failure 4.3.1 Primary failure and exhaustion As discussed above, some patients with chronic HCV infection lack strong and multispecific CD8+ T cell responses; however, it is difficult to know if virus-specific CD8+ T cell responses were not primed initially (primary CD8+ T cell failure) or responses were primed, but vanished quickly (CD8+ T cell exhaustion). Results obtained from the early phase of acute HCV infection in chimpanzees5,37 and in health care workers

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CD8 CD4+ CD25+

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PD1

regulatory CD4+ T cells

T cell dysfunction CD8+ FoxP3

regulatory CD8+ T cells

CD4+ CD4+ T cells

Figure 3. Mechanisms of Failure of HCV Immune Control. T cell dysfunction might be mediated by regulatory T cells, suppressive cytokines (e.g., IL-10) or receptors (PD-1), or by the lack of CD4+ help. In addition, T cell failure can be caused by the evolution of immune escape mutants.

infected through needlestick exposure32 support the hypothesis that CD8+ T cells are not primed in at least some patients with acute persisting HCV infection. An impaired priming of HCV-specific CD8+ T cells might be mediated by numeric and functional impairments of antigen-presenting cells, e.g., macrophages and dendritic cells.60–64 However this topic remains controversial.65,66 CD8+ T cell exhaustion might be explained by unspecific general as well as HCV-specific mechanisms. With respect to HCV-specific mechanisms of CD8+ T cell exhaustion, the core protein has been reported to impair CD8+ T cell activation, e.g., through interaction with membranebound complement receptor gC1qR.67,68

4.3.2 Lack of CD4+ help While CD8+ T cells are considered as the major effector cells against viral pathogens, the successful elimination of HCV probably also depends on sufficient CD4+ T cell help. Indeed, it has been demonstrated in the LCMV mouse model that CD4+ T cell help is needed to sustain cytotoxic

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CD8+ T cell responses during chronic viral infections.69 In chronic HCV infection, however, CD4+ T cell responses are either weak or even absent30 and functionally impaired, e.g., secrete low amounts of IL-2.51 Findings in the chimpanzee model support the central role of CD4+ help in CD8+ T cell-mediated viral clearance: When CD4+ T cells were depleted by neutralizing antibodies prior to viral re-challenge, HCV viremia was prolonged, CD8+ escape variants were selected and HCV finally persisted.29 Consistent with this concept, HCV-specific CD8+ T cell responses were seen almost exclusively in the face of a strong CD4+ T cell response in a study of acutely HCV-infected patients.42 A recent study demonstrated that the outcome of acute HCV infection was associated with efficient virusspecific CD4+ T cell responses. In this study, however, HCV-specific CD8+ T cell responses were induced irrespective of virological outcome or HCV-specific CD4+ T cell responses.44

4.3.3 Suppression by regulatory T cells In the last few years, the concept of regulatory T cells has undergone a comeback and different types of regulatory T cells have been characterized in different settings. In HCV infection, a possible role of CD4+ CD25+ Foxp3+ regulatory T cells as well as IL-10 producing CD8+ T cells has been suggested. In chronically HCV-infected patients, CD4+ CD25+ T cells have been found at a higher frequency compared to individuals with resolved HCV infection or healthy controls.70–72 These regulatory T cells suppress the proliferation as well as interferon-gamma secretion of virus-specific CD8+ T cells in vitro. The suppression by CD4+ CD25+ T cells was cell-cell contact-dependent70,71 and independent of suppressive cytokines such as IL-10 and TGF-β in some70,73 but not all studies.71 Interestingly, the suppression was not restricted to HCV-specific CD8+ T cells, but also included CD8+ T cells specific for other viruses, such as EBV or influenza.70,73 However, specificity in vivo might be mediated by the enrichment of CD4+ CD25+ T cells in the liver74 where they might limit immunopathology in the chronic phase of HCV infection. The mechanisms responsible for the induction of CD4+ CD25+ regulatory T cells are still poorly understood; however, it has been shown that they can be induced by certain HCV peptides

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from peripheral blood mononuclear cells (PBMCs) from HCV-infected, but not healthy individuals in vitro.75 Another type of regulatory T cells in HCV infection are virusspecific regulatory CD8+ T cells that express high levels of IL-10. These regulatory T cells have been detected in the liver of HCV-infected individuals and their suppression of virus-specific CD8+ effector T cells could be blocked by neutralizing IL-10 antibodies.76 The spectrum of regulatory T cells involved in HCV infection may further expand, since we recently described the induction of regulatory CD8+ T cells from the PBMC of HCV-infected patients which also expressed high levels of FoxP3 and CD25.77

4.3.4 Inhibitiory receptors: PD-1 The inhibitory receptor PD-1 (“programmed cell death 1”) has been demonstrated to be a strong marker for exhausted virus-specific CD8+ T cells in the LCMV mouse model. The antibody-mediated blockade of the interaction between PD-1 and its ligand PD-L1 led to the restoration of cytokine secretion, proliferation, and cytotoxicity by the exhausted virusspecific CD8+ T cells and a substantial reduction in viral load.78 Similar roles of PD-1 have been shown in human chronic viral infections (reviewed in Ref. 79). Indeed, in the acute phase of HCV infection, PD-1 is upregulated in HCV-specific CD8+ T cells, independent of outcome. However, in individuals with resolving infection, PD-1 expression decreases soon, while in patients with a chronic course of infection, HCVspecific CD8+ T cells remain PD-1 positive.80 This finding may help to explain the “stunned” phenotype of HCV-specific CD8+ T cells in the early acute phase of infection, which is restored in resolving infection but remains in persisting infection.32,35,58 In chronic HCV infection, virusspecific CD8+ T cells in the peripheral blood81 as well as in the liver82 have been shown to express high levels of PD-1. Blockade of PD-1/ PD-L1 interaction by antibodies restored cytokine production and proliferation of the exhausted CD8+ T cells from acute and chronic infection in vitro. It is important to note, however, that the antibody-mediated blockade of the PD-1/ PD-L1 pathway in chronically LCMV-infected mice did not result in viral clearance, although a significant reduction of viral load was

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achieved. Even more importantly, PD-L1−/− mice died due to immunopathologic damage after infection with a LCMV strain usually established persistent infection.78 These findings indicate that a subtle balance in the blockade of the PD-1/ PD-L1 pathway must be granted before it can be applied in clinical studies.

4.3.5 Inhibitory cytokines: IL-10 Recently, two reports about the role of IL-10 in the dysfunction of virusspecific T cells and viral persistence gained much attention. These reports showed that IL-10 was highly upregulated early in infection in mice with persistent LCMV infection and that this was associated with the dysfunction of virus-specific CD4+ and CD8+ T cells. The blockade of the IL-10/ IL-10 receptor (IL-10R) pathway by a genetic approach or by an anti-IL10R antibody early in infection, however, led to the restoration of T cell function and to clearance of infection.83,84 Of note, a role of IL-10 in HCV infection has been postulated for quite some time85 and IL-10 therapy has even been tested in clinical trials in HCV-infected patients. IL-10 administration led to a decrease in transaminases and reduced histological disease progression; however, viral titers strongly increased in some IL-10 treated patients.86 This indicates that IL-10 might not only mediate viral dysfunction and thus facilitate viral persistence in acute infection, but may also reduce immunopathology in the chronic phase of infection. In patients with acute persistent HCV infection, CD4+ T cells produce significantly more IL-10 (and IL-4) compared to patients with an acute resolving course.87 Blockade of the IL-10 pathway by anti-IL10R antibodies in vitro led to increased HCV-specific CD4+ and CD8+ T cell responses T cell responses. A direct inhibition of the IL-10 pathway, however, needs further careful evaluation in additional animal models before it can be transferred to man.

4.3.6 Viral escape HCV is an RNA virus with an enormous replication rate (approximately 1012 virions per day) with a RNA-dependent RNA polymerase that lacks

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a proofreading function. Therefore, multiple viral variants, called quasispecies, circulate in a single individual. It has been suggested that the selection of viral variants escaping from CD8+ T cell responses might facilitate the persistence of HCV infection. Indeed, the first evidence for viral escape in HCV infection came from chronically infected patients88 and experimentally infected chimpanzees.89 Indeed, it was shown that chronically infected patients harboured variant viral sequences in targeted CD8+ T cell epitopes which were non-immunogenic and not cross-reactive with the prototype peptides. These viral escape mutations remained fixed over a follow-up time of up to four years, indicating that escape mutations occur early in infection.88 In the chimpanzee model, it could further be demonstrated that viral escape mutations occurred during the first 16 weeks of infection and were associated with a chronic course of infection.89 Important additional information came from studies in acutely infected patients90,91 as well as population-based approaches.90,92 In these studies, viral escape from CD8+ T cell responses was demonstrated in patients developing persistent infection,90,91,93 but not in individuals with resolving infection.91,93 Interestingly, many mutations outside of targeted CD8+ T cell epitopes represented conversion to consensus,91 and the transmission of an HLA-B8 associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation.90 These results were supported by a study in a well-defined cohort of Irish women accidentally infected with HCV from a single source more than 20 years ago. Indeed, in this unique cohort, amino acid substitutions in known epitopes were directed away from consensus in women having the HLA allele associated with that epitope, and toward consensus in those lacking the allele.92 These findings were in agreement with the concept of viral fitness cost, indicating that viral escape mutations are often associated with a reduced replicative capacity of the virus.94 In the absence of the T cell pressure, e.g., upon transmission to an individual negative for the restricting HLA allele, the virus reverts to consensus and thus regains its full replicative capacity. More importantly, there might be some CD8+ T cell epitopes which are not affected by viral escape due to high functional constraints. For example, we have recently identified an HLA-A26 restricted epitope located

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at the NS5A/5B cleavage site which was targeted in all studied HLAA26+ patients (3/3) with acute HCV infection and a significant number of patients with chronic HCV infection (3/15). However, the epitope sequence was highly conserved in HLA-A26 positive and negative patients, indicating that viral escape did not occur in this functionally constrained region.95 Based on the finding that immunodominant CD8+ T cell epitopes leave their footprint in viral sequences in chronic HCV infection,90 viral genome sequencing studies were performed in order to identify footprints of additional potential CD8+ T cell epitopes.96,97 In addition to previously defined epitopes, these studies identified HLA allele-dependent polymorphisms and thus candidate CD8+ T cell epitopes. Importantly, the strongest association with any HLA allele in the study by Timm et al. was found for HLA-B27 in a region that was shown to contain an immunodominant HLA-B27 restricted CD8+ T cell epitope by an independent study from our group in another patient cohort.47 There are different molecular mechanisms through which a certain mutation mediates escape from the CD8+ T cell response. Especially those mutations located at the HLA binding anchors, usually P2 and the C-terminal amino acid, lead to the interruption of the peptide binding to the HLA molecule. Mutations in the center of the epitope, in contrast, are more likely to interfere with T cell receptor (TCR) recognition.98 Mutations in the flanking region, however, may prevent proteasomal epitope processing.90,99 The determinants of viral escape are less understood. In the chimpanzee model of HCV, it has been shown that upon depletion of CD4+ T cells in the acute phase of infection, viral escape from the CD8+ T cell response occurs and is associated with a persistent course of infection.29 This finding has led to the hypothesis that viral escape is caused by insufficient CD4+ help. Other studies indicate that a limited T cell receptor (TCR) diversity might be responsible for viral escape.100 Of note, viral escape does not occur in the context of dysfunctional CD8+ T cell responses.101 The strong association between HLA-B27 and viral escape within an immunodominant HLA-B27 restricted epitope,47,97 as well as the suggestion that escape variant epitopes might preferentially be restricted by HLA-B alleles,96,97 indicate that the restricting HLA

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allele background also plays an important role in determining viral escape.

4.3.7 Lack of homing to the liver Experimentally HCV-infected chimpanzees which progressed to viral persistence without temporary viral control lacked virus-specific CD8+ T cell responses in the liver despite detectable responses in the peripheral blood.32 This finding led to the tempting hypothesis that the failure of the virus-specific CD8+ T cell response might be caused by an insufficient homing to the primary location of infection, the liver. However, in chronically HCV-infected patients, virus-specific CD8+ T cells are detectable and even enriched in the liver.56,59,102,103 In a comprehensive study comparing the overall breath and vigor of CD8+ T cell responses in the peripheral blood and liver of chronically HCV-infected patients, we found that virus-specific CD8+ T cell responses were strongly enriched in the liver. Many responses were only detectable in the liver; however, few responses were limited to the peripheral blood.104 Therefore, it is possible that a defective homing of HCV-specific CD8+ T cells or their rapid deletion in the liver also contribute to T cell failure and viral persistence in a subset of patients.

5. CONCLUSIONS In the last few years, tremendous progress has been made in the characterization of the role that the different components of the immune system have in control of HCV infection. In addition, many concepts have evolved which explain why the host immune defence is only successful in some individuals, while in most individuals, HCV successfully evades the host immune response. The important question remains, and is currently controversially discussed, which branch of the immune system has the most important function in determining outcome of infection and thus might be a promising target for immunotherapeutic strategies. Hopefully, further characterization of the determinants of viral clearance in acute HCV infection will allow the introduction of a successful vaccine.

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80. Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G, Ferrari C (2006). J Virol 80, 11398–11403. 81. Penna A, Pilli M, Zerbini A, Orlandini A, Mezzadri S, Sacchelli L, Missale G, Ferrari C (2007). Hepatology 45, 588–601. 82. Radziewicz H, Ibegbu CC, Fernandez ML, Workowski KA, Obideen K, Wehbi M, Hanson HL, Steinberg JP, Masopust D, Wherry EJ, Altman JD, Rouse BT, Freeman GJ, Ahmed R, Grakoui A (2007). J Virol 81, 2545–2553. 83. Brooks DG, Trifilo MJ, Edelmann KH, Teyton L, McGavern DB, Oldstone MB (2006). Nat Med 12, 1301–1309. 84. Ejrnaes M, Filippi CM, Martinic MM, Ling EM, Togher LM, Crotty S, von Herrath MG (2006). J Exp Med 203, 2461–2472. 85. Mangia A, Santoro R, Piattelli M, Pazienza V, Grifa G, Iacobellis A, Andriulli A (2004). Cytokine 25, 103–109. 86. Nelson DR, Tu Z, Soldevila-Pico C, Abdelmalek M, Zhu H, Xu YL, Cabrera R, Liu C, Davis GL (2003). Hepatology 38, 859–868. 87. Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC (1997). Hepatology 25, 449–458. 88. Chang KM, Rehermann B, McHutchison JG, Pasquinelli C, Southwood S, Sette A, Chisari FV (1997). J Clin Invest 100, 2376–2385. 89. Erickson AL, Kimura Y, Igarashi S, Eichelberger J, Houghton M, Sidney J, McKinney D, Sette A, Hughes AL, Walker C (2001). Immunity 15, 885–895. 90. Timm J, Lauer GM, Kavanagh DG, Sheridan I, Kim AY, Lucas M, Pillay T, Ouchi K, Reyor LL, Zur Wiesch JS, Gandhi RT, Chung RT, Bhardwaj N, Klenerman P, Walker BD, Allen TM (2004). J Exp Med 200, 1593–1604. 91. Cox AL, Mosbruger T, Mao Q, Liu Z, Wang XH, Yang HC, Sidney J, Sette A, Pardoll D, Thomas DL, Ray SC (2005). J Exp Med 201, 1741–1752. 92. Ray SC, Fanning L, Wang XH, Netski DM, Kenny-Walsh E, Thomas DL (2005). J Exp Med 201, 1753–1759. 93. Tester I, Smyk-Pearson S, Wang P, Wertheimer A, Yao E, Lewinsohn DM, Tavis JE, Rosen HR (2005). J Exp Med 201, 1725–1731. 94. Altman JD, Feinberg MB (2004). Nat Med 10, 229–230. 95. Neumann-Haefelin C, Killinger T, Timm J, Southwood S, McKinney D, Blum HE, Thimme R (2007). J Gen Virol 88, 1986–1991.

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96. Gaudieri S, Rauch A, Park LP, Freitas E, Herrmann S, Jeffrey G, Cheng W, Pfafferott K, Naidoo K, Chapman R, Battegay M, Weber R, Telenti A, Furrer H, James I, Lucas M, Mallal SA (2006). J Virol 80, 11094–11104. 97. Timm J, Li B, Daniels MG, Bhattacharya T, Reyor LL, Allgaier R, Kuntzen T, Fischer W, Nolan BE, Duncan J, Schulze zur Wiesch J, Kim AY, Frahm N, Brander C, Chung RT, Lauer GM, Korber BT, Allen TM (2007). Hepatology 46, 339–349. 98. Soderholm J, Ahlen G, Kaul A, Frelin L, Alheim M, Barnfield C, Liljestrom P, Weiland O, Milich DR, Bartenschlager R, Sallberg M (2006). Gut 55, 266–274. 99. Seifert U, Liermann H, Racanelli V, Halenius A, Wiese M, Wedemeyer H, Ruppert T, Rispeter K, Henklein P, Sijts A, Hengel H, Kloetzel PM, Rehermann B (2004). J Clin Invest 114, 250–259. 100. Meyer-Olson D, Shoukry NH, Brady KW, Kim H, Olson DP, Hartman K, Shintani AK, Walker CM, Kalams SA (2004). J Exp Med 200, 307–319. 101. Urbani S, Amadei B, Cariani E, Fisicaro P, Orlandini A, Missale G, Ferrari C (2005). J Immunol 175, 7519–7529. 102. Grabowska AM, Lechner F, Klenerman P, Tighe PJ, Ryder S, Ball JK, Thomson BJ, Irving WL, Robins RA (2001). Eur J Immunol 31, 2388–2394. 103. He XS, Rehermann B, Lopez-Labrador FX, Boisvert J, Cheung R, Mumm J, Wedemeyer H, Berenguer M, Wright TL, Davis MM, Greenberg HB (1999) Proc Natl Acad Sci USA 96, 5692–5697. 104. Neumann-Haefelin C, Timm J, Spangenberg HC, Wischniowski N, Nazarova N, Kersting N, Roggendorf M, Allen TM, Blum HE, Trimme R (2008). Hepatology 47, 1824–1836.

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CHAPTER 20

Host Responses to Hepatitis C Virus Infection: Gene Expression Profiles and Viral Pathogenesis Melanie Lambotin, Françoise Stoll-Keller & Thomas F. Baumert

ABSTRACT Hepatitis C virus (HCV) is a member of the Flaviviridae family that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. With an estimated 170 million infected individuals, HCV has a major impact on public health. The current therapy for HCV infection is interferon-alpha in combination with ribavirin. However, this therapy is characterized by limited efficacy, high costs, and substantial side effects. Moreover, no protective vaccine is available to date. Thus, understanding of the pathogenesis of HCV infection plays a key role in the development of novel preventive and therapeutic approaches. Microarrays have been shown to represent a promising technique for elucidating and interpreting the mechanistic role of host gene expression in the pathogenesis of infectious diseases. In recent years, genomic studies have been performed to analyze host gene expression profiles during HCV life cycle in vitro as well as HCV infection in vivo. The results of these studies have significantly contributed to the understanding of virus-host interactions including the identification of distinct gene expression profiles associated with outcome of infection and response to antiviral treatment. In this chapter, we review recent findings on host gene responses to HCV infection in model systems for HCV infection and in the HCV infected patient. The contribution of gene expression profiling to the 471

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understanding of virus-host interactions within the viral life cycle, outcome of infection and response to antiviral treatment will be discussed.

1. INTRODUCTION: MOLECULAR VIROLOGY AND PATHOGENESIS OF HEPATITIS C VIRUS INFECTION Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped positive-stranded RNA viruses and is the only known member of the genus Hepacivirus.1,2 The HCV genome consists of an approximatively 9.4 kb linear, single-stranded, positive-sense RNA molecule that comprises a large open reading frame (ORF) of approximately 9 kb length flanked by structured 5′ and 3′ untranslated regions (UTR). Translation of the HCV ORF yields a polyprotein precursor that is co- and posttranslationally processed by cellular and viral proteases into mature structural and non-structural proteins.1,2 The structural proteins consist of the core protein which is the major component of the viral nucleocapsid, and the envelope glycoproteins E1 and E2, which are glycosylated and form a non-covalent complex believed to be the building block for the viral envelope.3 The non-structural proteins have various functions involved in viral RNA replication and proteolytic processing: there are two viral autoproteases NS2 and serine protease NS3; NS4A polypeptide, which is an essential co-factor for NS3 protease; NS4B and NS5A proteins; and finally, NS5B RNA-polymerase (review, see Refs. 1 and 2). The nonstructural proteins coordinate viral replication by the formation of a membrane-bound replication complex. Apart from these, an additional protein, termed F (frameshift)-protein, which is encoded by an overlapping reading frame in the core protein coding sequence, has been proposed.4 Initiation of the HCV infection is mediated by the docking of the viral envelope to the hepatocyte cell surface membrane, followed by entry of the virus into the host cell. Several lines of evidence have demonstrated that binding and entry of HCV is mediated by HCV envelope glycoproteins E1 and E2.5 Host cell proteins implicated in these very first steps of virus-host interaction include CD81, scavenger receptor BI (SR-BI),6,7 the LDL receptor,8 highly sulfated heparan sulfate1,9,10 and claudin-1.11 Once the virus is inside the cell, its life cycle is entirely cytoplasmic. Replication

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occurs through a minus-strand intermediate in a membrane-bound compartment termed the membranous web, yielding double-stranded RNA intermediates. Replicative intermediates are fully exposed to the cell’s dsRNA-sensing machinery and induce strong innate cellular responses following infection12,13 (Figure 1). The liver is the primary target organ of HCV and the hepatocyte is its primary target cell, but infection of B cells, dendritic cells and other cell types have also been reported.14–16 Similar to other viruses establishing persistent infection, HCV triggers an immune-mediated inflammatory response that either rapidly clears the virus or causes the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC).17,18

Figure 1. Model of the HCV life cycle within the infected hepatocyte. HCV infection is characterized by the following steps: Binding of viral envelope glycoproteins to host cellular membrane receptors (1); entry into the hepatocytes via clathrin-mediated endocytosis (2); fusion of the viral membrane with the endosomal membrane and release of viral RNA into the cytoplasm (3); translation of HCV RNA into the polyprotein precursor and processing of this protein both by viral and cellular proteases (4); viral replication in the membranous web (5); assembly and packaging of new virions (6); and finally maturation and release of the newly synthesized HCV virions (7). (Adapted from Ref. 18)

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HCV infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.19 Therapeutic options are improving but are still limited and a protective vaccine is not available to date. Fifty to 80% of patients develop a chronic HCV infection and 4% to 20% of patients with chronic hepatitis C will develop liver cirrhosis within 20 years. In patients with liver cirrhosis, the risk to develop HCC is 1–5% per year.20,21 The current treatment for chronic HCV infection consists of a combination of pegylated interferon and the oral antiviral drug ribavirin given for 24 or 48 weeks. However, this treatment is often difficult to tolerate and results in a sustained virological response in only some of the treated patients.20,21 Therefore, more effective and better tolerated therapeutic strategies are urgently needed.22 The development of novel antiviral strategies depends on a detailed molecular understanding of the viral life cycle. However, studies of HCV-host cell interactions and pathogenesis have been hampered for a long time by the lack of efficient cell culture systems and small animal models. Nevertheless, significant progress has been made over the past two decades using heterologous expression systems,23,24 functional cDNA clones that are infectious in vivo in chimpanzees,25 replicon systems,26,27 and functional HCV pseudo-particles (engineered retroviral particles bearing functional HCV envelope proteins) that enable the study of viral entry under reproducible and conveniently measurable conditions.28,29,30 Most recently, a major milestone was the production of recombinant infectious HCV particles in cell culture31–35 (for review, see Ref. 36). Microarrays have been shown to represent a promising technique for elucidating and interpreting the mechanistic role of host gene expression in the pathogenesis of infectious diseases. Microarrays studies have been employed to investigate gene profiling associated with HCV infection, thereby providing more insights into molecular mechanisms of viral infection and allowing identification of distinct gene expression profiles associated with the outcome of infection and response to antiviral treatment. In this chapter, we aim to review recent findings on host gene responses to HCV infection in state-of-the-art model systems and in HCVinfected patients (Table 1). Contribution of gene expression profiling for the understanding of HCV-host interactions during the viral life cycle,

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Table 1. Transcriptional analyses of host responses induced by HCV infection, protein expression or replication in various model systems. For each model system used, the regulated genes and their implications for both the virus and the host are indicated. Model System

Key Findings

Implications

Reference

Huh7 hepatoma cells expressing NS5A protein.

• Downregulation of TNF-α signaling. • Modulation of expression of genes involved in lipid metabolism and transport.

• Viral escape. • Development of liver steatosis.

62

• Facilitation of viral replication.

65 66

Different cells lines (HeLa cells, liver cell • Modulation of INF response. lines, and primary fetal hepatocytes) expressing NS5A protein, replicon model.

• Viral persistence.

50

Liver biopsies from SCID/uPA mice following HCV infection of transplanted human hepatocytes.

• Modulation of expression of genes involved in lipid metabolism.

• Pathogenesis of HCVinduced liver disease.

73

HCV infected chimpanzees.

• Induction of expression of ISG. • Modulation of expression of genes of the proteasome, apoptosis, innate immunity, lipid metabolism.

• Pathogenesis of HCV infection. • Prognosis markers of outcome of infection.

68 69 70

Liver biopsies before IFN therapy in HCV-infected patients.

• Modulation of innate IFN responses.

• Prognosis markers of response to IFN treatment.

88

PBMC from HCV-infected patients, sampled before and after IFN therapy.

• Downregulation of expression of chemokine and cytokine related genes.

• Prognosis markers of response to IFN treatment.

89 90 91

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• Protection against apoptosis. • Downregulation of expression of genes involved in innate immunity.

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HepG2 hepatoma cells expressing core protein.

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• Modulation of expression of genes involved • Condition the cells for in lipid metabolism and innate immunity. supporting viral propagation.

Host Responses to HCV

Binding of recombinant envelope glycoproteins to hepatoma cells.

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outcome of infection and responses to antiviral treatment in vivo will be discussed.

2. MODULATION OF HOST GENE EXPRESSION IN RESPONSE TO HCV INFECTION IN VITRO — UNDERSTANDING OF VIRUS-HOST INTERACTIONS 2.1 Host Responses to Cellular HCV Binding and Entry The first step of HCV infection is the attachment of viral envelope glycoproteins to cell surface receptors, which leads to the internalization of HCV into cells. In recent years, it has become clear that many viruses use the cell’s signaling pathways during entry, to induce changes in the cell that promote viral entry and early cytoplasmic events, to optimize later processes in the replication cycle, as well as to neutralize host defenses.37 To characterize cellular responses following binding of HCV envelope glycoproteins to host cells, a gene expression profile of hepatocytederived cell lines using recombinant envelope glycoproteins as well as HCV-like particles (HCV-LP; engineered insect-cell derived particles bearing functional HCV structural proteins [core, E1 and E2]38) as ligands for cellular binding was performed. This study demonstrated that binding of HCV envelope glycoproteins to host cells results in a cascade of intracellular signals modulating cellular gene expression, which may condition the cell for supporting viral propagation.39 The functional relevance and biological significance of the observed alteration of host cell expression was demonstrated by side-by-side analysis of findings obtained in the in vitro model systems with host cell responses induced by HCV infection in the human liver of HCV-infected patients. Interestingly, several identified genes have been associated with HCV infection, replication, gene expression or virus-host protein interaction. Several proteins encoded by these genes are involved in cell signaling, regulation of transcription, and immune response. Indeed, host responses included the upregulation of type II interleukin-1 receptor and high-affinity immunoglobulin G Fc receptor. Because type II interleukin-1 receptor plays an important role in mediating innate antiviral immune response,40

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HCV-mediated upregulation of its expression may interfere with the signaling linked to this receptor41, therefore counteracting innate antiviral defense strategies. Moreover, upregulation of the Fc γ receptor has been previously shown to be a strategy for HIV (human immunodeficiency virus) and other viruses to facilitate entry of virion-antibody complexes.42 Other changes induced by envelope glycoprotein binding that are detected in the liver of human HCV-infected individuals included the downregulation of defined chemokine receptors. This modulation may allow the virus to counteract innate antiviral defenses as previously shown for other viruses, such as HIV,43 human cytomegalovirus44 and herpes virus 6 and 7.45 The transcription of several genes associated with fatty acid biosynthesis and lipid metabolism also appear to be modulated in response to HCV binding to cells. Interestingly, HCV is known to induce formation of hepatocellular lipid droplets which colocalize with HCV proteins.46 Since molecules that block fatty acid biosynthesis inhibit replication, alteration of fatty acid biosynthesis and lipid metabolism observed following HCV binding could represent a strategy to facilitate viral replication. Noteworthy, expression of several genes induced by envelope glycoprotein binding and following HCV infection in vivo was different. Modulation of these genes is most likely implicated in virus-host interactions requiring ongoing productive viral infection. Antiviral immune responses within the liver, not present in the in vitro systems, may also contribute to the differential regulation of genes observed in vitro and in vivo. This study demonstrates that HCV initiates the induction of host cell responses immediately after the first contact of viral envelope glycoproteins with host cell surface molecules. It has been demonstrated that viruses take advantages of the cell’s own signal transduction systems to transmit signals to cells. Vesicular stomatitis virus and the simian virus 40 hijack the endocytic pathway to enter into cells,47 and the interaction between the HIV envelope glycoprotein gp120 and the cellular receptor CCR5 induces transcription and expression of factors that provide a conducive environment for HIV replication in resting PBMCs.48 All the cellular signals used by the viruses induce changes that may facilitate entry, prepare the cells to invade and neutralize host defenses. Thus, the

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transcriptional reprogramming of liver cells during HCV binding and internalization may be part of HCV strategy to facilitate viral infection and escape from innate host cell responses.39

2.2 Host Responses to HCV Replication and Protein Expression Following entry, HCV replication, translation and polyprotein processing occur within the membranous web. The HCV core protein is the first viral protein to be cleaved off the viral polyprotein and plays a major role in the formation of viral nucleocapsid. Microarray analyses of a human hepatoma cell line (HepG2) expressing HCV core protein from different viral genotypes have demonstrated that each core protein appears to induce its own gene expression profile. Furthermore, this analysis identified several induced genes being implicated in HCV replication and pathogenesis.49 In HepG2 cells expressing HCV core protein, significant changes were observed in the expression of genes, including genes regulating apoptosis, immune responses and cell cycle.50 First, the expression of core protein in hepatocytes correlated with the increased expression of factors known to protect cells against apoptosis.51,52 Expression of other cellular factors that may result in alteration of cell growth characteristics is also affected. Furthermore, HCV core protein leads to an increased expression of genes involved in innate immunity, among which is the major histocompatibility complex (MHC) class I polypeptiderelated sequence B and Iκ Bα. MICB is a ligand of NKG2D type II receptor that can activate the cytolytic response of NK cells, CD8αβ T cells and γδ T cells.53 By regulating the transcription factor NF-κ B, Iκ Bα has a crucial role in regulating immune response, inflammation and apoptosis.54 The increased expression of Iκ Bα by HCV core could result in a widespread dysregulation of NF-κ B transactivated genes. In contrast, other genes involved in immunity are downregulated, such as TRAIL (TNF super family member 10), liver activation regulated chemokine (LARC), and osteopontin. TRAIL functions as a strong mediator for immune regulation and inflammatory responses that induces apoptosis on diverse cell lines.55 Its presence was demonstrated in liver tissues of HCV-infected patients and

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cultures of hepatocytes,56 while LARC and osteopontin function as chemoattractants for recruitment of T lymphocytes.57,58 The modification of these genes suggest that the expression of HCV core protein could hamper the recruitment of immune cells by inhibiting the expression patterns of immune and chemokine factors. These findings suggest that, in addition to its role in forming the viral nucleocapsid, the core protein may also contribute to viral persistence by protecting infected hepatocytes from cell death by suppressing apoptosis and inflammatory reaction to HCV infection. HCV replication depends on the non-structural viral proteins NS3, NS4A, NS4B, NS5A and NS5B, which assemble in a membrane-bound replication complex, termed the membranous web. In addition to functions related to HCV replication, the non-structural viral protein NS5A appears to play an important role in modulating antiviral responses within the infected cell and in promoting long-term persistence of the virus. It could interfere with the function of the dsRNA-induced protein kinase R.59 NS5A has also been suggested to influence host signaling process such as PI3-kinase and apoptotic pathway60 and to possess transcriptional transactivation properties.61 Oligonucleotide assays have allowed the identification of numerous genes whose expression was modified in Huh7 cells expressing NS5A. These genes are involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and regulation of immune response.62 First, NS5A appears to act as a negative regulator in the TNF-α signaling cascade.62 Since TNF-related cytokines are crucial effectors of the innate and adaptative immune defenses and their receptors are targeted by many viruses, it is tempting to speculate that their downregulation by NS5A may enable the virus to escape immune responses, leading to persistent infection.62 Other genes that are involved in inflammation are modulated by NS5A: these include osteopontin, hepatocyte growth factor-like protein, calpain-1 and lipoprotein-associated phospholipase A2.62 Among them, STAT6, a mediator of IL-12 and IL-4 functions whose activation regulates liver inflammation injury,63 has been shown to be downregulated by NS5A, while proinflammatory chemokines like IL-8 are upregulated. Furthermore, expression of complement component-4-binding protein is decreased by NS5A. This molecule is an important regulator of the

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classical pathway of the complement system, which is mainly expressed in the liver. Its expression normally increases during inflammation. Modulating the expression of host genes involved in inflammatory processes therefore appears to be a strategy adopted by HCV to escape from the cellular antiviral response.62 Moreover, NS5A expression results in downregulation of the expression of genes involved in lipid transport and metabolism.62 Interestingly, it has been shown that HCV associates with low-density lipoprotein (LDL) including those containing the apolipoprotein E and B (ApoE; ApoB).64 Moreover, NS5A colocalizes with the HCV core protein on lipid droplets and interacts with ApoA1.46 Thus, the modification observed in the expression levels of these genes strongly suggest an important role for NS5A in modulating lipid homeostasis in hepatic cells that may affect the viral life cycle and/or contribute to the development of liver steatosis.62 Furthermore, almost all genes modulated by NS5A possess at least one binding site for nuclear factor κ B (NF-κ B), an activator of transcription.62 Interestingly, when blocking NF-κ B activity, the upregulation of genes induced by NS5A is inhibited, thus demonstrating that NF-κ B mediates NS5A effects on gene expression. Activation of NF-κ B then appears to play an important role in HCV pathogenesis and development of HCVrelated liver diseases.62 Finally, NS5A can affect the IFN response of target cells65: alpha and beta interferon (IFN-α /β ) are expressed by many cell types in response to viral or bacterial pathogens and induce a large number of genes, the IFN-stimulated genes (ISGs), many of which encode proteins with antiviral and antiproliferative functions. Interestingly, in cells expressing NS5A and treated with IFN, NS5A blocked the IFNmediated induction of some ISGs.65 Similar results have been obtained in cells supporting subgenomic HCV replication (Huh7/Rep), in which the expression levels of various ISGs is significantly lower than in naive Huh7 cells.66 Furthermore, in Huh7/Rep cells, replication of HCV is significantly suppressed by overexpression of some of these ISGs, such as PKR, MxA, IRF9, or IRF1. Conversely, knockdown of these same genes result in an increase of HCV replication. In contrast, the expression level of NS5A protein is decreased by the overexpression of these ISGs, thus confirming a key role of NS5A in the control of cellular IFN response. Taken together, these data strongly suggest that the downregulation of

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ISGs expression could be crucial for an efficient replication of HCV inside the host cell. Microarray studies have therefore demonstrated that, throughout the life cycle, the virus takes advantages of the host cell’s own signal transduction systems and that several HCV proteins are involved in this process complementing their function in viral entry, replication and assembly. The alterations of host gene expression induced by HCV may facilitate virus survival and dissemination but may also play a role in liver damage and pathogenesis of HCV. Finally, by identifying host genes targeted by HCV infection, functional genomics may also identify new targets for treatment of HCV infection.

3. GENE EXPRESSION PROFILING IN HCV INFECTION IN VIVO — IMPLICATIONS FOR PATHOGENESIS OF HCV INFECTION Although in vitro studies have provided interesting insights for the understanding of the mechanisms of HCV infection, all the modifications described occur in the absence of an active adaptive host immune response. However, the interaction between HCV viral components and the immune system ultimately determines the progression of infection, with two alternatives, clearance or persistent infection.17 Most recently, several studies have been performed in vivo, first to confirm the presence of host immune responses findings obtained in vitro, and also to correlate the host responses to both the outcome of infection and the response to IFN treatment.

3.1 Gene Expression Profiling and Outcome of Infection In the majority of infected individuals, HCV infection leads to viral persistence rather than viral clearance.17,67 However the mechanisms underlying this persistence are only partially understood. Mechanisms of viral persistence are reviewed in Chapter 19 by C. Neumann-Haefelin and R. Thimme. Events leading to viral clearance or persistence are difficult to examine in patients, since the time of infection is rarely known and access to

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serial liver samples is limited. Therefore, a considerable effort has been made to understand the course of infection in animal models in order to better understand the mechanisms of viral clearance. The chimpanzee represents the only naturally occurring in vivo model for HCV infection.68–70 The severe combined immunodeficiency disorder (SCID)-beige/albumin (Alb)-urokinase plasminogen activator (uPA) mouse containing a humanmouse chimeric liver represents the only small animal model for studying host response to HCV infection.71,72 DNA microarrays using the chimpanzee model have been performed to probe the liver for changes in gene expression associated with resolution or persistence of HCV infection.68 As in the in vitro models, gene expression profiles of HCV-infected cells are regulated in vivo and could be classified into four types of expression modification68: (i) changes due to hepatocytes-HCV protein interaction; (ii) changes induced by HCV replication; (iii) changes due to immune responses against HCV infection; and (iv) changes due to hepatocytes response to cytokines expressed by immune cells. According to data obtained in vitro, genes whose expression was affected by HCV infection included genes involved in metabolism, cell-cycle regulators, apoptotic markers, immune response genes, and numerous IFN responses genes.68 First, consistent with data obtained in vitro, genes involved in apoptosis are upregulated early in HCV infected chimpanzees.68 Apoptosis of infected hepatocytes represent an antiviral host mechanism contributing to the control of virus infection and viral clearance. In contrast, specific increases in genes encoding DNA-binding proteins and transcription factors indicate the presence of proliferative changes in the liver, which may result from liver regeneration to replace damaged hepatocytes.68 An early modification of expression of some genes involved in lipid metabolism is also observed in infected animals. In the SCID-beige/AlbuPA mouse, this modulation appeared to be host specific and tend to correlate with liver HCV RNA levels.73 In chimpanzees, some of these genes are differentially induced or repressed in animals that clear the virus versus animals that develop a persistent infection, and could therefore represent prognostic markers of outcome.69 As an example, the gene encoding UDP-glucose ceramide glucosyltransferase (UGCG) is selectively upregulated during the early onset of viremia before clearance of the virus. Although it has been shown that the apolipoprotein B (ApoB),

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a component of the very low density lipoprotein (VLDL), may be important for viral assembly,74 the exact role of all genes involved in lipid metabolism in HCV infection is not yet established. They could play a role in viral replication or represent an early hepatic response that contributes to viral clearance. As some of them are involved in fatty acid biosynthesis, they may also have implications in the development of steatosis in chronically infected patients. Additionally, several cytokines or immunomodulatory genes are upregulated in liver tissues. Increased levels of Mac2BP have been demonstrated in HCC and HCV-infected patients.75 However, its role in HCV outcome is not clear, since its expression levels appear not to correlate with the development of a chronic infection. Among the regulated genes is MIP-1β , a CCR chemokine induced by interleukin-1β , and a chemoattractant for macrophage, T cells and NK cells. Interestingly, expression level of MIP-1β increased during acute infection at the time of viral clearance but was not upregulated in chronic infection.70 However, assigning specific functions for these cytokines with regards to HCV infection requires further investigation.70 Most of the changes common to all animals in gene expression occur in known ISGs, indicating an ongoing IFN and/or dsRNA response to the virus.70 The increase of ISGs expression levels during both acute and chronic infection suggest that similar mechanisms may limit viral replication and percentage of infected hepatocytes during the two forms of infection.70 Interestingly, the expression patterns of the various ISGs regulated were temporally distinct, thus suggesting that different regulatory pathway and/or involvement of different cell types and IFN type (IFN-α /β or IFN-γ ) are operative over time.68,70 Moreover, this modification of gene expression profiles correlates with the outcome: analyses of liver biopsies from infected chimpanzees that developed persistent infection, transient viral clearance or sustained clearance, showed gene expression patterns consistent with an IFN-α induction that correlates with the magnitude and duration of infection.70 Additionally, viral clearance appears to be associated with the induction of IFN-γ -induced genes and of other genes involved in antigen processing and presentation, and in the adaptative immune response,69 potentially a result of the homing and activation of immune cells to the liver.70 Therefore, HCV could avoid

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recognition by the immune response, in order to establish a persistent infection, either by keeping its replication below a threshold level required for gene induction or by disrupting the induction of genes that control antigen processing and T cell recruitment.69,70 Additionally, several genes that were overexpressed in the chronically infected animals encode immunoglobulin or MHC-related polypeptides.70 This may reflect the humoral immune response to a constantly changing viral quasispecies. Alternatively, it may also suggest ongoing autoimmune pathologies that are often observed in HCV-infected patients.76,77 Among the genes whose expression is altered during infection are several genes that are of known importance in T-cell and/or NK-cell regulation or function, including HLA-G and NK cell activation protein 2B4 (CD244).70 HLA-G is a MHC gene that has been shown to inhibit lytic activity of NK cells78 and antigen-specific cytotoxic T lymphocytes,79 as well as inhibit allogenic proliferative responses.80 Its increase suggests that it may be important in modulating cellular responses during HCV persistence.70 CD244 is a cell surface glycoprotein that is expressed on all NK cells, CD8+ T cells, monocytes and basophiles. Its activation leads to NK cytolytic activity and IFN-γ secretion.81,82 Its expression decreased in HCV infected chimpanzees. Thus the combination of increased HLA-G and decreased CD244 expression may partially explain the phenotype of immune cells during HCV persistence and the reduction or absence of IFN-γ expression in animals with a persistent infection.70 Furthermore, an alteration of immunoproteasome gene expression has also been associated with HCV clearance.69 While chimpanzees with subsequent viral clearance exhibit a rapid and strong increase of the proteasome subunit LMP-2 expression during the first weeks of infection, chimpanzees with persistent infection were characterized by a flat curve with blunted response of LMP-2 expression.69 Proteasome-mediated degradation of viral antigens represents a key in the cascade of proteolytic processing required for the generation of peptides presented on the cell surface to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. Thus modulation of LMP-2 expression may play a role in HCV pathogenesis and outcome of viral infection, as shown for other viruses.69,83

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To conciliate data obtained from both in vitro and in vivo studies, a model has been proposed by Bigger and colleagues to explain the course of HCV infection.70 During acute infection, viral spread in the liver would occur rapidly and some hepatocytes become infected.70 IFN is then probably secreted by infected cells and the secreted INF induces zones of cells resistant to infection.70 The dsRNA response results in the induction of ISGs in infected cells. This leads to a loss of available replication space in the liver and a decrease in viral spread with no further increase in viremia.70 Infected cells quickly lose the ability to secrete IFN due to the accumulation of viral proteins that blocks this response. During acuteresolving infection, a T-cell response emerges to clear infected cells.70 IFN-γ may help by suppressing viral replication and possibly contributes to non-cytolytic clearance of viral RNA in some cells. The primary difference between acute and chronic infection may be the success of the T-cell response in eliminating infected cells, and the emergence of escape mutants may play an important role in determining whether the T-cell response is successful. In the absence of viral clearance, a dynamic equilibrium between newly infected cells, IFN secretion, inhibition of IFN secretion by viral proteins, cell death, cell proliferation and new susceptible cells sets up indefinitely.70 In addition to these studies investigating the different gene expression profiles linked to clearance or persistence, other analyses were performed, aiming to identify genes responsible for progression of the disease into cirrhosis and hepatocellular carcinoma (HCC). To identify a gene expression signature of HCV-induced liver disease, microarray analysis of RNA from HCV-infected cirrhotic livers was performed, allowing the identification of several potential gene markers of HCV-associated liver disease.84 These include (i) genes expressed in activated lymphocytes infiltrating the liver and activated macrophages; (ii) genes involved in cytoskeleton rearrangements and associated events; (iii) genes related to the anti-apoptotic signaling pathway; and (iv) genes involved with the interferon response and virus-host interactions.84 Furthermore, patients who progress into HCC exhibit an overexpression of genes involved in cell division and cell activity, concordant with the presence of the tumor. When analyzing the genes differentially expressed in HCV-HCC patients undergoing liver transplantation with HCC recurrence, compared to

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patients with the same explanted stage without recurrence, it appears that recurrence could be linked to an overexpression of genes associated with immune response and specific response to the virus. Some genes, acting as tumor suppressor genes or involved in the regulation of transcription and cell cycle, also appeared to be related to overall survival and cancerfree survival.85 This determination of genes predictive for progression of chronic HCV infection is of great importance since the identification of risk factors for developing cirrhosis and/or HCC may provide an opportunity to make an earlier diagnosis and intervene with more adapted preventive and therapeutic strategies.

3.2 Gene Expression Profiling and Treatment Response The current standard treatment for chronic HCV infection consists of the combination of pegylated interferon and the oral antiviral drug ribavirin given for 24 or 48 weeks. However, this treatment is often difficult to tolerate and results in a sustained virological response in only 50% of patients infected with HCV genotype 1.20–22 As a consequence, the number of patients presented with long-term sequelae of chronic hepatitis C, including HCC, is expected to further increase over the next 20 years.86 Given this scenario, there is an urgent need to develop more effective and better tolerated therapies for chronic hepatitis C.22,86 Several viral and host factors, such as HCV genotype, viral load, degree of fibrosis, body weight and ethnic origin have been shown to be useful predictors of IFN-α treatment outcome87 but are not sufficient to precisely predict the response to IFN treatment. Various studies have then been conducted to elucidate the underlying mechanisms that determine efficacy of IFN-α in different patients in order to predict treatment outcomes before initiating therapy. Based on the hypothesis that pretreatment non-responder and responder liver tissue would show consistent differences in gene expression levels that could explain and/or predict treatment outcome, Chen and colleagues performed a gene expression analysis on liver biopsy taken before therapy, from non-responder or responder patients. This study demonstrates that non-responder and responder patients differ fundamentally in their

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innate IFN response to HCV infection.88 Interestingly, all the identified genes are upregulated in non-responder patients while in liver from responder patients, their levels of expression are closer to the levels observed in non-infected liver.88 Moreover, many of these genes are IFN responsive genes, suggesting that the non-responder patients have adopted a different equilibrium in their host-virus immune response. In fact, it appears that patients showing an endogenous activated type I IFN induction pathway before therapy seem to be resistant to the beneficial antiviral effects of IFN administration.88 This study also defines for the first time a subset of genes, which could predict treatment responses before initiation of PEG-IFN plus ribavirin therapy.88 In addition to the great progress in the understanding of the mechanisms leading to viral persistence or viral clearance after IFN treatment, the possibility of predicting the chances of attaining a sustained virological response before initiation of therapy would limit exposure of persons with a low likelihood of therapeutic success to the toxicities of the treatment.88 Interestingly, similar data were obtained when analyzing the gene expression profiles in peripheral blood mononuclear cells (PBMC).89 The first aim of that study was to compare the gene expression profiles of PBMCs from patients with a chronic hepatitis C to non-infected individuals. Then, the authors went further by analyzing the relationship between PBMC gene expression profiles before the start of IFN therapy and IFN response. On the basis of changes in gene expression in PBMCs before therapy, a combination of genes predictive of an IFN response could be defined.89 Moreover, determination of changes in the gene expression profiles of PBMCs during the course of IFN therapy led to the identification of numerous genes whose expression profile differed before therapy and two weeks after the start of the treatment. These genes did not include as many IFN-α-stimulated genes as in liver, but they included several genes involved in immune regulation. Interestingly, very little change was observed two weeks after the beginning of the treatment in the pattern of non-responder patients. Therefore, the determination of expression levels of these genes, two weeks after the start of the IFN treatment, appears to be also useful in predicting therapeutic efficacy.89 These results are of particular interest since unlike liver-biopsy specimens, PBMCs can be easily collected, and sampling can be repeated as necessary.

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Aiming to elucidate the underlying host transcriptional response associated with interferon treatment outcomes, other studies found complementary results. The major observation is that patients who exhibited a vigorous early virological response to IFN with ribavirin treatment had concurrent vigorous alterations in PBMC gene expression levels.90,91 Indeed, the number of genes whose levels were induced or repressed is greater among responder than non-responder patients. The majority of genes that were downregulated in response to treatment encode products which are involved in translational regulation. In general, these genes were downregulated substantially in marked responder patients but only marginally in poor responder patients.86,87 Moreover, the clinical outcome of IFN therapy in chronic hepatitis patients is clearly associated with a transcriptional response to IFN.90,91 Patients who achieved long-term clearance of HCV after IFN-α therapy had a greater transcriptional response to IFN-α than patients who failed IFN-α treatment. Some analysis demonstrated that the difference in gene expression modifications observed between responder and non-responder patients is due to a global difference in the transcriptional response of ISGs rather than specific ones in a subset of ISGs.90,91 All these studies provide evidence that determining the gene expression modification before IFN treatment or during the therapy could help to predict the outcome of IFN treatment. Moreover, because analysis of gene expression profile in hepatocytes requires liver biopsy, an invasive procedure that is not always easy to do in patients, the finding that responses to IFN therapy could be predicted on the basis of changes in gene expression in PBMCs would facilitate prediction of treatment response in HCV-infected patients. However, further studies are needed to apply these findings for clinical practice.

4. CONCLUSIONS In conclusion, interaction of HCV with its host induces marked changes in host gene expression that could contribute to outcome of infection i.e., clearance or persistence. Some of these modifications, such as alteration of the expression genes involved in lipid metabolism or immunmodulation, could contribute to the pathogenesis of HCV-induced liver disease.

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The use of microarray technology to study gene expression during HCVhost interaction has provided valuable knowledge about genes that are regulated in response to HCV infection. Therefore, further studies are now required to pinpoint their role and the mechanism by which they contribute to pathogenesis of HCV infection. The genomic studies have also provided new interesting insights for the treatment of HCV infection since some identified regulated genes may be useful as predictors for treatment response. Ultimately, a more detailed understanding of virus-host interactions may result in innovative preventive and therapeutic strategies for HCV infection, one of the most common causes of chronic hepatitis, liver cirrhosis and HCC worldwide.

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68. Bigger CB, Brasky KM, Lanford RE (2001). J Virol 75, 7059–7066. 69. Su AI, Pezacki JP, Wodicka L, Brideau AD, Supekova L, Thimme R, Wieland S, Bukh J, Purcell RH, Schultz PG, et al. (2002). Proc Natl Acad Sci USA 99, 15669–15674. 70. Bigger CB, Guerra B, Brasky KM, Hubbard G, Beard MR, Luxon BA, Lemon SM, Lanford RE (2004). J Virol 78, 13779–13792. 71. Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, Addison WR, Fischer KP, Churchill TA, Lakey JR, et al. (2001). Nat Med 7, 927–933. 72. Meuleman P, Libbrecht L, De Vos R, de Hemptinne B, Gevaert K, Vandekerckhove J, Roskams T, Leroux-Roels G (2005). Hepatology 41, 847–856. 73. Walters KA, Joyce MA, Thompson JC, Smith MW, Yeh MM, Proll S, Zhu LF, Gao TJ, Kneteman NM, Tyrrell DL, et al. (2006). PLoS Pathog 2, e59. 74. Huang H, Sun F, Owen DM, Li W, Chen Y, Gale M Jr., Ye J (2007). Proc Natl Acad Sci USA 104, 5848–5853. 75. Correale M, Giannuzzi V, Iacovazzi PA, Valenza MA, Lanzillotta S, Abbate I, Quaranta M, Caruso ML, Elba S, Manghisi OG (1999). Anticancer Res 19, 3469–3472. 76. Manns MP, Rambusch EG (1999). J Hep 31 (Suppl 1), 39–42. 77. Schott P, Hartmann H, Ramadori G (2001). Histol Histopathol 16, 1275–1285. 78. Forte P, Pazmany L, Matter-Reissmann UB, Stussi G, Schneider MK, Seebach JD (2001). J Immunol 167, 6002–6008. 79. Fournel S, Huc X, Aguerre-Girr M, Solier C, Legros M, Praud-Brethenou C, Moussa M, Chaouat G, Berrebi A, Bensussan A, et al. (2000). Tissue Antigens 55, 510–518. 80. Lila N, Carpentier A, Amrein C, Khalil-Daher I, Dausset J, Carosella ED (2000). Lancet 355, 2138. 81. Nakajima H, Cella M, Langen H, Friedlein A, Colonna M (1999). Eur J Immunol 29, 1676–1683. 82. Nakajima H, Colonna M (2000). Hum Immunol 61, 39–43. 83. Rotem-Yehudar R, Groettrup M, Soza A, Kloetzel PM, Ehrlich R (1996). J Exp Med 183, 499–514. 84. Smith MW, Yue ZN, Geiss GK, Sadovnikova NY, Carter VS, Boix L, Lazaro CA, Rosenberg GB, Bumgarner RE, Fausto N, et al. (2003). Can Res 63, 859–864.

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CHAPTER 21

Differential Gene Expression in Coxsackievirus Infection and Its Effect on Viral Pathogenesis Huifang M. Zhang, Travis Lim, Honglin Luo, Bruce McManus & Decheng Yang

ABSTRACT Coxsackievirus, a positive single-stranded non-enveloped RNA virus, can infect multiple organs of humans and mice, and cause myocarditis, pancreatitis, meningitis and other diseases. Upon infection, this virus can alter the expression of numerous host genes, activate host defense mechanisms against viral infection or initiate immune/autoimmune responses leading to the destruction of infected cells. Altered gene expression has been profiled by a number of approaches and certain gene functions in cell survival, apoptosis, fibrosis, regulation of gene expression, immune responses and viral pathogenesis have been studied in cultured cells and genetically modified mouse models. In this chapter, we will focus our review on genes whose expression is altered during CVB3 infection, as identified by differential mRNA display, cDNA microarray and proteomic analysis. These gene functions will be discussed in terms of protective immune responses and disease induction in animal models. In addition, alteration of cellular gene expression by viral genes such as proteases 2A and 3C, as well as other non-structural proteins are also discussed in an attempt to summarize our understanding of the role of host-virus interactions in viral pathogenesis.

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1. INTRODUCTION Coxsackieviruses are members of the genus Enterovirus in the family Picornaviridae and are classified into two groups, A and B, on the basis of the type of paralysis caused in suckling mice. The group A viruses include 24 serotypes and cause generalized myositis. Group B viruses infect many tissues and organs in newborn mice, leading to slower, spastic paralytic death with destruction of pancreatic acinar cells, heart muscle cells and hepatocytes. They also cause focal myositis and lesions in the central nervous system and adrenal tissue.1 For this reason, group B coxsackieviruses have received more attention. To date, six serotypes have been discovered in coxsackievirus B (CVB) group. CVB3 is the most studied virus in this group as it was ranked the number one causal agent of viral myocarditis, particularly in children and young adults, by the World Health Organization (WHO).2 In addition, viral myocarditis can progress to dilated cardiomyopathy (DCM). Patients with DCM need heart transplantation.3 In North America, approximately 30% of viral myocarditis cases are caused by CVB3.4 CVB4 is another important pathogen in this group. It causes pancreatitis or insulin-dependent diabetes mellitus.5,6 During the past decade, CVB4-induced type 1 diabetes mellitus or pancreatitis received increasing attention.7 Thus, this article will review the literature mainly related to the infections caused by these two viral strains. The known mechanisms of CVB pathogenesis are two-fold: direct damage of infected cells as a byproduct of viral replication, and immune and/or autoimmune injury of the infected organs.8,9 However, knowledge of the molecular events leading to cellular injury is still incomplete. In recent years, researchers have attempted to uncover these determinants using differential mRNA display, cDNA microarray and proteomic analysis. In this chapter, we will review recent progress on the identification of these determinants and the understanding of gene function of selected up or downregulated genes in disease induction.

2. MOLECULAR BIOLOGY OF CVB 2.1 Genome Structure The CVBs are positive single-stranded non-enveloped RNA viruses. The genome is ∼7400 nucleotides (nts) long and encodes a single long open

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reading frame flanked by the 5′ and 3′ untranslated regions (UTRs). The 5′ end of the genome is unusually long (> 730 nts) and not link to the 7-methylguanosine triphosphate cap structure associated with eukaryotic mRNA, but instead with a viral encoded small peptide, VPg. The 5′UTR contains an internal ribosomal entry site (IRES) at nts 309 to 670.10,11 Thus, the coxsackievirus RNA, like all other picornaviruses, can be translated directly by a cap-independent IRES-driven mechanism into a single long polyprotein. The 3′UTR is approximately 100 nts long and contains three stem loops. These loop sequences interact each other to form kissing-pair tertiary structures, which play important roles in facilitating viral transcription and translation initiation.12 Upon translation, the single polyprotein is self-cleaved by viral protease 2A at cis-acting autocleavage sites into precursors P1, P2 and P3. These three precursors are further cleaved by 3C protease to produce individual proteins: P1 is processed into four structural proteins VP4, VP2, VP3 and VP1 that make up the viral capsid; P2 and P3 are processed into seven non-structural proteins including protease 2A, viroporin 2B, NTPase 2C, 3A, RNA replication primer 3B (uridylated VPg peptide),13 protease 3C, and RNA polymerase 3D.14,15 In addition, certain intermediate precursor enzymes such as 3CD can also be produced, which cleaves peptides in trans, releasing other virus components in their final form. Another intermediate precursor is 3AB, which binds 3D and stimulates its activity. Viral proteases are also responsible for cleaving a multitude of cellular proteins that lead to the shutdown of host cell cap-dependent translation and eventually, the cell death and viral particle release16,17 (see later discussion). Viral genome structure has been further studied by mutational analysis or using chimeric viruses to identify cardiovirulent determinants, which have been localized in the 5′UTR and the coding region. For the CVB3 5′UTR, a sequence within the stem-loop II region,18 particularly nts 484, 485, 473 or 475 at domain V, are critical for efficient viral translation and cardiovirulence.19,20 A recent report indicated that mutation of the GNRA motif within stem-loop V of the IRES of CVB3 can abolish the cardiovirulent activity.21 For the coding region, an asparagine-to-aspartate substitution demonstrated that the EF-loop or puff region of VP2 is crucial.22 Recently, a second mutation in the EF surface loop of VP2 as well as a mutation in the knob of VP3 has been found to significantly affect

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viral cardiovirulence.23 For CVB4, there is also strong evidence that the structural genes affect both tissue tropism and virulence. For example, mutations in both the VP1 and the VP4 structural genes have been shown to modulate the virulence of CVB4 in the pancreas.24–26 It is well documented that trace element deficiency, such as selenium, is a driving force for CVB3 mutation from an avirulent to a virulent strain.27 Recently, another factor, host aging, was found to promote the evolution of an avirulent CVB3 into a virulent strain.28

2.2 Viral Receptor and Tissue Tropism CVBs enter host cells through the coxsackie and adenovirus receptor (CAR).29 CAR is a member of the immunoglobulin superfamily of proteins, containing an amino-terminal V-like domain (D1 domain), followed by a C2-like domain (D2), a single membrane-spanning sequence, and a 107-residue cytoplasmic domain.30 However, neither the cytoplasmic domain nor the membrane-spanning domain are required for CAR to function as a receptor for adenovirus and CVBs.31,32 The 3D structure of CVB3 in complex with full length human CAR and also with the D1D2 fragment of CAR has been determined to ∼22Å resolution.33 It is believed that many, but not all, CVB isolates also interact with a second receptor or co-receptor, the decay accelerating factor (DAF, CD55).34,35 One of the differences in the function between CAR and DAF is their ability to induce conformational changes of viral particles. Attachment of CVB to CAR induces A particle formation, leading to release of viral RNA, but attachment of CVB to DAF does not.36 Unlike DAF, CAR is a component of the tight junction (TJ) and is inaccessible to virus approaching from apical surface.37 DAF is abundant on the apical surface of polarized epithelial cells and is accessible to pathogens in the intestinal lumen. A recent report revealed that attachment to DAF triggers two independent intracellular signals required for virus entry: activation of AbI kinase initiates Rac-dependent actin rearrangements that deliver the virus to the TJ, where an obligatory interaction with CAR results in A particle formation; in parallel, activation of Fyn initiates signals that lead to internalization of A particles from the TJ and permit subsequent events in uncoating and replication.38

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CVB3 tissue tropism largely depends on receptor expression levels of target cells. However, mRNA expression levels of CAR in certain organs of mice do not correlate well with the susceptibility or viral load in CVBinfected tissues,39,40 implying that other intracellular protein factors may also affect viral tissue tropism — for example, through interactions with viral RNAs. These proteins include polypyrimidine-tract binding protein (PTP), La autoantigen, GAPDH and poly-A binding protein (PABP) (see review Ref. 17). Recently, we and others have further demonstrated that La protein can specifically bind to multiple sites within CVB3 RNA with differential affinity,41,42 and that La can bind the 3′UTR independently of the polyA tail.43 In a study of host protein and viral RNA interaction using a mouse model, we found that kidney and older heart (but not young heart) are resistant to CVB3 infection and that this resistance correlates well with the strong interaction of a 28 kDa mouse protein with the nts 210–529 in the CVB3 5′UTR.39 Whether this 28 kDa protein confers tissue resistance to CVB3 infection needs to be further studied. As mentioned earlier, the molecular determinants of tissue tropism can also be located in viral genome, particularly at the 5′UTR and the structural gene VP1 region.44 The amino acid residues of capsid protein VP1 that are responsible for recognition/binding of the viral receptor have been mapped to residues K78, A80, A91, and I92. The presence of these amino acids is sufficient to induce lytic infection in HuFi H cells.45 However, derived CVB3 variants and constructed hybrid viruses showed different affinity to CAR and DAF, indicating that cell surface molecules other than CAR and DAF may be involved in attachment of these viruses to the cell surface, and therefore in viral tissue tropism. This hypothesis is supported by a recent study which revealed that human cardiomyocytes might express up to five proteins distinct from CAR and DAF that can act as receptors for CVB3.46

3. DIFFERENTIAL HOST GENE EXPRESSION PROFILES IN CVB INFECTIONS CVB infection of host cells alters the expression of numerous genes. This differential expression plays a crucial role in host defense and pathogenesis. To identify these up or downregulated genes, a number of studies have

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been conducted using different approaches, such as differential mRNA display,47 cDNA microarray and others, in cultured cells and in mouse models.48–51 The identified differentially expressed genes can be divided into groups involved in immune responses, gene expression regulation, signal transduction (see next chapter), prosurvival or prodeath, inflammation, target cell fibrosis, etc. Selected groups will be discussed below.

3.1 Host Genes Involved in Immune Responses 3.1.1 Cytokine and chemokine profiles It has long been known that cytokine activity plays an important role in determining the histological and immunological responses in animal models of CVB3-induced myocarditis.52–55 However, a comprehensive analysis of cytokine and chemokine profiles was carried out only in recent years. Gemosa and colleagues analyzed cytokine expression profiles by RT-PCR in the heart, spleen, and thymus of NMR1 mice during CVB3 infection and revealed that cytokines IL-1α, IL-6, IL-10, IL-12, IFN-β, INF-γ and IFN-α were produced in the heart or spleen but not in the thymus. However, IL-2 and IL-4 were not detectable in these tissues.56 Another interesting observation is the persistent upregulation of IL-10 and its leading role in acute and chronic myocarditis by subverting immune responses.56 Later studies from the same research group revealed that elevated expression of TNF-α, IL-1α, IFN-γ , IL-10, IL-18, macrophage inflammatory protein-1α (MIP-1 α ), and transforming growth factor-β (TGF-β ) persisted as long as 98 days post infection (pi).57 The role of upregulation of IL-18 and IL-1β in viral myocarditis was further confirmed using IL-12Rβ 1- and TLR-4- deficient mice, in which toll-like receptor 4 (TLR4) and IL-12Rβ 1 were shown to share common downstream pathways that directly influence IL-β 1 and IL-12 production.58 TNF-α and its receptor p55 were found to play a role in the upregulation of CD1d, a non-polymorphic MHC I-like molecule often associated with innate immunity and essential for pathogenicity of CVB3-induced myocarditis.59,60 As intense mononuclear leukocyte infiltration is a characteristic of viral myocarditis, it is of interest to know the production of cytokines in monocytes. An early report indicated that human monocytes

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can produce low amounts of proinflammatory cytokines upon CVB3 infection.61 Recently, a study reported that CVB3 infection of human monocytes induced transcription of proinflammatory cytokines IL-1, IL-6 and TNF-α, but only very little cytokine protein could be detected. In striking contrast, IL-10 expression was strongly and persistently induced by CVB3 at both the mRNA and protein levels.62 CVB3 infection also upregulates a number of chemokines. Besides MIP1α as mentioned above, many other chemokines are also expressed either constitutively [e.g., monocyte chemoattractant protein 1 (MCP-1), MCP-2, MCP-3, MCP-5, macrophage-derived chemokine (MDC) and lymphotactin], or through CVB3 induction [e.g., MIP-2, monokine induced by IFN-γ and interferon-γ -inducible protein 10 (IP10/CXCL10)] in the heart.63,64 However, the expression of MCP-1, MCP-2, MCP-3, MCP-5, MDC and lymphotactin could increase 1.2–3.7 folds when mice were challenged with CVB3. Our data obtained by differential mRNA display and cDNA microarray indicated that IP10/CXCL10 had a 5-fold increase at day 7 post CVB3 infection in mice.47,49 In transgenic CXCL10 mice, we further showed that CXCL8 and CXCL9, as well as their receptor CXCR3 were also upregulated following CVB3 infection (unpublished data). Although infiltrating immune cells are the major source of cytokines and chemokines, recent reports indicated that non-immune cells can also produce chemokines or cytokines in the heart. For example, upon CVB3 infection cardiomyocytes can produce a large amount of MCP-165 and cardiac fibroblasts can produce IL-6 and IL-8.66 These chemokines produced in the heart will subsequently lead to migration of mononuclear cells to the heart.

3.1.2 Interferon stimulated genes (ISG) Interferons are widely expressed cytokines that have potential antiviral and growth-inhibitory effects. During early viral exposure, cells at the local site of viral entry rapidly produce and secrete type I IFNs (IFN-α , β , ω and τ ). Type II IFN, consisting only of IFN-γ , is elicited at slightly later stage of infection. This cytokine is secreted by activated natural killer (NK) cells, CD4+ Th cells and CD8+ cytotoxic T lymphocytes,67 and its production is influenced by the expression of β 2-microglobulin.68

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Members of both types of IFNs contribute to the host’s antiviral defense by upregulating MHC I expression and activating NK cells, macrophages and T cells.69,70 In addition, IFNs act in auto-, para- and endocrine fashions to trigger intracellular signaling necessary for activation and expression of ISGs. These upregulated genes play a critical role in modulating cell survival or death, thus limiting viral replication and dissemination. The protective role of IFNs in CVB infection has long been known from studies using cultured cells71,72 and confirmed by immunizing CVB3-susceptible mice with recombinant CVB3 variants overexpressing IFN-γ protein.73,74 The roles of the IFN system in viral infection were further investigated by recent studies using genetically modified mouse models. Wessely et al. used CVB3-infected IFN receptor-deficient mice and found that type I but not type II signaling is essential for the prevention of early death due to CVB3 infection. Interestingly, neither type I nor type II IFN signaling has a dramatic effect on early viral replication in the heart. However, lethal viral replication in the liver is controlled by type I IFNs. These results demonstrated that the IFN system is capable of modulating both viral pathogenicity and tissue tropism.75 One of the mechanisms by which IFNs enhance the host antiviral state is their regulatory interactions with other cytokines or chemokines. For example, IFNs can induce overproduction of certain chemokines such as IP10 in CVB3-infected mouse heart.47 IP10 has proapoptotic activity and induces HeLa cell apoptosis via a p53-dependent pathway.76 The induction of host cell death by upregulated IP10 may limit viral replication and further dissemination. It was also reported that the antiviral effect of IFNs (β and γ ) is due to their downregulation of IL6, IL8, TGF- β 1, IL1β and IL4, which are important cytokines in the pathogenesis of viral myocarditis.77,78 Further, IFN expression is regulated by other cytokines during CVB3 infection. For example, studies using mice deficient in IL-12p35 demonstrated that IL-12 deficiency did not prevent the development of acute myocarditis, but allowed a significant increase of viral replication.79 In addition, signal transducer and activator of transcription 4 (STAT4) or IFN-γ deficiency also resulted in significantly increased levels of viral replication, indicating that IFN-γ produced by IL-12 and STAT4 transcription is important in reducing CVB3 levels in the heart.79 This notion is supported by experiments using a mouse model of chronic pancreatitis

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treated with IL-12 during CVB4 infection.80 In our own recent study using transgenic IP10 mice, we found that overexpression of IP10 in the heart upregulated IFN-γ production and Th1 cytokines (unpublished data). In search of further molecular mediators of the protective role of IFNs in viral myocarditis, several subsets of ISGs have been identified by a number of laboratories, which will be briefly discussed below. 3.1.2.1 2′-5′ oligoadenylate synthetase (OAS)/RNAse L and dsRNA-dependent protein kinase (PKR) OAS is a thermostable isozyme that uses ATP to produce 2′,5′-oligoadenylates with 5′-terminal triphosphate residues.81 IFN induces activation of OAS only in the presence of dsRNA. Activated OAS can activate latent endonuclease RNAse L that is responsible for degrading viral RNAs.82 In addition to OAS, IFNs can induce the expression of PKR. In response to viral dsRNA, PKR (serine/threonine protein kinase) can catalyze the phosphorylation and inactivation of eIF2α , resulting in inhibition of viral protein synthesis.83,84 Both OAS/RNase L and PKR are IFN inducible and have been found to function in the IFN-orchestrated antiviral defense against CVB infection. Deonarain et al. used IFN-β deficient mice to identify ISGs during CVB3 infection. They found that in contrast to wild-type mice, the IFN-β deficient mice had an increased susceptibility to infection and more severe injury in the heart. Further analysis found a downregulation of ISG targets including the 2′-5′OAS and a serine/threonine kinase.85 A similar observation was reported in CVB4-infected islet cells.86 This study demonstrated that pancreatic islet cells express RNase L, OAS and PKR and that the expression of OAS and PKR is increased following exposure to IFN-α and IFN-γ . They also found that the 2′5′-OAS/RNase L pathway is required for IFN-α and IFN-γ -mediated islet cell resistance against CVB4 infection. Further, the INF-γ -mediated repression of CVB4 infection of islet cells requires an intact PKR pathway. 3.1.2.2 Mx GTPase and IGTPase Another example of an IFN-regulated protein with demonstrated antiviral activity is Mx GTPase.67,87 Mx proteins are GTPases that belong to the

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superfamily of dynamin-like GTPase.88 The intrinsic GTPase activity of Mx proteins is required for their antiviral activity.89 Mx proteins function via self-association and importantly, with viral proteins. The highly conserved tripartite GTP binding motif is present within the N-terminal region of the ∼70–∼80 kDa proteins.67,90 Animal model studies suggested that Mx alone is sufficient to block the replication of virus in the absence of any other IFN-α /β -inducible proteins.90 Mx expression is induced by IFN-α and IFN-β but not by IFN-γ.90,91 The antiviral activity of Mx protein depends on the specific Mx protein, its subcellular localization, and the type of challenge virus examined. To date, there is only one report on the antiviral function of Mx protein in myocarditis induced by CVB3 infection using IFN-β deficient mice. These mice had decreased Mx GTPase expression and increased myocarditis severity.85 Intriguingly, we identified an IFN-inducible GTPase (IGTP) in a differential mRNA display of CVB3-infected vs. uninfected mouse hearts. We found that IGTP was significantly upregulated at day 7 pi.47 This protein is predominantly located in the endoplasmic reticulum.92 Further functional studies demonstrated that IGTP overexpression activates the PI3/AKT survival pathway and inhibits virus-induced apoptosis by delaying the cleavage of eIF4GI, a eukaryotic translation initiation factor.93 In contrast to Mx GTPases, IGTP is induced by IFN-γ and its molecular weight is ∼47 kDa, which is much smaller than that of the Mx proteins. Whether IGTP should be classified as an associated member of the Mx GTPases needs to be studied. However, its role in antiviral immunity is similar to that of Mx GTPases. 3.1.2.3 Inducible nitric oxide synthase (iNOS) Another upregulated IFN-inducible protein is IFN-γ -inducible NOS.94 iNOS can catalyze the synthesis of nitric oxide (NO) from L-arginine. NO is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. To investigate the role of iNOS in CVB-induced myocarditis, several studies have been conducted using different model systems. One of the early studies using CVB3-infected NMRI mice demonstrated that high iNOS mRNA levels appeared at day 4 and remained high until day 28 pi. The mRNA of inflammatory cytokines TNF-α, IL-1α and IFN-γ also increased at different time points pi.

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They also found that iNOS is located in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart.95 Another study using iNOS knockout mice revealed that without iNOS expression, mice had an increased mortality rate following CVB4 infection in comparison to normal mice.96 The protective role of iNOS in CVB3-induced myocarditis was confirmed by in vitro and in vivo studies using a recombinant CVB3 variant expressing IFN-γ .74,97 However, this direct effect was not restricted to the homologous virus.74 The mechanisms by which iNOS protects tissue against CVB infection were further studied. Several reports indicated that iNOS-catalyzed production of NO can inhibit the function of viral proteases 2A and 3C,98,99 two important viral proteases involved in host-virus interactions (see discussion below). For CVB3 2A, dystrophin proteolysis is inhibited through S-nitrosylation.100 Recently, a study aimed to elucidate the relationship among IFN-γ , iNOS and IL-10 in ongoing myocarditis found that coordinated secretion of IFN-γ and IL-10 is critical for the effective resolution of CVB3 myocarditis. Moreover, lack of regulatory IL-10 leads to uncontrolled iNOS production, thus contributing to ongoing myocardial injury.101

3.1.3 Major histocompatibility complex (MHC) proteins In addition to antiviral effects exerted at single-cell levels that reduce viral transcription and translation, IFNs modulate a number of immunoregulatory interactions between cells, e.g., those of NK cells and Th cells with virus-infected cells. Cytotoxic T lymphocytes recognize and kill infected cells exposing viral peptides presented by MHC proteins.67 NK cells play an important role in the early host response to some viruses by killing infected cells and/or producing cytokines, e.g., IFN-γ and TNF-α. NK cells are activated by proinflammatory cytokines, e.g., IL-12 and type I IFNs.102 Whether MHC molecules are upregulated or downregulated during CVB3 infection is controversial. One report indicated that CVB4 infection of human fetal thymus organ culture upregulated HLA class I expression103 which exacerbates CVB4 pathogenesis. These results are supported by another study of CVB4 infection in recent-onset type 1

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diabetic patients.6 However, two other studies demonstrated the downregulation of HLA class I cell surface expression in CVB3 infected HeLa cells.70,104 One of the two reports further indicated that the lowered HLA class I expression in target cell does not correlate with an increased susceptibility to NK cell-mediated killing. Instead, NK cells responded with a robust production of IFN-γ. Thus, NK cells contribute to the host antiCVB3 immune response by production of IFN-γ rather than by directly killing infected cells.70

3.2 Gene Expression Involved in Acquired Immunity and Apoptosis The host immune response cannot always successfully repress viral replication and in certain conditions, it loses the battle against virus infection, resulting in disease. This is largely due to the balance of innate immunity tilting toward pathogenic gene expression. Several such genes have been identified in different studies. The sarcoma (src) family of kinases (p56lck) is a T-cell receptor tyrosine kinase. Mice lacking the p56lck gene were completely protected from CVB-induced acute pathogenicity and chronic heart disease, indicating that p56lck is crucial for both CVB3 proliferation and targeting of T-cells to the heart.105 Further, p56lck could trigger extracellular signal-related kinase (ERK1/2) activation in host target cells and play an important role in viral pathogenesis.106 In addition, it was demonstrated that CD45 knockout animals were resistant to viral myocarditis. After analysis, it was demonstrated that CD45 is an important tyrosine, as well as the Janus kinase/STAT phosphatase, and that virus-triggered CD45 activation can shut down IFN production.107 Myeloid differentiation factor (MyD88) is a key adaptor protein that plays a major role in the innate immune pathway. Using MyD88 deficient mice, Fuse et al. found that MyD88 could activate downstream inflammatory signals including IL-1β receptor-associated kinase-4 (IRAK-4) and tumor necrosis factor receptor-associated factor-6 (TRAF-6), and could also induce nuclear translocation of NF-κ B, leading to increased production of cardiac cytokines such as IL-1β , TNF-α , and IFN-γ (Figure 1).108 In addition, MyD88 increases tissue CAR and p56lck expression, leading to increased virus entry. This combination leads to increased virus titer,

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Figure 1. Cellular responses to CVB3 infection. Upon infection or IFN stimulation, a number of genes involved in antiviral affects, inflammation, apoptosis or fibrosis are differentially expressed. Arrows represent activations between proteins in signaling pathways; lines ending in bars represent inhibitory effects on viral replication or gene expression. The figure does not indicate that these proteins are present in all cells at the same time.

tissue inflammation and cardiac damage. However, absence of MyD88 confers host protection possibly through novel direct activation of interferon regulatory factor-3 (IRF-3) and IFN-β. In addition to immune and autoimmune-mediated destruction of the myocardium, apoptosis of infected cardiomyocytes is also involved in viral pathogenesis. A number of proapoptotic genes are upregulated in CVB-infected cells, such as proapoptotic Bcl-2 family proteins, caspases, perforin, granzyme B, Fas/LasL, iNOS, proapoptotic cytokines (TNF-α, TGF-β ), certain ISGs and many others (see reviews Refs. 16 and 109). Here we limit our discussion to several host genes that were reported

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recently in the context of CVB infections. These genes include mBNip21, Bag-1, Cyr61, Siva, activating transcription factor 3 (ATF3), β-catenin, and glutathione. Since the first three genes were identified by genomewide transcriptional analysis, we will discuss them in the next section. Siva is an upregulated gene in CVB3-infected heart. This zinc-containing protein induces cell apoptosis via interactions with the CVB3 VP2 protein.110 Recently, a new Siva interacting protein, the peroxisomal membrane protein PMP22, was pulled from the screening of a human heart cDNA library.111 This protein may be involved in host response to CVB3 infection. Another differentially expressed protein is ATF3. This factor is an early-induced gene involved in diverse cellular functions in response to various stresses, including viral infection. Its role is controversial since it is both anti-apoptotic112,113 and proapoptotic.114 A recent study of CVB3 showed that infection of HeLa cells markedly reduced ATF3 expression at both the mRNA and protein levels in parallel with degradation of p53. Further, overexpression of ATF3 stimulated apoptotic cell death following CVB3 infection, accompanied with augmented eIF2α phosphorylation. However, ATF3 overexpression did not affect viral protein synthesis but promoted virus progeny release. Thus, ATF3 is under the control of p53 in part, and ATF3 downregulation via p53 degradation may contribute to effective viral production as a modulation mechanism of CVB3-induced cell death.115 β-catenin, a transcription factor and substrate of glycogen synthase kinase 3β , is also a downregulated gene in CVB3 infection. Inhibition of glycogen synthase kinase 3β suppresses CVB3-induced cytopathic effect and apoptosis via stabilization of β-catenin.116 Another downregulated protein is glutathione, a pro-survival gene. This protein is an intracellular reducing agent that helps to maintain the redox potential of the cell and is important for immune function. It was reported that CVB3 infection of BALB/c mice progressively decreased plasma glutathione levels, which coincided with cardiomyocyte apoptosis.117 This data implies that glutathione has pro-survival activity and in turn benefits viral replication in host cells. This notion is supported by a recent study demonstrating that picornaviruses require glutathione for efficient production of mature infectious virions, as pharmacological inhibition of glutathione synthesis strongly inhibited viral replication in HeLa cells.118

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4. GENOME-WIDE GENE EXPRESSION PROFILES 4.1 CVB3-Infected Mouse Heart A genome-wide differential gene expression display, comparing expression levels in infected versus uninfected samples, has been analyzed by several laboratories. However, each laboratory employed a different approach and has distinct emphases. Our laboratories initially conducted an analysis by differential mRNA display47 and later by cDNA and oligo microarrays.49,119 In the differential mRNA display, a total of 28 genes with an altered expression level were identified. In a cDNA microarray containing ∼7000 clones, a total of 169 genes were up or downregulated in CVB3-infected mouse hearts, which were further divided into eight groups according to gene function. Several of these genes from the above experiments have been further functionally characterized by establishing Tet-On inducible HeLa cell lines and transgenic mouse models. Mouse BNip21 (Bcl-2 and nineteen kDa interacting protein) is a BH3 (Bcl-2 homologous domain 3) only proapoptotic protein of the Bcl-2 family and induces HeLa cell apoptosis through a mitochondria-dependent pathway.120 IGTP and IP10 are two interferon-inducible proteins. IGTP is a member of the p47 kDa family of GTPases. Our recent study revealed that IGTP activates the PI3K/Akt survival pathway through the mediator focal adhesion kinase (FAK), subsequently activating NF-κB [Liu et al., Cell Microbiology, in press]. The chemokine IP10, however, seems to play a role in enhancing cell death. In transfected Tet-On HeLa cells, IP10 overexpression can induce cell apoptosis through a p53-dependent pathway initiated by suppression of human papillomavirus type 18 E6 and E7 expression.76 Another cDNA microarray analysis was performed by Peng et al. using an array carrying 588 known mouse genes. 42 genes had altered expression in the heart.50 One of the downregulated genes, Bag-1, an inhibitor of apoptosis and modulator of chaperone activity, was further studied.50 Downregulation of Bag-1 during CVB3 infection promoted apoptosis, suggesting the reduction of anti-apoptotic protein may be an important mechanism by which CVB3 causes cardiomyocyte death. Three other microarray analyses focused on the identification of extracellular matrix (ECM) genes with altered expression in CVB3-infected

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mice. The first used a cDNA microarray containing 8192 genes and identified nine overexpressed genes in the heart. Northern hybridization confirmed four (Fin15, ILk, Lamr1 and ADAMTS-1) of the nine genes. However, the functions of these four genes were not studied further in this report.121 The second analysis was conducted using CVB3-infected HeLa cells. Although the total altered gene expression profile has not been published, they reported an upregulated expression of cysteine-rich protein gene (cyr61).48 Cyr61 is an early-transcribed gene secreted into the extracellular matrix. Cyr61 expression facilitates CVB3 growth in the cells and promotes host cell death by viral infection. The third analysis was aimed at identifying the mediators of virus-induced cardiac fibrosis in viral myocarditis and DCM. In this study, connective tissue growth factor (CTGF) was highly expressed in infected mouse hearts, particularly in fibroblasts.51 Further, in the course of myocarditis, CTGF upregulation coincided with increased mRNA transcription of cardiac TGF-β and procollagen type I, preceding the formation of fibrotic lesions, suggesting that CTGF is a crucial molecule in the development of fibrosis. This finding is consistent with a previous report, except for their observations on whether or not cardiomyocytes can express CTGF during CVB3 infection.122 Another upregulated, fibrosis-causing gene is PDGF, which was identified in CVB3-infected mouse heart by RT-PCR, and its expression was localized with viral RNA and inflammatory infiltrates, adjacent to fibrotic areas.123 The ECM is a complex structural entity surrounding and supporting cells to maintain the cardiac system in addition to performing various other important functions. Matrix metalloproteinases (MMPs) are enzymes responsible for the degradation of the ECM during the myocardium remodeling process. Studies using BALB/c H-2d mice demonstrated upregulation of MMPs 1, 3, 8 and 9 at the protein level in the heart after infection with CVB3.124,125 Our laboratory has shown that MMPs 2, 9 and 12 are upregulated in male A/J mice 9 days after infection, and further, MMPs 2 and 9 demonstrated enhanced activity at this time point.126 Conversely, the tissue inhibitors of MMPs (TIMPs) 3 and 4 were found to be downregulated during CVB3-induced myocarditis in these mice.124,126 We further demonstrated that knockout of MMP-9 expression in CVB3infected mice lead to an increased severity of myocarditis, suggesting a

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beneficial role of MMPs in limiting viral infection and in attenuating cardiac damage.127

4.2 CVB4-infected Pancreas The Global transcriptional profile has been analyzed in CVB4-infected pancreas islet cells. One such study used two CVB4 variants to conduct a comparative investigation of gene expression profiles in virus-infected mice.128 Pancreas tissue damage caused by non-virulent CVB4-P could be resolved, but damage caused by virulent CVB4-V infection was permanent. To correlate the gene expression maps to the pathological changes occurring in the pancreas, Affymetrix MG-U74Av2 mouse GeneChips were compared to identify differentially expressed genes. Hundreds of genes were up or downregulated, and further divided into nine functional groups. Between the two CVB4 variants, acute pancreatitis that resolved was associated with tissue regeneration, which was accompanied by increased expression of genes involved in cell growth, inhibition of apoptosis, embryogenesis and by increased division of acinar cells. However, permanent damage during CVB4-V infection was due to the lack of tissue repair and the expression of genes involved in apoptosis, acinoductular metaplasia, remodeling of the extracellular matrix, and fibrosis.128 Another analysis of genome-wide gene expression in human islet cells focused on CVB4-induced inflammatory cytokine production. In this study, the transcriptional profiles of two CVB4 variants were compared by probing Atlas Cytokine/receptor nylon membrane arrays (Clontech).129 The CVB4 lytic variant (V89-4557) increased MCP-1 and RANTES while the CVB4-nonlytic variant (VD2921) increased IL-1β , IL-6, IL-8 and MCP-1, and decreased IL-13. In addition, the IL-5R-α and IL-2Rα were up and downregulated, respectively, in this strain. Furthermore, a higher level of cytokine production was seen in islets infected by the VD2921 strain than those infected by the V89-4557 strain. Surprisingly, none of the type I IFNs was increased after infection with either of the virus strains, but VD2921 increased the expression of IFN-γ antagonist. The global profiling of gene expression in human islet cells in response to CVB5 infection or cytokine stimulation was conducted by two

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laboratories using similar approaches. One laboratory used islet cells infected with CVB5 or treated with IL-1β plus IFN-γ to probe for altered gene expression. A total of 13077 genes were detected, with 945 and 1293 single genes found to be modified by exposure to virus and the indicated cytokines, respectively.130 Four hundred and eighty-four genes were similarly modified by the cytokines and viral infection combined together. The most marked and consistent increase hits were observed in genes encoding cytokines, chemokines and related receptors, various signaling molecules, and molecules involved in cell defense, repair and immune responses. Thus the authors indicated that several chemokines, IL-15 and the intercellular adhesion molecule ICAM-1 might contribute to the homing and activation of mononuclear cells in the islets during viral infection and/or an early autoimmune response, which leads to beta cell dysfunction and death. In another study, human islet cells were treated with IFN and screened for differentially expressed genes involved in antiviral defense. Twenty three and six upregulated genes were identified in islet preparations treated with IFN-α and IFN-γ , respectively.131 These genes include many proteins known to act as antiviral defense such as Viperin,132 ISG15,133 IFITM1,134 2′,5′-OAS, and many others that have been mentioned above. In addition, several intracellular sensors for viral RNA were upregulated in IFN-treated islets, including melanoma differentiationassociated gene-5 (MAD-5), toll-like receptor 3 (TLR3) and retinoic acidinducible gene 1 (RIG-1) (Figure 1). These three genes induce type I IFN production in infected cells and provide human islets with powerful protection from virus-induced type 1 diabetes.131

5. PROTEOME-WIDE ANALYSIS CVB-induced gene expression profiles have also been analyzed with a proteomic approach. CVB3-infected and non-infected HeLa cells and HepG2 cells were used to perform this study. Cellular proteins were separated on 2D gels and peptide sequences were analyzed by fingerprint and MS/MS sequencing using a Proteomics analyzer 4700 (MALDI-TOF/ TOF) mass spectrometer.135 This study identified more than 230 differentially regulated and modified proteins. The modified proteins were found in the gels of both control cells and CVB3-infected cells, but on

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different points within each. The authors predicted that these electrophoretic mobility changes might be due to post-translational modifications such as phosphorylation and/or proteolytic onset. The identified genes were organized into eight functional groups, which include gene expression, cell structure and associated proteins, protein metabolism, stress response, nuclear proteins, cell cycle, cell signaling, and energy metabolism. Among these, several proteins including nucleophosmin, lamin, the RNA-binding protein UNR and p38 MAP-kinase were interpreted in the context of myocarditis.135

6. VIRAL PROTEINS AFFECTING HOST GENE EXPRESSION It is well documented that picornavirus proteases 2A and 3C contribute to viral pathogenesis by downregulating host gene expression through cleavage of cellular translation and transcription factors17,136 (Figure 2). Viral proteases 2A and 3C also cleave and activate caspases, PARP and Bid, leading to apoptosis.137,138 Recently, CVB3 3C was found to cleave the inhibitor of κ Bα (Iκ Bα ). After cleavage, a proteolytic fragment of IκBα can form a stable complex with NF-κ B, translocate to the nucleus, and inhibit NF-κ B transactivation, increasing apoptosis and decreasing viral replication.139 In shutting off host mRNA translation, CVB protease 2A palys a major role by cleavage of eIF4GI,137,140 but no report thus far indicates that it can also cleave eIF4GII. Our recent study indicated that transfection of CVB3 2A or 3C, or both together cannot cleave another functional homologue of eIF4GI, DAP5/p97/NAT1, even though CVB3 infection causes indirect cleavage of this protein.137 It is known that the cleavage of eIF4GI significantly inhibits cellular mRNA translation but benefits IRES-driven viral RNA translation initiation. The mechanism for this alternative translation initiation requires a properly configured mRNA template rather than the disruption of host cell translation initiation factors.141 Another important protein for translation initiation, poly-A binding protein (PABP), is cleaved by CVB3 2A.142,143 In fact, most PABP in poliovirus-infected cells is processed by protease 3C, but not 2A.144,145 It was found that only a subset of cellular PABP is cleaved, suggesting that compartmentalization or

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Figure 2. Interactions of CVB3 proteins with host transcriptional and translational machinery. CVB3 proteases 2A and 3C cleave host structural proteins (e.g., dystrophin) and non-structural proteins involved in cellular gene expression at transcription and translation levels, resulting in inhibition of cap-dependent translation and promotion of IRESdriven translation initiation. CVB3 2B and 3A suppress protein secretion and trafficking from the ER to the Golgi complex. Certain cleavages of transcription factors are known to occur in poliovirus, a very close relative of CVB3. Small arrows represent activations between proteins in signaling pathways; lines ending in bars represent inhibitory connections; big open arrows indicate cleavage by viral proteases or caspase-3.

alternate conformation may regulate PABP cleavage. Indeed, PABP in the ribosome-enriched fraction was preferentially cleaved by 3C as compared to PABP in other fractions. Enteroviruses inhibit host gene expression not only at translational levels, but also at transcriptional levels. In poliovirus infection, 3C cleaves and inactivates transcription factors and activators, including TATA-binding protein (TBP),146 transcription factor IIIC2,147 activator Oct-1148 and cyclic AMP-responsive element-binding protein (CREB).149

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CREB has anti-apoptotic activity if phosphorylated by IGF-I in a PI3Kand MEK1-dependent manner.150 Our recent work has shown that transfection of CVB3 2A or 3C in HeLa cells could reduce CREB protein expression and induce apoptosis.137 The cleavage of these transcription factors requires 3C to act in the nucleus. A recent study151 showed that 3C enters the nucleus in the form of its precursor 3CD, where it presumably undergoes autocatalysis to generate 3C and cleaves its target proteins. CVB3 proteins 2B, and 3A can also affect host protein production, but at the level of protein secretion and trafficking (Figure 2). 2B inhibits protein trafficking from ER to Golgi complex via its targeting of these organelles and alterations of their membrane permeability.152,153 3A inhibits protein secretion and trafficking by destroying the Golgi complex itself and blocking activation of the ADP-ribosylation factor 1 (Arf1) protein, a key factor in protein transport and organelle structure and maintenance.154,155

7. CONCLUSION CVB-induced gene expression profiles have been analyzed using a number of approaches, and the identified genes have been successfully studied to gain new insights into the mechanisms of viral pathogenesis. However, when comparing the data obtained from different approaches, the up or downregulated gene profiles were not very consistent. This may be due, at least in part, to the experimental systems used, the focus of gene groups selected, the time points used for analysis or the experimental errors generated. Therefore, to ensure the accuracy of the analysis, changes in expression should be confirmed by qRT-PCR and/or Western blot or other quantitative measurements. The function of the gene product should be further analyzed by gene transfection of cultured cells or using genetically modified animal models. Presently, most of the data on gene expression changes during CVB infection are from cDNA or oligo microarray analyses; proteomic approaches are still in the beginning stages. This latter approach not only identifies differentially expressed genes but also discovers post-translational modifications. With the progress of proteomic analysis, additional interesting data will be generated. In addition, most previous profiling studies focused on genes induced by wild-type virus or

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recombinant CVB expressing a cytokine or a growth factor, or by transfection of cells with an individual viral gene. There is lack of study on regulation of gene expression by microRNA (miRNA), a significant, recently characterized mechanism of gene expression regulation. In other words, it is not known whether CVB RNA is targeted by cellular miRNAs, which could result in translational silencing or miRNA-mediated degradation of viral RNA. On the other hand, it is also unknown if enteroviruses encode miRNAs which could interact with cellular mRNAs to activate RNAi mechanisms be beneficial to viral replication. A microarray analysis of miRNA expression profiles of host and virus will likely to bring many new surprises.

ACKNOWLEDGMENTS This work was supported in part by grants from the Canadian Institutes of Health Research and Heart and Stroke Foundation of BC and Yukon.

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CHAPTER 22

Host Signaling Responses to Coxsackievirus Infection Guang Gao, Decheng Yang, Bruce M. McManus & Honglin Luo

ABSTRACT Coxsackievirus has evolved various strategies to manipulate the host cell’s metabolic and regulatory systems to create a favorable environment for its own benefits. Prior to immune injury of target organs, infection of target cells induces signaling/transcriptional responses that include activation of numerous intracellular signaling pathways. These pathways have been demonstrated to be involved in the regulation of viral replication and viral pathogenesis, and activation of the ubiquitin-proteasome system, triggering aberrant host protein degradation and/or viral protein modification. In this chapter we will review the changes of host response profiling induced by coxsackievirus infection, with specific focus on the host signaling pathway and the ubiquitin-proteasome system, and discuss the consequences of these coxsackievirus-mediated modifications in viral infection.

1. INTRODUCTION 1.1 Coxsackievirus and Viral Myocarditis Coxsackieviruses (CVB) are typical human enteroviruses in the family of Picornaviridea, a genus that includes the well-studied and closely related polioviruses.1 Six CVB serotypes (CVB1-6) have been found so far. 525

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Among them, CVB3 has been considered as the primary causative agent of viral myocarditis. Viral myocarditis and its main sequelae, dilated cardiomyopathy (DCM), are the most prevalent cause of morbidity and mortality worldwide, particularly in children. The concept that myocarditis contributes to the pathogenesis of a subset of DCM is strengthened by the detection of viral genome in hearts with DCM. Viral RNA is present in the heart muscle of approximately 25% of patients with DCM.2,3 The only definitive treatment currently available for DCM is heart transplantation. Global surveillance data compiled by the World Health Organization indicate that CVB is the most common pathogen associated with clinical cardiovascular disease. Nearly 50% of clinical myocarditis cases in North America are reported to be attributable to picornaviral infection, with the CVB3 serogroup accounting for the most significant portion of such infections.4 CVB3 is a non-enveloped, cytolytic, single-stranded RNA virus with a short replication cycle. During the replication cycle, viruses invade into cytoplasm of host cell through coxsackieviral receptors, which include the coxsackievirus and adenovirus receptor (CAR) and the decay accelerating factor (DAF) co-receptor. Viral genomic RNA can be employed directly as mRNA template for translation of a single viral polyprotein, which encodes 11 proteins, including four capsid proteins VP1-VP4 and seven non-structural proteins. Virus-encoded proteases 2A and 3C process the CVB3 polyprotein post-translationally so that no full-length viral polyprotein is observed. In the early infection, these viral proteases have also been shown to cleave multiple host proteins directly, including regulatory proteins involved in host transcription and translation.5,6 CVB protein 3D is an RNA-dependent RNA polymerase, which is essential for viral RNA replication. Viral RNA can also function as a template for viral RNA transcription to synthesize more copies of parental RNA through a negative strand intermediate, which is transcribed from CVB3 positive sense genome by processing of viral protein 3D.

1.2 Coxsackievirus-induced Early Direct Injury of Myocardium The mechanism of cardiac muscle damage in virus-induced myocarditis remains not fully understood. Traditionally, viral myocarditis was considered

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to be an immune system-mediated disease of the heart; however increasing evidence suggests that early direct virus-induced injury occurring prior to infiltration of the immune response also contributes significantly to the pathogenesis of CVB3-induced myocarditis. It has been shown that CVB3 infection induces a direct cytopathic effect (degenerative changes in cell morphology) and cell apoptosis.7 CVB3 infection leads to the activation of a mitochondria-mediated caspase cascade, causing host cell death.7,8 Further studies suggested that apoptosis during the late phase of virus infection facilitates viral progeny release. Apoptosis, but not cytopathic effects, could be prevented by inhibition of caspases or overexpression of mitochondrial anti-apoptotic protein Bcl-2 and Bcl-xL, indicating that the two cellular responses are regulated separately.7,8 In virus-infected hearts, it was shown that infection of severe combined immune-deficient mice with CVB3 resulted in increased myocyte damages and death as compared to immunocompetent controls, reinforcing the importance of direct viral-induced damage.9,10 Previous studies have suggested that early host gene responses to viral infection play a key role in determining the severity of myocarditis and progression of DCM.11–14 Considering such an important role of early direct injury, in this chapter, we shall focus on the early determining factors, in particular, the interaction between virus and host ubiquitin-proteasome system, as well as the signaling pathways. It is believed that a better understanding of the molecular basis of early direct effect of viral infection on cardiac myocytes will provide valuable insights into the mechanisms by which viral infections injure heart muscle.

2. THE UBIQUITIN-PROTEASOME SYSTEM IN COXSACKIEVIRUS INFECTION 2.1 The Ubiquitin-Proteasome System (UPS) The UPS is a major intracellular pathway for protein degradation, with over 80% of all cellular proteins being recycled through this pathway.15,16 Abnormalities of the UPS have been associated with several human diseases, including cancer, neurodegenerative, and cardiovascular diseases.17,18 For UPS-mediated proteolysis, protein substrates are first conjugated to

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multiple ubiquitins, and then degraded by the proteasome. Ubiquitin is a small, highly conserved 76-amino acid protein, which is activated in an ATP-dependent process by the ubiquitin-activating enzyme (E1), and subsequently transferred to a ubiquitin-conjugating enzyme (E2). Final transfer of ubiquitin to the target protein requires ubiquitin-protein ligase (E3). The structure of the ubiquitin conjugation system is hierarchical: there is only one E1 enzyme in human. Dozens of E2 enzymes have been identified, each of which interacts with one or several E3 enzymes.19,20 The human genome encodes 500–600 E3 enzymes. Each E3 targets one or a few substrates. Therefore, the substrate specificity of the ubiquitin conjugation system is conferred by the E3s. After several rounds of ubiquitination, a polyubiquitin chain is formed through an isopeptide bond between the C-terminal glycine 76 of ubiquitin and a lysine residue of the adjacent ubiquitin or the protein substrate. The ubiquitinated substrate is quickly recognized and subsequently degraded by the 26S proteasome, and ubiquitin is recycled via the action of deubiquitinating enzymes.15,16 The process of polyubiquitination and degradation by proteasome is depicted in Figure 1A. The 26S proteasome consists of one 20S catalytic core and two 19S regulatory complexes. The 20S core is a barrel-shaped cylinder composed of two outer α-rings and two inner β -rings. The α -rings modulate the entrance of protein substrates, whereas the β -rings execute the action of proteolysis. The 19S complex plays an important role in regulating the function of the 20S. Its activities include ubiquitin recognition, deubiquitination, substrate unwinding, and substrate translocation to the 20S core.19 In addition to well-established role of polyubiquitination in protein degradation, ubiquitination has been reported to be involved in the regulation of many important cellular functions, including cell cycle regulation, apoptosis, signal transduction, and transcriptional regulation. Monoubiquitination of some proteins, such as histones, calmodulin, actin, and some transmembrane proteins, serves as a signal for receptor internalization, gene regulation, and virus budding without targeting for degradation15,16 (Figure 1B).

2.2 UPS and Viral Infection Accumulating studies have suggested that viruses can evolve different strategies to utilize or manipulate the host UPS to achieve successful viral

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Figure 1. The ubiquitin-proteasome system. A. Polyubiquitination and protein degradation. Substrate protein is labeled with at least four ubiquitins in the presence of ubiquitinactivating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The polyubiquitinated substrate is then recognized and subsequently degraded by the proteasome, and ubiquitin is recycled via the action of deubiquitinating enzymes (DUBs). B. Monoubiquitination and protein function regulation. Target protein is tagged with one ubiquitin. Monoubiquitination serves as a signal for the regulation of protein function, without targeting for degradation.

infection. The mechanisms by which UPS regulates viral infection involve degradation of intracellular proteins or excessive viral proteins that are against efficient viral replication and modulation of viral protein function through ubiquitin-mediated modification.21,22

2.2.1 UPS-mediated proteolysis and viral infection The UPS has been reported to play a critical role in almost every step of viral life cycle, including viral entry, replication, budding, progeny release, as well as reactivation of latent virus.21,22 Moreover, several viruses have been shown to utilize the host UPS to escape host immune surveillance.23,24 A number of viral and host protein substrates of the UPS have been identified.21 Here we use p53 as an example to illustrate the role of protein degradation in viral infection. p53 is an important transcription factor

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which plays a key role in cell growth control by modulating processes leading to apoptosis and DNA replication.25 Intracellular tumor suppressor p53 inhibits the replication of several viruses by promoting cell death and through direct inhibition of viral replication. Many viruses have been shown to evolve different strategies to inactivate p53 during early viral infection to suppress apoptosis. For instance, the human papillomavirus E6 protein inhibits apoptosis by interacting with the cellular ubiquitin ligase E6-associated protein to form a complex, targeting p53 for polyubiquitination and degradation.26 In adenovirus-infected cells, the level of p53 is also markedly reduced. E1B 55K and E4orf6, two adenovirus gene products, have been shown to regulate the function of p53 by directing it for degradation.27 In addition to its role in apoptosis, p53 has been reported to directly interfere with the replication of several viruses, such as human immunodeficiency virus type 1 (HIV-1),28 Simian virus 40 (SV40),29 hepatitis B virus30 and herpesviruses31 via various mechanisms. In the case of SV40 infection, wild-type p53 binds to SV40 large T antigen and blocks its function in mediating viral replication.29 p53 regulates HIV-1 gene expression by suppressing transcriptional activation of the long terminal repeat.28 It has been recently demonstrated that CVB3 facilitates p53 degradation in HeLa cells32 and in murine cardiomyocytes. It is speculated that CVB3 infection promotes UPS-mediated proteolysis of p53 to maximize its own replication by attenuating the inhibitory effect of p53 on viral replication.

2.2.2 Ubiquitin and ubiquitin-like modification in viral infection Viral protein modification by ubiquitin and/or ubiquitin-like proteins has also been suggested to play an important role in the regulation of viral protein function. Monoubiquitination of some viral proteins directly regulates viral transcriptional activities without targeting for degradation.33,34 It was reported that monoubiquitination of the Gag protein of retroviruses is required for virus budding.35–37 Depletion of free ubiquitin by proteasome inhibitors prevents Gag ubiquitination and subsequently blocks virus progeny release/budding. In addition, ubiquitination of HIV-1 Tat protein38 and human T-cell leukemia virus type 1 Tax protein39 has been shown to modulate their transactivation activities.

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Additionally, a number of ubiquitin-like proteins have been found to function as protein modifiers as well. These proteins, including SUMO proteins and autophagic proteins Atg8 and Atg12, regulate a variety of cellular functions, such as transcription, DNA repair, signal transduction, autophagy, and cell cycle control, by post-translationally modifying the target proteins.40–42 SUMO proteins (SUMO-1, 2 and 3) are a family of small proteins that are structurally similar to ubiquitin.43,44 Protein modification by sumoylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. SUMO E1-activating enzyme is a heterodimeric complex consisting of Aos1 and Uba2. While Ubc9 is the only known SUMO E2conjugating enzyme, three classes of SUMO E3-ligases have been identified, RanBP2, PIAS and the Polycomb protein Pc2. The sumoylation target is a lysine that occurs in the consensus motif ΨKXE where Ψ is a hydrophobic amino acid and X is any residue.45 Like monoubiquitination, sumoylation regulates proteins function and subcellular localization, instead of targeting proteins for degradation. The role of sumoylation in modification of viral proteins has been recently recognized.32 For example, retroviral Gag protein was shown to be modified by SUMO and this modification of Gag regulates viral infectivity.46 SARS (Severe Acute Respiratory System) coronavirus N protein also undergoes sumoylation which might play an important regulatory role in the viral replication cycle.47,48 Autophagy is a cellular process by which cells recycle cytoplasm and dispose of excess or damaged organelles.49,50 Two ubiquitin-like modifiers, Atg8 (also called microtubule-associated protein light-chain 3 (LC3) in mammals) and Atg12, have been shown to be required for autophagy. Autophagy begins with the sequestration of organelles or cytoplasm within a double-membrane vesicle (autophagosome). The outer membrane of the autophagosome then fuses with the lysosome to release the inner membranous vesicle for degradation. Two ubiquitination-like conjugation systems, which involve ubiquitin-like coupling of either Atg12 to Atg5 or Atg8/LC3 attachment to the phosphatidylethanolamine, are essential for the formation of autophagosome. Autophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.51 Recent studies also suggested that autophagy plays a major role in the control of viral replication.49,50 Autophagy has been primarily recognized as an antiviral host response by clearing viruses out

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of the cells.49 As a result, some pathogens try to escape autophagy. For example, it has been shown that herpes virus encodes a neurovirulence gene product that blocks autophagy.52 However, increasing evidence suggests that autophagy may also be utilized by viruses to enhance their efficiency of replication. It has been shown that some viruses induce autophagosomes to generate sites of viral replication without triggering cellular defenses.49,50 Replication of positive-stranded RNA viruses requires intracellular membrane surfaces on which to assemble their replication complexes. Autophagosome, a double-membrane vesicle, may provide a physical scaffold to concentrate viral components, thereby increasing viral replication. It has been reported that coronaviruses colocalize with the ubiquitin-like autophagy proteins, Atg8 (or LC3 in mammals) and Atg12.53 Viral replication is impaired in autophagy knockout (Atg5−/−) embryonic stem cell lines.53 Additionally, poliovirus proteins have also been shown to colocalize with a co-transfected GFP-LC3 protein.54 Stimulation of autophagy increases poliovirus yield, and inhibition of the autophagsomal pathway decreases viral yield.

2.3 UPS-Mediated Protein Degradation/Modification in Coxsackievirus Infection It has been recently demonstrated that the UPS-mediated protein degradation/modification plays a critical role in CVB3 replication. Cell culture studies using murine cardiomyocytes showed that treatment with the proteasome inhibitor MG132 or lactacystin markedly decreases CVB3 viral RNA and protein levels, and inhibits CVB3 progeny release.55 It was further demonstrated that inhibition of the UPS does not affect virus entry and has no direct influence on the proteolytic activities of viral proteases, suggesting that reduction of viral replication by proteasome inhibitors is unlikely due to the blockade of virus entry and cross-inhibition of virus protease proteolytic activities.55 These results suggest that the UPS may be utilized during CVB3 infection to control host protein degradation and promote viral replication and infectivity. Indeed, it was found that CVB3 infection promotes ubiquitin-mediated proteolysis of several host proteins, including cell cycle protein cyclin D1, tumor suppressor p53 and transcription activator β -catenin.32,56

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Using HeLa cell model of CVB3 infection, it was shown that there is a reduction of cyclin D1 and p53 expression.32 This reduction is abrogated when specific UPS inhibitors MG132 and lactacystin are used. Most excitingly, it has been found that CVB3 facilitates the ubiquitination of cyclin D1, which suggests a direct mechanism of protein degradation. β -catenin is one of the major targets of glycogen synthesis kinase 3β (GSK3β ) and has been implicated to play a key role in cell survival and cytoskeleton organization.57 GSK3β phosphorylates and targets β -catenin for ubiquitination and degradation through the UPS pathway.57 It was reported that CVB3 infection stimulates GSK3β activity via a tyrosine kinase-dependent mechanism.56 Blockage of GSK3β activation attenuates both CVB3-induced cytopathic effect and apoptosis.56 It was further shown that CVB3 infection results in reduced β-catenin protein expression, and that inhibition of GSK3β or proteasome function leads to the accumulation and nuclear translocation of β-catenin.56 CVB3-induced cytopathic effect and apoptosis are significantly reduced in β-catenin overexpressing cells.56 These results demonstrated that coxsackievirus infection stimulates GSK3β activity, which contributes to virus-induced cytopathic effect and apoptosis through ubiquitin-mediated degradation of β-catenin. The function of the UPS during the course of CVB3 infection was examined to further understand the underlying mechanisms by which the UPS regulates viral replication. There are no noticeable changes in proteasome activities following CVB3 infection. However, viral infection leads to an increasing accumulation of protein-ubiquitin conjugates, accompanied by a reduced protein expression of free ubiquitin, suggesting an important role of protein ubiquitination in UPS-mediated viral replication.58 Using a small-interfering RNA (siRNA), it was demonstrated that gene-silencing of ubiquitin significantly reduces viral protein synthesis and viral titers, possibly through downregulation of protein ubiquitination and subsequent alteration of protein function and/or degradation.58 Several ubiquitin-related signature motifs, such as P(T/S)AP, YP(X)nL and PPXY, have been identified.59 These motifs act as consensus binding sites for WW domain-containing E3 ligases. Using a function motif search, it was recently found that viral protein 3A carries a proline-rich

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motif PPXY at its N terminus, raising a possibility that 3A may promote protein ubiquitination by recruiting host ubiquitin ligase to the viral replication complex. The function of this motif in CVB3-mediated protein ubiquitination and in viral infectivity is currently under investigation. As discussed above, viral protein modification by ubiquitin and/or ubiquitin-like proteins plays a critical role in the regulation of viral protein function. It has been recently demonstrated that CVB3 polymerase 3D is post-translationally modified by ubiquitination.58 Such observations raise an interesting possibility that the UPS may regulate CVB3 replication through ubiquitinating 3D, which is essential for initiating viral RNA replication. In addition to ubiquitination, it was found that 3D contains a highly conserved sequence of VKDE that matches the ΨKXE consensus motif for sumoylation, suggesting that sumoylation also may be required for the regulation of 3D transcriptional activities. Mitogen-activated protein kinase phosphatase (MKP-1), which dephosphorylates and inactivates ERK signaling, has been reported to be post-translationally regulated via the UPS.60,61 It has been previously demonstrated that ERK activation during CVB3 infection is required for CVB3 replication and contributes to virus-mediated pathogenesis (please also see the next section).62 Thus, it is postulated that UPS regulates CVB3 replication may be partially, by modulating ERK signaling pathway. It was found that ERK phosphorylation is reduced in the presence of proteasome inhibitor, which is correlated with an increase in MKP-1 expression.63 It was further demonstrated that knockdown of MKP-1 partially recovers the loss of ERK phosphorylation, and subsequently restores viral replication, suggesting that inhibition of the ERK signaling pathway contributes, at least in part, to proteasome inhibitor reduction of coxsackievirus replication.63 In searching for new antiviral agents against CVB3 infection, it was found that treatment with pyrrolidine dithiocarbamate, an antioxidant, strongly inhibits CVB3 replication.64 It was further demonstrated that the antiviral inhibitory effect is independent of its antioxidant property as it is due to the selective inhibition of host protein degradation.64 Curcumin, a natural compound derived from turmeric, is also reported to be a potent inhibitor of CVB3, most likely through its function in the regulation of the UPS.65 It was shown that curcumin regulates the UPS function by reducing both

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the 20S proteasome proteolytic activities and cellular deubiquitinating activities.65 Using a mouse myocarditis model, it was demonstrated that dysfunction of the UPS induced by CVB3 infection may contribute to the pathogenesis of myocarditis.66 Furthermore, application of a proteasome inhibitor attenuates myocardial damage induced by CVB3 infection, suggesting that the UPS may be an attractive and novel therapeutic target for viral myocarditis. However, the potential toxicity of a general inhibition of proteasomes was recognized to function as a therapeutic means. Further investigation to identify specific targets of the UPS by CVB3 infection will allow for even more precise targeting of drug therapy. Most recently, an important role of autophagy in the regulation of coxsackievirus infection was demonstrated.67 It was found that CVB3 infection induces formation of autophagosome, and inhibition of autophagy by 3-methyladenine or by siRNA knockdown of the genes (ATG7, Beclin-1 and VPS34) critical for autophagosome formation significantly reduces viral protein expression and viral progeny titer. Conversely, treatment with rapamycin (a known autophagy inducer) or nutrient deprivation, prior to CVB3 infection results in an increased viral protein and progeny synthesis. It was further demonstrated that blockage of autophagosome-lysosome fusion by gene silencing of lysosome protein LAMP2 enhances viral replication. These data suggested that autophagic machinery may not have a destructive role in CVB3 infection; it rather contributes to the formation of viral RNA replication complexes by providing sites for CVB3 replication. In summary, despite the demonstrated importance of the UPS in CVB3 replication, the underlying mechanisms by which CVB3 subverts the UPS to insure its own replication are still not fully understood. It is speculated that ubiquitin or ubiquitin-like modifications of viral proteins are required for viral replication, such as viral polymerase 3D which is essential for initiating viral RNA replication. Alternatively, UPS-mediated proteolysis of certain intracellular proteins may target specific aspects of the viral replication process and thus control its replication. For example p53, which is largely regulated through the UPS, has been suggested to be involved in replication in a number of viruses. Based on these hypotheses,

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Figure 2. A proposed model for the role of the UPS in coxsackievirus infection. Coxsackievirus infection facilitates protein ubiquitination, which subsequently increases intracellular protein degradation by the proteasome and/or viral protein modification by ubiquitin and ubiquitin-like proteins. Degradation of intracellular proteins that are against viral infection provides a favorable environment for virus to achieve successful replication, meanwhile ubiquitin or ubiquitin-like modification of viral proteins is required for viral protein function. Ub, ubiquitin; DUBs, deubiquitinating enzymes.

a model system on the role of the UPS in CVB3 replication is proposed in Figure 2.

3. HOST INTRACELLULAR SIGNALING IN COXSACKIEVIRUS INFECTION 3.1 Signaling Pathway and Viral Infection Many viruses are known to manipulate host signaling machinery to regulate virus replication and host gene responses. The mitogen-activated protein kinases (MAPKs) constitute a ubiquitous superfamily of highly related serine/threonine kinases that are activated in response to a variety of extracellular stimuli, to mediate distinct cellular functions.68,69 Three major types of MAP kinase cascades have been reported in mammalian cells: extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun

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NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAPKs.68,69 ERK is the most well characterized member of the MAPK family and plays a central role in the regulation of a variety of cellular processes including cell proliferation, cell differentiation, cell death, and stress responses.68,69 The ERK signaling cascade consists of three kinase modules, composed of Raf, MEK1/2, and ERK1/2. The best known mechanism for ERK activation is the activation of a small GTP-binding protein Ras by growth factor receptors or tyrosine kinases, such as Src. Ras activates the Raf/MEK/ERK cascade by inducing Raf targeting to the membrane, where it is phosphorylated and activated by other kinases. A Ras-independent mechanism has also been reported. Upon activation, ERK translocates to the nucleus and phosphorylates a wide variety of substrates, including transcription factors (e.g., c-myc, c-jun, NF-IL6, ATF-2, AP-1, and elk-1), protein kinases, cytoskeletal proteins, and phospholipase A2, leading to cell proliferation, differentiation, or survival. The second most widely studied MAP kinase cascade is the JNK/ SAPK.68,69 This cascade is activated following exposure to UV radiation, heat shock, or inflammatory cytokines. The activation of these MAPKs is mediated by two small G-proteins: Rac and cdc42. Activated JNK/SAPK binds to the N-terminal region of c-Jun and induces its phosphorylation. The p38 kinase is activated in response to inflammatory cytokines, endotoxins, and osmotic stress. Following its activation, p38 translocates to the nucleus and phosphorylates activating transcription factor 2 (ATF-2). Another known target of p38 is MAPK-activated protein kinases (MAPKAPK) that is involved in the phosphorylation and activation of heat-shock proteins.68,69 Various viruses have been shown to evolve diverse mechanisms to stimulate intracellular signaling pathways that may promote viral replication, regulate host inflammatory response, or induce viral oncogenic transformation of cells. Several viruses, such as human cytomegalovirus,70 simian virus 40,71 HIV-1,72 and influenza virus73 have been reported to manipulate the ERK signaling machinery to regulate viral gene expression and viral replication. Signaling pathways involved in HIV-1 infection have been studied extensively. HIV-1-encoded proteins were shown to interact with intracellular kinases such as tyrosine kinase Lck74 as well as cellular

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serine/threonine kinases.75 HIV-1 binding to CD4, the major HIV receptor, can also activate the MEK/ERK signaling pathway.76 Activation of ERK enhances HIV-1 infectivity through phosphorylation of viral protein Vif and Vif-independent mechanisms.72

3.2 Intracellular Signaling Pathway in Coxsackievirus Infection The critical role of the host signaling pathways in the regulation of coxsackievirus infectivity has been increasingly recognized. CVB3, like other viruses, has the ability to take over multiple intracellular signaling pathways of host cells, benefiting its replication. There is evidence that tyrosine kinases, phosphatases and MAPKs may regulate viral replication. It was shown that CVB3 infection induces tyrosine phosphorylation of a cytosolic 48 kDa protein, and a membrane-bound 200 kDa protein occurring 4 hours postinfection.77 Herbimycin A, a protein tyrosine kinase inhibitor, reduces tyrosine phosphorylation and consequent production of progeny virions.77 The Src family kinase Lck was reported to be required for CVB3 replication in T-cell lines and mice lacking this gene were completely protected from CVB3 myocarditis.12 IFN-α regulates susceptibility to CVB3 infection and such an effect is likely to be mediated by the Janus kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) pathway.78 It was also shown that protein tyrosine phosphatase CD45 is involved in CVB3 infection by directly dephosphorylating JAK, a key mediator of cytokine-sensitive signal transduction. CD45-/- mice were completely protected from lethal CVB3 infections and showed absence of histological lesions in myocytes or inflammation in the heart.78 Our group and others have demonstrated the involvement of ERK1/2 pathway in CVB3 infection.62,79–81 We showed that CVB3 infection triggers biphasic early transient and late sustained activation of ERK1/2. Exposure of HeLa cells to CVB3 stimulates ERK1/2 activity, with a first peak at 10 minutes and a return to baseline at 30 min; a second peak of ERK1/2 activation is apparent at 7 hours, and activation remains elevated until 9 h postinfection.62 The early transient activation of ERK possibly results from the engagement of CVB3 with its native receptor CAR, and co-receptor DAF.62,81 Meanwhile, the late activation of ERK requires

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active replication of CVB3 and is necessary for the replication of CVB3. This activation is possibly mediated by cleavage of RasGAP by CVB3 viral protease such as 3C. Notably, the importance of ERK pathway has been demonstrated by inhibition of MEK, immediately upstream protein kinase ERK, which results in significant reduction in CVB3 viral progeny production and viral protein synthesis.62 This inhibition was also shown to prevent CVB3-mediated cytopathic effect and apoptosis, and block CVB3-induced RasGAP cleavage, suggesting that ERK activation may be achieved through a positive-feedback mechanism. As such, ERK activation enhances viral replication, resulting in a cleavage of host signaling protein RasGAP, further promoting Ras activity and subsequent activation of the ERK cascade.62 The significance of ERK1/2 in CVB3 infection is reported in both immune cells and cardiac myocytes as well.81 Multiple events in the course of the viral life cycle may depend on ERK phosphorylation of viral or host proteins. ERK activation may be involved in viral entry by increasing viral endocytosis or facilitating virus propagation, and release of viral particles by regulating cytoskeletal organization. Activation of ERK1/2 also may be required for viral RNA transcription or viral protein translation. It is speculated that CVB3 activation of ERK may directly phosphorylate viral component(s) or affect host gene expression which is essential to viral replication and dissemination. For example, viral polymerase 3D is a potential substrate to be phosphorylated by CVB3 infection. The ERK cascade also may enhance viral replication by regulating host cytokine expression. Affymetrix GeneChip technology was used to screen possible genes downstream of ERK pathway and found that a number of genes induced by CVB3 infection are significantly decreased due to U0126 inhibition of MEK (unpublished data). These include a variety of genes associated with the inflammatory response, such as interleukin-8 (IL-8) which exhibits the most significant differential transcriptional profile. Aberrant expression of inflammatory cytokines observed during progression of CVB3-induced diseases has been implicated in the pathogenicity of myocarditis.82,83 Elevated levels of IL-8 are detected in the myocardium of patients suffering from myocarditis84,85 and in human myocardial fibroblasts infected by CVB3.86 It has been reported that IL-8 increases cytopathic effects of enterovirus replication and suppresses the antiviral activity of IFN-α 87 which is normally

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active against CVB3 replication.88 IL-8 also stimulates HIV-1 replication.89 Such observations imply that ERK activation may enhance CVB3 replication, at least in part, through upregulation of the IL-8 gene. As alluded to earlier, other protein kinases in the family of MAPKs include JNK/SAPK and p38 MAPKs, both of which belong to the stressactivated protein kinases and are activated when cells are under stress, such as osmotic stress, inhibition of protein synthesis, or exposure to UV radiation, inflammatory cytokines, heat shock, or endotoxins.68,69 Our group and others have reported that both JNK and p38 pathways are activated during the active replication steps of CVB3 life cycle,90,91 and activation of these SAPKs is required for efficient viral replication and viral progeny release.91,92 It was shown that activation of JNK induces cell death via upregulation of the cysteine-rich protein gene.90 It was found that JNK activation may also play a role in host defense machinery through the phosphorylation and activation of ATF-2, a component of the AP-1 transcription complex.91 This cascade reaction may mediate signaling regulation of expression and secretion of proinflammatory cytokines in the progression of CVB3-induced pathogenesis. It was also shown that p38 MAPKs are necessary for CVB3-induced caspase-3 activation and subsequent viral progeny release.91 Caspase-3 activation leads to prominent production of reactive oxygen species in infected cells and activation of NF-κ B.64 In addition to the evidence of the involvement of MAPK signaling pathways in CVB3 infection, studies also demonstrated that the phophatidyl-3-kinase (PI3K)/Akt pathway plays a role in CVB3 replication.93,94 It was found that CVB3 infection leads to the activation of the PI3K/Akt pathway, and this activation can also be mediated by upregulation of the interferon-gamma inducible GTPase.95 Inhibition of this pathway using specific pharmacological inhibitors or dominant-negative mutants of Akt or integrin-linked kinase (ILK) significantly impair virus production yet increase apoptosis of infected cells.93,94 Most recent reports have revealed an important signaling mechanism by which coxsackievirus enters through epithelial tight junctions.96,97 Coyne et al.96 reported that coxsackievirus interaction with DAF in polarized epithelial cells triggers activation of Abl and Fyn kinases, which leads to the movement of virus to the tight junction and subsequent entry

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into the host cells. They further demonstrated that CVB entry requires the activation of small GTPases, Rab34, Ras, and Rab5.97 In summary, CVB3 infection induces activation of multiple intracellular signaling pathways. Such activations are required or necessary for viral life cycle, including viral entry, replication and progeny release, or against host cell defense, e.g., repressing immune system and inducing apoptosis, all of which point to an outcome of being beneficial for viral replication in host cells.

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38. Bres V, Kiernan RE, Linares LK, Chable-Bessia C, Plechakova O, Treand C, Emiliani S, Peloponese JM, Jeang KT, Coux O, et al. (2003). Nat Cell Biol 5, 754–761. 39. Peloponese JM, Jr., Iha H, Yedavalli VR, Miyazato A, Li Y, Haller K, Benkirane M, Jeang KT (2004). J Virol 78, 11686–11695. 40. Hay RT (2006). Ernst Schering Res Found Workshop, 173–192. 41. Kerscher O, Felberbaum R, Hochstrasser M (2006). Annu Rev Cell Dev Biol 22, 159–180. 42. Welchman RL, Gordon C, Mayer RJ (2005). Nat Rev Mol Cell Biol 6, 599–609. 43. Boggio R, Chiocca S (2006). Curr Opin Microbiol 9, 430–436. 44. Hilgarth RS, Murphy LA, Skaggs HS, Wilkerson DC, Xing H, Sarge KD (2004). J Biol Chem 279, 53899–53902. 45. Seeler JS, Dejean A (2003). Nat Rev Mol Cell Biol 4, 690–699. 46. Gurer C, Berthoux L, Luban J (2005). J Virol 79, 910–917. 47. Fan Z, Zhuo Y, Tan X, Zhou Z, Yuan J, Qiang B, Yan J, Peng X, Gao GF (2006). J Med Virol 78, 1365–1373. 48. Li YP, Chen Y, John J, Moylan J, Jin B, Mann DL, Reid MB (2005). Faseb J 19, 362–370. 49. Kirkegaard K, Taylor MP, Jackson WT (2004). Nat Rev Microbiol 2, 301–314. 50. Wileman T (2006). Science 312, 875–878. 51. Shintani T, Klionsky DJ (2004). Science 306, 990–995. 52. Talloczy Z, Jiang W, Virgin HWt, Leib DA, Scheuner D, Kaufman RJ, Eskelinen EL, Levine B (2002). Proc Natl Acad Sci USA 99, 190–195. 53. Prentice E, Jerome WG, Yoshimori T, Mizushima N, Denison MR (2004). J Biol Chem 279, 10136–10141. 54. Jackson WT, Giddings TH, Jr., Taylor MP, Mulinyawe S, Rabinovitch M, Kopito RR, Kirkegaard K (2005). PLoS Biol 3, e156. 55. Luo H, Zhang J, Cheung C, Suarez A, McManus BM, Yang D (2003). Am J Pathol 163, 381–385. 56. Yuan J, Zhang J, Wong BW, Si X, Wong J, Yang D, Luo H (2005). Cell Death Differ 12, 1097–1106. 57. Hardt SE, Sadoshima J (2002). Circ Res 90, 1055–1063. 58. Si X, Gao G, Wong J, Wang Y, Zhang J, Luo H (2008). PLoS ONE 3, e2585. 59. Macias MJ, Wiesner S, Sudol M (2002). FEBS Lett 513, 30–37.

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60. Lin YW, Chuang SM, Yang JL (2003). J Biol Chem 278, 21534–21541. 61. Orlowski RZ, Small GW, Shi YY (2002). J Biol Chem 277, 27864–27871. 62. Luo H, Yanagawa B, Zhang J, Luo Z, Zhang M, Esfandiarei M, Carthy C, Wilson JE, Yang D, McManus BM (2002). J Virol 76, 3365–3373. 63. Wong J, Zhang J, Si X, Gao G, Luo H (2007). Biochem Biophys Res Commun 358, 903–907. 64. Si X, McManus BM, Zhang J, Yuan J, Cheung C, Esfandiarei M, Suarez A, Morgan A, Luo H (2005). J Virol 79, 8014–8023. 65. Si X, Wang Y, Wong J, Zhang J, McManus BM, Luo H (2007). J Virol 81, 3142–3150. 66. Gao G, Zhang J, Si X, Wong J, Cheung C, McManus BM, Luo H (2008). Am J Physiol Heart Circ Physiol 295, 401–408. 67. Wong J, Zhang J, Si X, Gao G, Mao I, McManus BM, Luo H (2008). J Virol Jul 2 [Epub ahead of print]. 68. Chang L, Karin M (2001). Nature 410, 37–40. 69. Johnson GL, Lapadat R (2002). Science 298, 1911–1912. 70. Chen J, Stinski MF (2002). J Virol 76, 4873–4885. 71. Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby M (1993). Cell 75, 887–897. 72. Liu Z, Carthy CM, Cheung P, Bohunek L, Wilson JE, McManus BM, Yang D (1999). Virology 265, 206–217. 73. Pleschka S, Wolff T, Ehrhardt C, Hobom G, Planz O, Rapp UR, Ludwig S (2001). Nat Cell Biol 3, 301–305. 74. Collette Y, Dutartre H, Benziane A, Ramos M, Benarous R, Harris M, Olive D (1996). J Biol Chem 271, 6333–6341. 75. Luo T, Garcia JV (1996). J Virol 70, 6493–6496. 76. Manna SK, Aggarwal BB (2000). J Immunol 164, 5156–5166. 77. Huber M, Selinka HC, Kandolf R (1997). J Virol 71, 595–600. 78. Irie-Sasaki J, Sasaki T, Matsumoto W, Opavsky A, Cheng M, Welstead G, Griffiths E, Krawczyk C, Richardson CD, Aitken K, et al. (2001). Nature 409, 349–354. 79. Opavsky MA, Martino T, Rabinovitch M, Penninger J, Richardson C, Petric M, Trinidad C, Butcher L, Chan J, Liu PP (2002). J Clin Invest 109, 1561–1569. 80. Lim BK, Nam JH, Gil CO, Yun SH, Choi JH, Kim DK, Jeon ES (2005). Virus Res 113, 153–157.

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81. Cunningham KA, Chapman NM, Carson SD (2003). Virus Res 92, 179–186. 82. Kelly RA, Smith TW (1997). Circulation 95, 778–781. 83. Lane JR, Neumann DA, Lafond–Walker A, Herskowitz A, Rose NR (1992). J Exp Med 175, 1123–1129. 84. Chow LH, Gauntt CJ, McManus BM (1991). Lab Invest 64, 55–64. 85. Satoh M, Tamura G, Segawa I, Tashiro A, Hiramori K, Satodate R (1996). Virchows Arch 427, 503–509. 86. Heim A, Zeuke S, Weiss S, Ruschewski W, Grumbach IM (2000). Circ Res 86, 753–759. 87. Khabar KS, Al-Zoghaibi F, Al-Ahdal MN, Murayama T, Dhalla M, Mukaida N, Taha M, Al-Sedairy ST, Siddiqui Y, Kessie G, et al. (1997). J Exp Med 186, 1077–1085. 88. Heim A, Brehm C, Stille–Siegener M, Muller G, Hake S, Kandolf R, Figulla HR (1995). J Mol Cell Cardiol 27, 2199–2208. 89. Lane BR, Lore K, Bock PJ, Andersson J, Coffey MJ, Strieter RM, Markovitz DM (2001). J Virol 75, 8195–8202. 90. Si X, Luo H, Morgan A, Zhang J, Wong J, Yuan J, Esfandiarei M, Gao G, Cheung C, McManus BM (2005). J Virol 79, 13875–13881. 91. Kim SM, Park JH, Chung SK, Kim JY, Hwang HY, Chung KC, Jo I, Park SI, Nam JH (2004). J Virol 78, 13479–13488. 92. Hirasawa K, Kim A, Han HS, Han J, Jun HS, Yoon JW (2003). J Virol 77, 5649–5656. 93. Esfandiarei M, Luo H, Yanagawa B, Suarez A, Dabiri D, Zhang J, McManus BM (2004). J Virol 78, 4289–4298. 94. Esfandiarei M, Suarez A, Amaral A, Si X, Rahmani M, Dedhar S, McManus BM (2006). Circ Res 99, 354–361. 95. Zhang HM, Yuan J, Cheung P, Luo H, Yanagawa B, Chau D, Stephan-Tozy N, Wong BW, Zhang J, Wilson JE, et al. (2003). J Biol Chem 278, 33011–33019. 96. Coyne CB, Bergelson JM (2006). Cell 124, 119–131. 97. Coyne CB, Shen L, Turner JR, Bergelson JM (2007). Cell Host Microbe 2, 181–192.

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CHAPTER 23

Endothelial Damage Response by Dengue Virus Infection Irene Bosch, Katherine J. Martin, Aniuska Becerra Artiles & Rajas V. Warke

ABSTRACT Dengue virus has reemerged as a major global health problem in the tropics, particularly among children. This mosquito-borne flavivirus causes an estimated 50 million infections annually. Most dengue infections result in a febrile illness. Less frequently, infections cause dengue hemorrhagic fever, a potentially fatal vascular leakage syndrome. Here we review the events of dengue vascular leakage and hypotheses that have been put forth to explain these events. Genomic technology has enabled the identification of genes induced by human host cells in response to dengue virus. The results define a dengue common response observed in many cell types and composed of type-I interferon (IFN)-induced genes. Responses of vascular cells are probed in vitro by using human umbilical vein endothelial cells. Vascular specific responses to dengue include increased endothelial cell proliferation and angiogenesis, wound healing, cell adhesion changes, T cell inhibition and complement activation. These processes are likely to participate in dengue-induced vascular leakage or regulation of the immune response during the acute phase of the disease. This approach has the potential to detect new antiviral or pathophysiologic mechanisms as well as disease markers in dengue. In particular, we discuss Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL), 547

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Interleukin-1 Receptor Like-1/ST2 (IL-1RL1/ST2) and Indoleamine 2,3-dioxygenase (IDO).

1. INTRODUCTION Dengue is the most rapidly expanding arboviral disease in the tropics and subtropics. Almost half of the world’s population is estimated to be at risk, with 50 million infections a year and one million severe dengue cases reported for the Americas in 2005. There are currently close to 70 countries reporting dengue cases to the World Health Organization from Asia, Africa and the Americas.1 Due to the large proportion of people suffering from this disease, for which there is no vaccine or antiviral treatment, dengue is considered a public health emergency of international concern.2 The critical issues in dengue pathogenesis today are: a. Finding the physiological and molecular causes of vascular leakage and hemorrhage. b. Finding the mechanisms of viral entry, and cellular and tissue tropism. c. Finding host genetic factors associated with dengue severity (genetic predisposition). d. Finding dengue viral factors related to virulence. e. Understanding T and B cell responses and their relation to pathology and protection. f. Developing antiviral strategies, since currently no vaccine, preventive or curative therapy (only an intravenous rehydration based support therapy to prevent hemoconcentration and possible hypotensive shock), nor predictive diagnostics are available for this severe disease. g. Developing predictive diagnostics, since currently no clinical differentiation of dengue infections in the clinic is possible due to the lack of viral diagnostics and laboratory tests that can be implemented early in the disease. Our research is aimed at describing the changes observed upon infection of human endothelial cells with dengue virus in vitro and comparing these specific cellular events with changes observed in the acute phase of the disease in vivo. In this chapter, we will describe the results of gene

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expression analyses of human umbilical vein endothelial cells (HUVECs) that show how gene expression changes in endothelial cells exposed in vitro to dengue virus. Further, we will specifically address some of the individual genes discovered that play a central role in the gene expression network in vitro and are also relevant for in vivo regulation of the disease. Defining HUVECs as a model system to study virus-induced pathology of the endothelium, we will review briefly the current knowledge on vascular leakage associated with dengue disease.

2. DENGUE AND VASCULAR LEAKAGE Dengue virus is transmitted to humans by the bite of a mosquito (Aedes aegypti) infected with the virus. The disease manifestations can range from an acute, self-limiting febrile syndrome (Dengue Fever, DF), to a severe disease (Dengue Hemorrhagic Fever, DHF) that can result in a lifethreatening shock syndrome.3 Among the symptoms observed in dengue are: high fever, headache, retro-ocular pain, myalgias/arthralgias, and skin rash. Dengue is also characterized by thrombocytopenia, leucopenia, coagulation alterations, hepatic inflammation, hemorrhagic manifestations and vascular leakage. Dengue virus is a positive single-stranded RNA virus that belongs to the Flaviviridae family. All four serotypes of dengue virus (DENV1, DENV2, DENV3 and DENV4) are able to infect and produce all grades of the disease. A major characteristic of dengue virus is that infection with one of the serotypes induces an immune response that is able to control and generate protection only against the infecting serotype. The great variation in amino acid sequence among the four serotypes (65–70% homology) results in the lack of protection against infections with a different (heterologous) serotype. Furthermore, consecutive infections with heterologous serotypes (secondary infections) are associated with more severe manifestations of the disease. Many researchers have previously reported on this specific topic, and their valuable reviews are available to cover this topic.4–7 In addition to the complications in secondary dengue infection due to modulation of the memory immune response, the complement system is also activated by the presence of immune complexes (ICs) formed

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between viral particles and antibodies and also by cytokines. Both mechanisms contribute to the clearance of virus. Moreover, it has been reported that ICs can mediate tissue injury characterized by vascular leakage, circulatory shock, reduction of blood platelet count and leukocyte margination.8,9 Interactions between the ICs and the Fc receptor are likely to trigger an array of downstream effects, each with its own distinct signaling pathway. Animal models for a related virus, West Nile virus (WNV) infections, using C1q or Fc gamma receptor-deficient mice, showed that complement limits WNV spread and Fc gamma receptor is necessary for protection mediated by some antibodies.10 Recently, immune modulation of the antibody-dependent entry of dengue virus in vitro and in patients suggested that modifications in innate and adaptive responses of secondary dengue infections were due to the presence of antibody-antigen complexes.11 The Figure 1A was inspired by the model of M. Diamond,12 dengue infection is represented by an arrow. It is proposed that the virus first encounters dendritic cells (DCs) in the skin, then enters the lymphatic system and expands there. Once in the blood stream, the virus becomes systemic. Other tissues and organs could serve as targets for dengue virus. These targets may also include cardiac/skeletal muscle cells, neurons, endothelium and liver, though demonstration of their infection would be difficult.

2.1 Endothelium, Coagulation and Vascular Leakage The endothelium constitutes an important tissue in the immune system13 that is in contact with blood cells, platelets and microvesicles as well as circulating cytokines, growth factors and serum components of the complement system. Overall, endothelial cells are key players during inflammatory reactions.14,15 They can control the adhesion, rolling and infiltration of different leukocytes into the inflamed tissues. The expression of adhesion molecules and the production of cytokines and chemokines are involved in the process of leukocyte infiltration. The endothelium is also a major component of the coagulation system, as it is able to express several molecules involved in coagulation and fibrinolysis. The normal endothelium constitutively produces inhibitors of blood coagulation and modulators of fibrinolysis (thrombomodulin and

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Figure 1. Models of Dengue Infection. A) Route of infection of dengue into the blood is preceded by a lymphatic expansion of the virus. The first cells likely to expand the virus coming from the mosquito bite are the dendritic cells located in the skin; migration to lymph nodes and spleen would follow. Other tissues such as muscle or neurons could be involved in expansion, but currently they have not been conclusively shown in vivo; ultimately, the infection is systemic. B) Systemic blood components hypothetically involved in the acute phase of dengue infection. The various phases of the immune cells during the acute phase are indicated by numbers I–IV to indicate each of the events. The infection (phase I) of blood cells (infection has been demonstrated for monocytes, B cells and even mastocytes, platelets and T cell lines) indicates further expansion of the virus. The virus travels with serum proteins and immunoglobulins (IgMs and IgGs), forming immune complexes. The immune complex is bound by FcR II for uptake. The secretion of cytokines and procoagulant activity of platelets are part of the immune activation (phase II). Sequestration of platelets gives rise to thrombocytopenia, and plasma leakage is a consequence of the multifactorial process, allowing plasma components and even erythrocytes to exit the bloodstream (phase III). Immune suppression (phase IV) will occur in a sequential manner to control the cell activation. Secreted PDL2 by dendritic cells, PD-1 by memory T cells (grey colored T cell) and secretion of sST2, increased activity of 2,3-indoleamine dioxygenase (IDO) and TRAIL (this chapter) are among the immune controlling events. The presence of immune complexes is more prominent in secondary dengue infections, where more severe forms of the disease are observed including more severe plasma leakage. (Original artwork inspired by published graphics.)

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plasminogen activators).16 The protective, non-thrombogenic properties of the endothelium can be lost after activation by cytokines or pathogens, leading to the expression of tissue factor, which initiates the coagulation cascade. If this response is excessive or uncontrolled, it results in coagulopathy and alterations in the fibrinolytic system. Vascular leakage is defined as the passing of blood components through the vessel walls to different cavities and interstitial spaces throughout the body. The infiltration of cells across the endothelial barrier has been studied extensively.17 Vascular leakage18 has been suggested to be a mechanism to control infection as it allows effector cells and plasma components to reach the space outside the blood vessels19 and infiltrate tissues where pathogens exist. In dengue illness, vascular leakage is associated with more severe manifestations (DHF/DSS) of the disease. Disease severity is reflected in the hemoconcentration phenomena, measured as an increase in the hematocrit, and also in the accumulation of liquid in tissues, organs and cavities. The mechanism by which the vessels become more permeable during severe disease is not known, but it is likely a result of the inflammatory response, as many factors that can cause leakage, such as several proinflammatory cytokines, chemokines and complement molecules, are detected at higher levels in the serum during acute phase of the disease.20,21 The coagulant state along with fibrinolysis and the reduction in the number of platelets likely contributes to the bleeding phenomena associated with dengue virus infections. Moreover, the virus could cause the endothelium to be in a proinflammatory state, indirectly or directly by infection, adding to the severe disease outcome. Previous studies have confirmed the association of high levels of von Willebrand factor antigen (vWf) multimers with severe disease,22 indicating endothelial damage in acute dengue infections. The inflammatory response induced in dengue infection and the procoagulation and fibrinolytic state during acute disease, defines the proclivity to endothelial cell damage and increased permeability. The magnitude of the leakage could be associated with irreversible shock and ultimately, death. A diagram of systemic dengue infection is shown in Figure 1B. The steps shown from top to bottom are infection, immune activation, vascular leakage and

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immune suppression. We will give support through the data presented below to this chronology of events.

3. THE HYPOTHESIS: ENDOTHELIAL DAMAGE The existence of four serotypes of dengue virus, the lack of immunity in young children, and the presence of partial immunity in older children and adults, all favor the appearance of the severe form of the disease during secondary dengue virus infections, which is characterized by vascular leakage. The endothelium which is an integral part of the immune and coagulation systems participates in the control of immune and coagulation responses in dengue virus infection. Also, endothelial cell dysfunction is considered the cause for vascular leakage which is a clinical parameter for manifestation of severe dengue virus infection. We decided to study the role of endothelial cells in direct contact with the virus as one of the likely possible scenarios of the in vivo situation. We proposed an experimental approach to identify and study novel factors potentially induced in response to dengue virus, especially those factors that could be involved in the onset and/or regulation of the vascular leakage.23–25 We postulated that the changes in gene expression observed in this in vitro model of endothelial cell infection could be extrapolated to the in vivo host response against the virus, which led us to propose that part of the mechanism of disease severity may reside in the endothelium. To address this hypothesis, we performed genome-wide, microarray gene expression analyses in vitro using HUVEC cultures infected with dengue virus. We compared these results with those of other cell types infected with dengue and with HUVECs infected with different DNA and RNA viruses. These analyses resulted in lists of genes that were co-regulated and differentially regulated in different cell types in response to different viruses. Then, in the context of a “common response” or a “dengue-specific response”, we studied the expression of some of the genes in our lists using an ex vivo model, in which peripheral blood mononuclear cells (PBMC) were infected with dengue virus. Furthermore, we investigated the possible roles of these gene products in controlling the spread of the disease and the inflammatory response. We also looked for correlations between these gene products with

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disease severity, and have proposed several candidates as disease markers. Molecules that represent examples of both situations include the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and the Interleukin-1 Receptor-Like 1/ST2 (IL-1RL1/ST2).

4. GENOME-WIDE ANALYSIS 4.1 Common Dengue Virus Response Our first objective was to identify genes that were coordinately induced in response to dengue virus in all cell types. We included four cell types that are known to be permissive to dengue infection: endothelial cells (HUVEC), monocytes, B-cells and DCs.25 In expression profiles of these cells, we identified all genes that were induced at least 2.5-fold following dengue infection. A total of 79 genes comprised the “Dengue Common Response” signature (Figure 2). The identities of these common dengueinduced genes showed the strong activation of the type-I IFN signaling network. We proposed that the induction of this set of genes provides evidence of cellular infection with dengue virus. The 79 dengue common response gene identities included genes in several different Gene Ontology (GO) categories: – – – –

Virus response: CCL8, CD38, CXCL10, IFI44, IRF7, MX1, MX2, OAS1, RSAD2 and TRIM22. RNA binding: ADAR1, HEM45, MDA5, OAS1, OAS3, OASL, PKR, and RIG1. Apoptosis: CASP3, TRAIL/TNFSF10, FAS, FADD, RIP1, MyD88, CASP7 and CASP10. Anti-apoptosis: IAP1, c-FLIP.

A recent genome-wide study addressed how coordinately processed adenoviral signaling networks can lead to cell-type specific outcomes.26 This study used mathematical modeling to identify a common effectorprocessing mechanism, allowing cell-specific responses to be predicted. It was concluded that different cell types process signals similarly through common networks and effector proteins and it is the initiator signals that

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Figure 2. Expression levels of dengue common response signature (67 genes) in endothelial cells (HUVEC), monocytes (Mo), B-cells (B), and dendritic cells (DC). Genes were normalized to the expression level of mock-infected (C636 insect cell supernatant treatment of the cells for 48 h) cells for each cell type independently. Normalized expression levels are represented according to the color key shown. Only gene upregulation is shown. Affymetrix GeneChip U133A was utilized and data obtained were analyzed using GeneSpring software (Agilent).

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are more likely to be highly cell-specific. Thus, cell-specific signal activation, i.e., differing cell-specific receptors, adaptors and kinases, are likely to represent the primary mechanism for achieving distinct phenotypes. This common-processing model was also useful in predicting pharmacological candidates in that study. The dengue common response signature, characterized by a common type-I IFN signaling network, represented one approach that we utilized to find antiviral candidates. One gene, TRAIL, was identified as central player in the network and we proposed it as a potential common linker of the type-I IFN inducible genes. Upon further study, we were able to demonstrate that TRAIL has potent antiviral activity against dengue virus.25 Recently, it has been proposed that TRAIL may act as a potential antibacterial agent through its protective physiological role to cells targeted for destruction.27 We performed studies to assess the timing of the dengue common response. We looked at expression profiles of HUVECs infected with dengue virus for 8, 24, and 48 hours. The majority of the dengue common response genes were induced at the earliest time points of dengue infection (Figure 3). We also examined the expression of the dengue common response signature in endothelial cells in response to a panel of viruses including five RNA viruses (Dengue, West Nile, Hantaan, Sin Nombre, and Yellow Fever virus) as well as two DNA viruses (Epstein-Barr and Vaccinia virus). We found that the dengue common response genes were induced in response to all five RNA viruses, with the highest levels of induction of all genes by dengue virus, WNV and Hantaan virus. The common response genes were not induced by either of the DNA viruses.

4.2 Endothelial-specific and Dengue Virus-specific Responses Though a variety of different cell types may respond to dengue infection via the common type-I IFN signaling network, we still need to account for observations that different cell types respond differently to the virus. For example, recent work has shown that dengue virus infection of myeloid DCs results in activation of the early innate immune response, incomplete DC maturation, as well as production of antiviral cytokines

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Figure 3. Expression levels of dengue common response signature (67 genes) in endothelial cells (HUVEC) at three time points (8 h, 24 h, 48 h) of dengue infection, and in response to other viruses. Viruses tested included West Nile (W), Hantaan (HN), Sin Nombre (SN), Yellow Fever (YF), Epstein-Barr (EB) and Vaccinia.96 Expression levels in response to polyIC exposure (pIC) are also shown. All conditions represent HUVECs. Samples were normalized to untreated cells.

TNF-α and IFN-α.28,29 In contrast, vascular endothelial cells have been shown to respond to dengue infection by producing proinflammatory cytokines such as IL-6, IL-8, and RANTES,25 actin cytoskeleton rearrangement and increased permeability to small molecules.30,31 To investigate cell-type specific responses to dengue virus, we analyzed all genes whose expression changed by more than 3-fold in response to dengue virus in at least one out of four different cell types including endothelial, monocytes, dendritic and B-cells. Specifically, we analyzed the endothelial genes (90 probe sets) using hierarchical clustering. The endothelial-specific genes were highly enriched in GO classes that included cell proliferation, cell cycle, angiogenesis, complement activation, and cell adhesion genes (Table 1). Cell adhesion genes may be particularly relevant to dengue vascular pathology involving plasma leakage.32 These genes function to maintain the integrity of the endothelial cell layer and, at the same time, are important in the movement, chemotaxis, and transmigration of leukocytes across the endothelial cell layer as reviewed by others.33 Among these

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Table 1. Identities of Gene Ontology (GO) classes induced preferentially in endothelial cells in response to dengue virus, which are absent or less induced in other cell types. Cell Proliferation Cyclin D2 IL15 IL15RA IL6 TNFSF4 LRRFIP2 MDK growth factor

Cell Cycle

Angiogenesis

Complement

Cell Adhesion

cyclin D2 RGS2 inhibin β A CDKN1A/p21/Cip1 TNFSF15 — —

ARTS-1 CEA-CAM1 — — — — —

CI factor TFPI2 — — — — —

PDZD2 CEA-CAM1 CX3CL1 galectin 8 Laminin gamma 2 serpin B1 ERBB2IP V-CAM1 I-CAM1 desmoplakin

genes encoding for chemokines and cytokines were CCL20, CCND1, and IL-6; and adhesion molecules, ICAM1 and VCAM1; growth factors present were vascular endothelial growth factor (VEGF) and angiopoietin 1; T cell regulators, 2,3-indoleamine dioxygenase (IDO) and complement subunit 1 (CS1). IL-6 may be a key candidate in inducing dengue vascular pathology.34 IL-6 induces an increase in endothelial permeability by altering actin filaments and changing endothelial cell shape,35,36 similar to the action of VEGF.37 But IL-6 has potent anti-inflammatory and protective properties including the ability to inhibit the production of TNF-α.38 TNF-α, a cytokine known to induce vascular permeability,15 was not detected among the endothelial-specific dengue-induced genes. We detected other genes39 such as CXCL1, Kynureninase (KYNU), Laminin gamma 2 (LAMG2), tissue factor protein inhibitor (TFPI),40 and regulator of G protein 2 (RGS2),24 in addition to TNFSF4 encoding OX40-ligand, which is an important co-activator in efficient T cell function of binding to endothelial cells.41–45 We found that besides dengue virus, all RNA viruses tested induced this common IFN response signature in endothelial cells. We subsequently asked if dengue virus also elicited a unique, virus-specific response from

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endothelial cells. We hypothesized that a “Dengue-specific Response” could be useful in the development of virus detection assays and virusspecific therapy. We performed gene expression analysis in endothelial cells in response to dengue and other viruses and poly-IC. A set of 30 genes was identified to be induced only in response to dengue virus infection. Identities and expression levels of the 30 most significantly denguespecific genes are shown (Figure 4). A GO analysis was also performed for 571 probe sets that were significantly overexpressed in dengue infections compared with six other viruses or pIC (Welch t-test, p < 0.05). (Table 2). Of the 30 genes listed in the dengue specific response, CS1 and MCP-2/ CCL8 are known to be upregulated as a direct response to IFN.46 C1S is the first component of the classical pathway of the complement system. Complement is a highly regulated system that plays a crucial role in host defense, but uncontrolled activation can lead to an anaphylaxic shock. Several other genes encode for: T, NK cell activator IL15 and its receptor,47 cell adhesion and migration protein (CEACAM1-4L),48 proinflammatory IL-1β converting enzyme (CASP1)49 and extracellular matrix remodeling protein (LGMN).50 In future work, biological testing of the properties of the genes comprising the common-response and endothelial cell dengue-specific response could be performed in vitro by utilizing specific siRNA strategies and techniques of gene knockdown similar to those applied in other studies.51 With the increasing success of inhibitory RNA as a tool for repressing gene expression, and the capacity to measure dengue genomes by qRT-PCR as a fast assessment for determining viral loads, we envision that high throughput testing of antiviral cellular proteins can be conducted. As an example of successful discoveries in the field of virus infections utilizing the techniques described in this chapter are those previously reported in the detection of RNA helicases, such as MDA-5 in infected monocytes using Differential Display and Affymetrix GeneChip analysis.52 Another set of important genes expressed during dengue viral infections are the oligoadenylate synthetases (OASs) which produce 2′,5′-A oligonucleotides, required for RNaseL activation.53,54 RNase L is known to inhibit WNV.55 We report in the present analysis the expression of OASs 1, 2, 3 and OASL isoforms contributing to RNase L activation through the stimulated type-I IFN responses.

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Figure 4. Expression levels of 30 genes specifically expressed at significantly higher levels in dengue infections of endothelial cells (HUVEC) and at lower levels in infections by other viruses. Viruses tested included West Nile (WN), Hantaan (HN), Sin Nombre (SN), Yellow Fever (YF), Epstein-Barr (EB) and Vaccinia (VC).96 Expression levels of these genes were selected based on their similarity with a hypothetical perfectly dengue-specific gene (ttest, variances calculated using GeneSpring cross-gene error model, p < 0.0002, multiple testing correction: Benjamini and Hochberg False Discovery Rate). All conditions represent HUVECs. Samples were normalized to untreated cells. UDP-glucose ceramide glucosyltransferase (UGCG), Interleukin 1 receptor-like 1 or ST2 (IL1RL1), Poliovirus receptor (PVR), chemokine ligand 2 (CXCL2), lysosomal-associated membrane protein 3 (LAMP3), IL-6, Filamin A interacting (FILIP1L), heparin EGF-like growth factor (HBEGF), KKR1 small subunit processome component homolog, CCL8, Kynureninase (L-kynurenine hydrolase, KYNU), superoxide dismutase 2 (mitochondrial) SOD2, chemokine ligand 3 CXCL3, complement component 1 (CS1), Tumor necrosis factor ligand superfamily member 4 TNFSF4 (OX-40L), SCL15A3, Interleukin 15 receptor alpha (IL15RA), carcinoembryogenic antigen-related cell adhesion molecule 1 (CEACAM1), Caspase 1 interleukin 1 beta convertase (CASP1), legumain cystein protease (LGMN), golgi membrane protein 1 (GOLPH2/GOLM1), endothelial cell growth factor 1 (ECGF1), purinergic receptor P2X, ligand-gated ion channel, 4 (P2RX4), platelet-derived growth factor receptor-like (PDGFRL), torsin family 1 member B (TRO1B), serpin peptidase inhibitor clade B member 1 (SERPINB1), c-FLIP caspase 8 and FADD-like apoptosis receptor (CFLAR), dickkopf homolog 3 (DKK3), laminin gamma 2 (LAMC2), chemokine ligand 5 CXCL5.

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Table 2. Identities of Gene Ontology (GO) classes preferentially induced in endothelial cells by dengue virus in comparison to other viruses. Apoptosis

Angiogenesis

Survivin BNIP1 BNIP2 BNIP3 BNIP3L Caspase 3 Caspase 6 FADD TNFSF5 TNFSF10 IL1 α

angiopoietin 2 angiopoietin L4 CEA-CAM1 ECGF1 IL-8 ARTS-1 VEGFRP

Chemokines CCL2 CCL20 CCL5 CCRL2 CXCL1 CXCL2 CXCL3 CXCL5 CXCL6 IL1 α IL8

Blood Coagulation annexin A7 PLAU PLAUR

Fever IL1 α IL1 β

In other literature, global gene expression of whole blood of dengue patients has been recently reported.56,57 Fink et al.56 reported gene expression from in vitro infections of HepG2 cells that match with many of our in vitro findings using primary human cells. They also used the approach for predicting gene expression in patient samples based on gene responses found in vitro. We have used HUVECs in a similar way, to create PCR-based arrays for the analysis of patient samples. INF type-I responses in dengue patients from Thailand and Venezuela were detected by our analysis (data not shown). Similar to the results from Simmons et al., we also found severe dengue cases associated with attenuation of the innate immune gene expression response. Also in agreement with the current knowledge derived from Global gene expression profiling of PMBCs, Simmons reported an overall upregulation of type-I IFN response genes.57 In Figure 5, the interactions of genes in dengue virus in vitro infections were used to generate a bibliographic linkage map using a Pathway Architect (Stratagene). The upstream regulators predicted by pathway interactions analysis are shown in blue. The red ovals indicate the genes detected by our Affymetrix analysis of in vitro infected primary human cells. This figure represents TRAIL gene as a major regulator of the cellular response to dengue virus.

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Figure 5. Network analysis using Pathway Architect analysis (Stratagene) of the genes of the dengue common response. The blue circles are predicted upstream regulators; the orange circles are upregulated genes collected from the Affymetrix GeneChip analysis done using GeneSpring.

5. ANTIVIRALS AND MARKERS OF DISEASE IN DENGUE INFECTION In the previous section, we described the common response of RNA viruses, here we will focus on molecules that appeared central to that response and those that were more specifically observed in the endothelial cells.

5.1 New Antiviral Mechanism Against Dengue Virus Detected by Global Gene Expression: TRAIL TRAIL is a member of the TNF family that specifically promotes neuroprotection and proliferation of growth in non-cancerous cells,58–61 but promotes apoptosis in cancer cells, by binding to and activating the death receptors DR4 and DR5.62 TRAIL has also been shown to negatively regulate innate

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immune responses independent of apoptosis.63 In vitro and in vivo studies have demonstrated the tumoricidal and antiviral activity of TRAIL without significant toxicity towards normal cells or tissues.64 IFNs enhance expression of TRAIL, while on the other hand, TRAIL treatment enhance expression of IFN-inducible genes like IFITM1, IFIT1, STAT1, LGal3BP, PRKR as well as IFN-β itself.65 The molecular cross-talk and functional synergy observed between TRAIL and IFN signaling pathways may have implications for the physiologic role and mechanism of action of TRAIL protein in non-tumor cells. We have recently found that recombinant TRAIL (rTRAIL) treatment strongly inhibits dengue virus replication25 and dengue virus antigen levels in dengue-infected DCs by an apoptosis-independent mechanism. Furthermore, rTRAIL treatment of dengue-infected DCs inhibited the expression of proinflammatory cytokines and chemokines (IL-6, TNF-α, MCP-2, IP-10, MIP-1β ) (unpublished data). These data suggest that TRAIL plays a dual benefical role of antiviral and proinflammatory cytokine suppression during dengue virus infection. The regulation of MCP-2 and IP-10 by TRAIL has been reported in HUVECs.66 Further investigation of TRAIL as an anti-inflammatory protein in the context of dengue virus infection will be conducted, as we propose that TRAIL is able to control the proinflammatory response triggered after dengue virus infection.

5.2 A Potential Dengue Disease Marker with Anti-Inflammatory Effects Detected by Global Gene Expression in Infected Endothelial Cells 5.2.1 Interleukin-1 receptor-like 1 precursor (IL1RL1)-ST2 gene IL-1RL1/ST2 is a member of the interleukin-1 receptor (IL-1R) family of proteins. Alternative splicing of the gene generates three mRNAs, corresponding to a longer membrane-anchored form (ST2L), a shorter released form (sST2) and a membrane-bound variant form (ST2V).67–69 ST2L has been found to be selectively expressed on Th2-polarized T lymphocytes70 and mast cells,71 and it has been described as an activation marker for Th2 cells.

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It has been shown that sST2 can inhibit IL-1R and TLR4 signaling, through the sequestration of MyD88 and Mal proteins.72 Proinflammatory stimuli, including LPS and cytokines, induce the expression of sST2 in human and mouse cells.73–75 The administration of soluble ST2 or ST2-Fc fusion protein is able to suppress the production of proinflammatory cytokines in vitro and in vivo and recently reviewed72,76 attenuate the inflammatory response in vivo. Elevated levels of sST2 have been found in diseases including Th2associated inflammatory disorders, autoimmune diseases,77 asthma,78 sepsis79 myocardial infarction80–83 and dengue virus infection.23 The ST2L molecule was recently described as part of receptor complex for the cytokine IL-33.84,85 A dual role has been suggested for IL-33: as a nuclear factor with transcriptional regulation activity and as a proinflammatory cytokine.86 In a mouse model of cardiac disease, the beneficial anti-hypertrophic effect of IL-33 is blocked by sST2, suggesting that sST2 could be acting as a decoy receptor.80 ST2 and IL-33 binding recruits MYD88, IRAK1, IRAK4, and TRAF6, followed by phosphorylation of MAPK kinases.86 In a small cohort of patients, mostly classified as DF, we found higher sST2 levels in serum from dengue virus-infected patients, when compared to patients with other febrile illness (OFI); we also found that the levels of sST2 were higher in secondary infections compared to primary infections (Figure 6).23 The increased levels of sST2 were observed at the late febrile stage and especially at defervescence. We also found correlations in dengue patients between sST2 levels and other parameters associated with disease severity. These results prompted us to propose that serum levels of the sST2 protein could be a marker for dengue infection, a parameter that could indicate dengue infection when the levels of circulating virus are dropping. Serum sST2 levels could also be an indicator of the inflammatory response and, as suggested by others,87 could be a downregulatory mechanism triggered to control the exacerbated inflammatory response.

5.2.2 Specific endothelial response to dengue virus: Indoleamine 2,3-dioxygenase (IDO) IDO is an enzyme ubiquitously distributed in mammalian tissues and cells, including DCs88 and T cells.89 It catalyzes the initial and rate-limiting

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Figure 6. Soluble ST2 levels in serum from patients during the course of the disease quantified using an ELISA kit. Illness day 0 is the day of defervescence. Illness days −2 and −1 are febrile days. Illness days +1 and +2 are post-febrile days; conv is the convalescent day. Upper panel: Other Febrile Illness (OFI) (N = 11) and dengue patients (N = 24); significant differences is sST2 levels between OFI and dengue patients at days −1 (p = 0.0088) and 0 (p = 0.0004) are shown. Lower panel: dengue patients classified as Primary (N = 10) or Secondary (N = 13) infections; significant differences in sST2 levels between primary and secondary infections at days −1 (p = 0.047) and 0 (p = 0.030) were found. Normal donors: 15.9 ± 4.4 pg/ml (N = 14). Non-parametric statistical analysis using Mann-Whitney U test was utilized.

step in the catabolism of L-tryptophan along the kynurenine pathway.90 In vivo, IDO activity in serum is increased under pathological conditions such as toxoplasmosis,91 viral, and bacterial infections,92,93 as well as allograft rejection.88 IFN-γ is the most potent known inducer of IDO expression.94

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IDO-expressing cells can inhibit T cell proliferation and function by depleting L-tryptophan in the surrounding microenvironment. This process is termed “immunosuppression by starvation”.95 Further studies will be needed to investigate whether tryptophan metabolites released by dengue virus-infected endothelial cells are involved in inhibiting CD8 T cells. We hypothesize that IDO is involved in the establishment of an immunosuppressive condition during dengue virus infection, as described by others.87 Our gene expression analysis showed that kynureninase expression in endothelial cells infected with dengue virus was elevated. This finding suggested that IDO (the rate limiting step in the catabolism of Tryptophan) might play a role in dengue virus-induced pathophysiology. Using Mass Spectrometry methods to measure amounts of kinerenine and Tryptophan, we studied their levels in serum from dengue-infected patients and patients with other febrile illnesses (OFI). Preliminary results indicated diminished levels of L-Tryptophan and increased levels of kynurenine in dengue infected patients compared to OFI, during the acute stage of the disease (Figure 7). This result supports the hypothesis of T cell and other immune cell inhibition during dengue infection.

6. SUMMARY Important new players of the innate immune response in dengue infection can be identified by using global gene expression analysis of infected cells in vitro, followed by complementary assays for gene expression and further protein function testing as potential antiviral or serum markers in acute phase of the disease, using patient’s samples. Based on this approach, we recently identified TRAIL as an antiviral25 and sST2 as a differential marker of primary versus secondary dengue virus infection.23 The genes proposed here represent valuable target candidates for future research addressing functional confirmation in dengue disease pathogenesis. We encourage the rational search for infection markers using relevant in vitro cell models.

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Figure 7. Tryptophan and kynurenine levels in serum measured using a high performance liquid chromatography-mass spectrometry (HPLC-MS) method. We analyzed samples from healthy donors (N = 5), OFI (N = 5) and dengue patients (N = 6) at febrile, post-febrile and convalescent days. Bars represent mean values (−/+ SEM) for each patient group at different disease days. Un-paired t-test analysis to compare OFI vs. dengue was performed.

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ACKNOWLEDGMENTS This work was funded by NIAID grants P01 AI34533 and U01 AI45440. We thank our clinical study colleagues at the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand (David Vaughn, Timothy Endy, Mammen Mammen, and Ananda Nisalak), the Queen Sirikit National Institute of Child Health, Bangkok, Thailand (Siripen Kalayanarooj), the Banco Municipal de Sangre, Caracas, Venezuela (Norma Bosch), and the University of Massachusetts Medical School (Francis Ennis, Alan Rothman, Sharone Green, and Daniel Libraty). We thank the Proteomics Facility at UMASS Medical School for assistance in IDO activity determination and the Affymetrix Facility at UMASS for processing of GeneChips. We are in debt with Kris Xhaja for her excellent technical assistance. The adapted drawings were created by artist Mary Lynn Davis.

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71. Moritz DR, Rodewald HR, Gheyselinck J, Klemenz R (1998). J Immunol 161, 4866–4874. 72. Sweet MJ, Leung BP, Kang D, Sogaard M, Schulz K, Trajkovic V, Campbell CC, Xu D, Liew FY (2001). J Immunol 166, 6633–6639. 73. Kumar S, Tzimas MN, Griswold DE, Young PR (1997). Biochem Biophy Res Comm 235, 474–478. 74. Tajima S, Oshikawa K, Tominaga S, Sugiyama Y (2003). Chest 124, 1206–1214. 75. Oshikawa K, Yanagisawa K, Tominaga S, Sugiyama Y (2002). Clin Exp Allergy 32, 1520–1526. 76. Gadina M, Jefferies CA (2007). Sci STKE 2007, pe31. 77. Kuroiwa K, Arai T, Okazaki H, Minota S, Tominaga S (2001). Biochem Biophy Res Comm 284, 1104–1108. 78. Oshikawa K, Kuroiwa K, Tago K, Iwahana H, Yanagisawa K, Ohno S, Tominaga SI, Sugiyama Y (2001). Am J Respir Criti Care Med 164, 277–281. 79. Brunner M, Krenn C, Roth G, Moser B, Dworschak M, Jensen-Jarolim E, Spittler A, Sautner T, Bonaros N, Wolner E, et al. (2004). Intensive Care Med 30, 1468–1473. 80. Sanada S, Hakuno D, Higgins LJ, Schreiter ER, McKenzie AN, Lee RT (2007). J Clin Iinvest 117, 1538–1549. 81. Brown AM, Wu AH, Clopton P, Robey JL, Hollander JE (2007). Ann Emer Med 50, 153–158, 158 e151. 82. Shimpo M, Morrow DA, Weinberg EO, Sabatine MS, Murphy SA, Antman EM, Lee RT. (2004). Circulation 109, 2186–2190. 83. Weinberg EO, Shimpo M, De Keulenaer GW, MacGillivray C, Tominaga S, Solomon SD, Rouleau JL, Lee RT (2002). Circulation 106, 2961–2966. 84. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X, et al. (2005). Immunity 23, 479–490. 85. Chackerian AA, Oldham ER, Murphy EE, Schmitz J, Pflanz S, Kastelein RA. (2007). J Immunol 179, 2551–2555. 86. Carriere V, Roussel L, Ortega N, Lacorre DA, Americh L, Aguilar L, Bouche G, Girard JP (2007). Pro Natl Acad Sci USA 104, 282–287. 87. Mathew A, Kurane I, Green S, Vaughn DW, Kalayanarooj S, Suntayakorn S, Ennis F, Rothman AL (1999). J Immunol 162, 5609–5615. 88. Mellor AL, Munn DH (2004). Nat Rev Immunol 4, 762–774.

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89. Fallarino F, Grohmann U, Vacca C, Orabona C, Spreca A, Fioretti MC, Puccetti P (2003). Adv Exp Med Biol 527, 183–190. 90. Takikawa O, Truscott RJ, Fukao M, Miwa S (2003). Adv Exp Med Biol 527, 277–285. 91. Daubener W, Spors B, Hucke C, Adam R, Stins M, Kim KS, Schroten H (2001). Infect Immun 69, 6527–6531. 92. Obojes K, Andres O, Kim KS, Daubener W, Schneider-Schaulies J (2005). J Virol 79, 7768–7776. 93. Oberdorfer C, Adams O, MacKenzie CR, De Groot CJ, Daubener W (2003). Adv Exp Med Biol 527, 15–26. 94. Musso T, Gusella GL, Brooks A, Longo DL, Varesio L (1994). Blood 83, 1408–1411. 95. Liu Z, Dai H, Wan N, Wang T, Bertera S, Trucco M, Dai Z (2007). J Immunol 178, 4260–4266. 96. Brovchenko I, Geiger A, Oleinikova A (2005). J Chem Physics 123, 044515.

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CHAPTER 24

Molecular Pathogenesis of Human Norovirus Ming Tan, Tibor Farkas & Xi Jiang

ABSTRACT Advances in molecular virology have significantly impacted the research of noroviruses. The application of molecular diagnostic methods and the wide surveillance of gastroenteritis disease have led to the recognition of noroviruses as the major cause of epidemics of gastroenteritis worldwide. The high frequency of norovirus disease can be explained by the low infectious dose, the wide genetic and antigenic variations, the high titer and prolonged shedding of viruses by ill and asymptomatic patients, the high environmental stability of virions, and the possible short-lived, type-specific immunity of the host. Application of molecular techniques also resulted in new understanding in the replication, virus-host interaction and pathogenesis such as the development of cell culture and reverse genetics systems and the identification of norovirus receptors. In addition, studies in molecular virology of the murine norovirus and other animal caliciviruses provide additional, useful information about the virologic features of human noroviruses. This chapter will summarize these advances with an emphasis on the molecular pathogenesis of human noroviruses.

1. INTRODUCTION Norovirus, one of the four genera in Caliciviridae, contains non-enveloped, small round-structured (Φ ≈ 38 nm) viruses with a single-stranded, positive 575

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Figure 1.

Electron micrograph of norovirus. Bar = 50 nanometers.

sense RNA genome of ∼7.5 kb. Noroviruses have been recognized as the major cause of epidemic and sporadic acute gastroenteritis affecting people of all ages in both developing and developed countries. Previously, Norovirus was called “small round structured viruses” (SRSV) and “Norwalk-like viruses”, referring to their appearance under electron microscopy (Figure 1) and the prototype “Norwalk virus” isolated from Norwalk, Ohio, respectively. Noroviruses are genetically diverse and according to phylogenetic analyses they are classified into > 30 genetic types within five genogroups (Gs). GI, GII and GIV contain human isolates, while GIII represents the bovine while GV, the murine noroviruses. Viruses that cluster within GII and GIV have also been isolated from swine and feline, respectively. Because of the relative high frequency of recombination among norovirus genomes, classification of recombinant strains can vary based on the regions analyzed.

2. NOROVIRUSES DEFINED BY MOLECULAR APPROACHES The cloning of the viral genome of the prototype Norwalk virus1 and subsequently many other noroviruses since the early 1990s have greatly facilitated

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the molecular characterization of noroviruses. Sequence comparisons of the full length viral genomes with other members of caliciviruses have resulted in the description of the genomic organization which solidified the classification of noroviruses in the calicivirus family. The availability of these full-length genomic cDNAs also allowed further characterization of the genome and individual structural and non-structural proteins by in vitro expression and mutagenesis studies. The generation of the recombinant norovirus virus-like particles (VLPs) in insect cells using the baculovirus system is an excellent example. Empty VLPs assemble spontaneously when the noroviral capsid protein is expressed in the insect cell culture.2 These VLPs are morphologically and antigenically similar to the authentic viruses, and since noroviruses cannot be propagated in tissue culture, the VLPs played a critical role in studying the structure, immunology, and virus-host interaction, which led to the elucidation of the 3-D structure of norovirus capsid, the determination of the host immune responses to noroviruses, and the discovery of human histo-blood group antigens (HBGAs) as noroviral receptors. In addition, the recombinant VLPs are also valuable reagents for the development of immunological assays for diagnosis and candidate vaccine against noroviruses.

2.1 Genomic Structure Norovirus virions contain a full-length RNA genome of ∼7.5 kb and a subgenomic RNA of ∼2.5 kb, both with a poly (A) tail at the 3′ end (Figure 2). The viral genome is linked with a small protein at the 5′ end [VPg, virus protein genome linked3,4]. The genomic RNA is organized into three opening reading frames (ORFs). The largest ORF (ORF1), starting at the 5′ end of the genome, encodes a polyprotein precursor that is cleaved by the viral protease into at least six non-structural proteins (Figure 2), including an N-terminal protein with a yet unknown function, an NTPase, a picornavirus 3A-like protein with a potential function of mediating Golgi disruption, the VPg, the protease, and the RNA-dependent RNA polymerase.4 ORF2 encodes the ∼60 kDa capsid protein (VP1), and ORF3 encodes a minor structural protein (VP2) of 23 kDa. VP2 is a basic protein.

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ORF1

A

P48

NTPase QG 398

ORF2

p22 VPg pro QG EG 762 923

EA 1101

ORF3

RdRp

VP1

VP2

(A)n

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EA 1275

Figure 2. Organizations of the Norwalk virus genomic (A) and subgenomic RNA (B) with indications of the three ORFs and the proteins coded by various ORFs. ORF1 encodes a polyprotein precursor that is cleaved after translation by proteinase into six non-structural proteins: p48, NTPase, p22, genome-linked viral protein (VPg), protease (pro), and RNA-dependent RNA polymerase (RdRp). The protease cutting sites are shown by arrows. ORF2 and 3 encode the major (VP1) and the minor (VP2) structural proteins, respectively. The VPg that links to the 5′ end of the genomic and subgenomic RNA is indicated by a circle. (A)n denotes the poly(A) tail at the 3′ end. Adapted with permission from Ref. 4.

Limited evidence suggested that VP2 may play a role in the stability of the viral capsid,5,6 but the mechanism remains unknown. The ∼2.5 kb subgenomic RNA contains ORF2 and ORF3, and shares a ∼30 nucleotide highly conserved region at the 5′ ends with the full-length genomic RNA, which possibly has a role in translation initiation. Both genomic and subgenomic RNA are packed in virions and can be found in infected cells.3 The subgenomic RNA may facilitate the expression of structural proteins necessary for progeny virus assembly.

2.2 Virion Structure The norovirus capsid contains 180 capsid protein monomers that are further organized into 90 dimers. The atomic structure of recombinant Norwalk virus capsid exhibits a T = 3 icosahedral symmetry.7 Structural studies of other noroviruses and caliciviruses by cryo-electron microscopy (cryo-EM) confirmed this basic structural feature.8,9 Each capsid protein has two major domains, the N-terminal shell (S) and the C-terminal protruding (P) domains, linked by a flexible short hinge.7 The S domain constitutes the shell, whereas the P domain forms

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dimers building up the arch-shaped protrusions emanating from the shell. The dimerization of the P domains not only stabilizes the structure of the viral capsid, but also provides the structural basis for viral receptor interaction.10 The P domain can be further divided into two subdomains, P1 and P2, which correspond to the leg and the head of the protruding arch of the P dimer respectively. The P2 subdomain constructs the outermost surface of the viral capsid and contains the most variable sequences, suggesting its importance in virus-host interaction and host immune response.

3. NOROVIRUS-ASSOCIATED GASTROENTERITIS Norovirus-associated acute gastroenteritis lasts for 1–3 days with 1–2 days of incubation period. The major clinical manifestations include a sudden onset of watery diarrhea, vomiting, nausea, abdominal cramps/pain, anorexia, malaise, and low-grade fever.3,11 The disease is mild to moderate and self-limited; however, severe illness leading to dehydration can occur in the elderly, infants and the immunocompromised.3 Human volunteer studies indicated that one third of the infected individuals are asymptomatic.12 Virus shedding in the feces reaches the maximum level around the onset of clinical symptoms and can continue up to 2–3 weeks, which has important implications in disease control.12,13 In the immunocompromised, prolonged virus shedding lasting for over a year has been described.14

3.1 Diagnosis Several approaches are available for diagnosis of noroviruses. The most commonly used is reverse transcription-PCR (RT-PCR) for detection of the viral RNA in stool specimens. RT-PCR followed by sequencing of the RT-PCR products is widely used in molecular epidemiology of noroviruses. Due to the wide genetic diversity of noroviruses, selection of primers for broad detection is critical. A number of primer sets based on conserved regions of the RNA polymerase or the capsid genes have been used.15–18 Continual improvement of primers may be necessary because new strains with unique sequences are still being found.

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RT-PCR is highly sensitive and specific but it involves multiple steps and needs sophisticated skills and equipment that are not available in all clinical laboratories. Multiplex and real time RT-PCR assays which provide faster results and quantitation of virus load, and assays using specific primers that are able to distinguish genogroups have also been developed for norovirus detection.19–21 A genetic microarray using multiple genotype-specific probes is under development for rapid genotyping which may be useful for outbreak control and environmental monitoring.22 Immunological tests to detect the viral antigens in stool samples have also been developed for the diagnosis of norovirus gastroenteritis. These enzyme immunoassays (EIA) are based on antibodies generated against recombinant norovirus VLPs. Several EIA kits using such antibodies have been commercially available for diagnosis of noroviruses, including the IDEIA (DakoCytomation Ltd, Ely, United Kingdom), the SRSV (II)-AD (Denka Senken Co. Ltd, Tokyo, Japan), and the RIDASREEN (RBiopharm AG, Darmstadt, Germany) kits. Although these assays are useful for outbreak investigation in research laboratories, they are not widely used for clinical diagnosis due to their low sensitivity and specificity as compared to RT-PCR.23–26 The major challenge of EIA development is the wide antigenic variation of noroviruses and the limitation of antibodies used in the assays to capture all antigenic types. Efforts to overcome this problem include the generation of monoclonal antibodies against type-common epitopes.27–29 Antibodies cross-reactive to a number of strains within or between genogroups have been found.30,31 Alternatively, assays based on pooled hyperimmune antibodies generated by cross-immunization of animals with VLPs of different antigenic types have also been developed.32,33 With the continual improvement, immunoassays are expected to be more widely used for clinical diagnosis of norovirus disease in the near future.

3.2 Transmission Noroviruses are highly contagious viruses that can spread quickly through the fecal-oral route by person-to-person contact and through contaminated environmental surfaces. Contaminated food and water are common sources of infection which usually lead to large outbreaks that mainly

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occur in closed or semi-closed settings such as child care centers, schools, restaurants, summer camps, hospitals, nursing homes, cruise ships, battleships, and military troops.3 Viral features related to the widespread nature of norovirus gastroenteritis include the large volume of watery excretion contaning infectious viruses shed by infected patients, the long excretion time, the high environmental stability of the virions and the low infectious dose. A recent volunteer study showed that the prototype Norwalk virus can be shed in stools for up to 56 days after innoculation, with a median peak of millions of genomic copies per milligram stool.34 In another volunteer study, Norwalk virus has been found to remain infectious after 61 days of storage in ground water at room temperature.35 The minimium dose leading to norovirus infection has not yet been thoroughly studied. One volunteer study showed that administration of noroviruses accounted for less than 104 viral genome copies could result in clinical illness,36 while another study37 showed that 850 to 2350 virions in shellfish could lead to an outbreak of norovirus infection. Reports of norovirus infection trasmitted by airborne aerosol38,39 further suggested that the infection dose of noroviruses is very low. As a common source of large outbreaks, food and drink could be contaminated by norovirus through food handlers at any step during food processing.40,41 For example, uncooked foods such as salads, cakes, sandwiches, and icing could be contaminated by food handlers at the site of food preparation. Previous studies have recorded many such foodborne norovirus outbreaks caused by sick food handlers.42–44 More importantly, asymptomatic food handlers may be a critical factor of transmission that could easily be neglected.45,46 A recent study on norovirus-associated gastroenteritis at food catering settings in Japan47 revealed that a large number of asymptomatic food-handlers were infected with noroviruses. Thus asymptomatic food handlers are a big challenge in disease control for norovirus outbreaks and need to be considered by public health authorities.

3.3 Epidemiology Increasing epidemiologic studies indicated that noroviruses are the most important cause of epidemic acute gastroenteritis. Noroviruses cause more

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than 85% of non-bacterial acute gastroenteritis outbreaks in the USA and Europe.48–50 The US CDC estimated that up to half of the foodborne outbreaks are attributable to noroviruses and approximately one in ten Americans become ill with norovirus gastroenteritis each year. Foodborne pathogens infect 76 million people annually and cause 325 000 hospitalizations. Norovirus alone causes $350 to $750 millions in losses each year due to care for illness and lost revenue from recalled foods.51 Surveillance in many other countries revealed similar pictures suggesting that norovirus gastroenteritis is a global public health problem representing a large economic burden. People of all ages are susceptible to norovirus infection, although the young, the elderly, and the immunocompromised may suffer more severe disease consequences. In temperate countries, norovirus infection occurs throughout the year with a peak season in the winter months, and it is known as “winter vomiting disease”.11 Epidemiologic studies showed distinct prevalence and seasonalities between genogroups.48,52–54 By analyzing 250 published norovirus outbreaks, occurred in the period between 1981 and 2006, Moe and colleagues55 found that 70% of the outbreaks were caused by GII and 18% by GI noroviruses. In the remaining 12% both GI and GII noroviruses were involved. The outbreaks caused by GII noroviruses mainly occurred in winter (56%), while the GI outbreaks showed no seasonal trends. In addition, the GI noroviruses had a higher attack rate (median 57%) than GII (29%). Higher attack rates were also associated with foodborne outbreaks. Increased surveillance has also elucidated epidemic patterns of norovirus gastroenteritis. For example, since the mid 1990s, the GII-4 strains have became the most predominant type in many countries and new variants of GII-4 viruses emerged every 1–2 years, which are usually accompanied with an increased epidemic. According to the HBGA binding specificity of the GII-4 viruses, the majority (secretors, ∼ 80%) of the populations are susceptible to GII-4 strains, which at least partially explains their high prevalence. However, the accompanying high epidemic also indicates a selection pressure on the viruses for better fitness. One hypothesis is the antigenic drift driven by the host herd immunity. Alternative selection factor could be the higher affinity of the host, higher environmental stability, or higher virulence of the viruses. Future studies to test these hypotheses are necessary.

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3.4 Host Immune Response Knowledge of host immunity to norovirus infection has been derived mainly from volunteer challenge studies. Homotypic immunity to Norwalk virus infection was observed but volunteers who initially revealed a protective immunity became susceptible to Norwalk virus when they were rechallenged 27 to 42 months later,56,57 indicating the lack of a long-term immunity to norovirus. This may explain why adults are susceptible to norovirus infection and why norovirus-associated gastroenteritis is so widespread in every population throughout the world. The role of humoral immunity in norovirus infection is controversial. Early volunteer studies found that pre-existing antibody levels to the prototype Norwalk virus did not correlate with protection, and in some cases preexisting antibodies seemed to be an indicator of host susceptibility to Norwalk virus.56,58,59 In other volunteer and outbreak studies, however, homotypic preexisting antibodies were correlates of protection.36,60 The role of host cellular immunity in norovirus infection has not been studied until recently. Lindesmith et al.61 reported a significant increase of gamma interferon (INF-γ ) and interleukin 2 (IL-2), but not IL-6 or IL-10, in sera of volunteers following challenge with SMV (GII-2). Similar results were obtained in another study where volunteers received recombinant Norwalk virus VLPs orally.62 The INF-γ production was CD4+ celldependent, denoting a predominant T-helper 1 (Th1) immune response.61 To explore fecal immune responses, a more direct marker of gut mucosal immunity, cytokine profile of stool specimens collected from travelers with naturally acquired norovirus infection were investigated.63 Again, increased INF-γ and IL-2 levels were detected in the norovirus-associated diarrhea specimens, confirming a predominant Th1 immune response. Noroviruses are antigenically highly diverse. A number of studies have been performed to elucidate homo vs. heterotypic immune responses following norovirus infection. Using samples from 13 outbreaks caused by four different norovirus genotypes (GII-1, GII-3, GII-4, and GII-7), Rockx and colleagues64 detected both homologous and heterologous serological responses. However, the avidity of antibodies could not be used to differentiate between homologous and heterologous antibody responses. Instead, a homologous blocking response but not a heterologous blocking

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response was observed after a GII-4 infection as determined by an in vitro receptor-binding/blocking assay.64,65 Similar homologous blocking effects have also been observed in sera of laboratory animals immunized with various norovirus VLPs.32,66

4. MOLECULAR PATHOGENESIS OF NOROVIRUS ASSOCIATED GASTROENTERITIS The elucidation of the pathogenesis of norovirus-associated gastroenteritis has been severely hampered by the lack of an effective cell culture system or animal model. Despite these difficulties, significant progress in understanding the pathogenesis of norovirus gastroenteritis has been made. The application of recombinant norovirus VLPs as a probe to study virus-host interaction has resulted in the discovery of human HBGAs as the receptors. In addition, the recent development of the murine norovirus model, the 3-D organoid and the primary duodenal culture systems, the gnotobiotic pig model and the different reverse genetics systems of noroviruses have provided promising new approaches to study virus-host interaction and pathogenesis.

4.1 Virus-Host Interaction Virus attachment is the first step in viral infection which may trigger downstream processes such as penetration and uncoating. One or more receptor(s)/co-receptor(s) are involved in this step. In noroviruses, at least in some strains, a carbohydrate receptor that is related to the human HBGAs plays a critical role in this step.

4.1.1 Polymorphic human HBGA systems HBGAs are complex carbohydrates existing at the outermost part of N- or O-linked glycans of many glycoproteins or glycolipids on the surface of red blood cells and mucosal epithelia of the respiratory, genitourinary and digestive tracts [reviewed by Le Pendu et al.,67 Ravn and Dabelsteen68]. HBGAs are also present as free oligosaccharides in biological fluids, such as saliva, intestinal contents, milk, and blood. These antigens are synthesized

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by sequential addition of monosaccharides to carbohydrate precursors by variable glycosyltransferases encoded by the ABO, secretor, and Lewis gene families. The frequent occurrence of gene silencing in each of the three gene families results in highly polymorphic HBGA phenotypes. For example, silent alleles of the secretor gene (FUT2) are found in about 20% of European and North American populations, resulting in secretornegative status. Similarly, a silent FUT3 gene is found in about 10% of the same populations, leading to a negative status of Lewis blood types. Silent genes in the ABO family are variable and present in about 40–60% of populations depending on geographical locations, resulting in different distribution patterns of ABO blood types. Thus, the combined polymorphism of all three gene families results in an extreme complexity of human HBGAs.

4.1.2 Recognition of HBGAs by noroviruses The recognition of HBGAs by noroviruses has been demonstrated by a number of binding, blocking and hemagglutination experiments using saliva, milk, synthetic oligosaccharides and monoclonal antibodies specific to human HBGAs. Eight distinct receptor-binding patterns have been described and all antigens in the ABO, secretor (H) and Lewis families seem to be involved in norovirus binding.33,65,69 The eight binding patterns represent two major binding groups, the A/B binding group and the Lewis binding group.33 All strains in the A/B binding group recognize the A and/or B epitopes with variable binding activities to the H epitope, while strains in the Lewis binding group mainly recognize the Lewis epitope and exhibit variable binding activities to the H epitope. The H epitope seems to be involved in binding for both binding groups but alone it is not recognized by the Lewis binding strains. In addition to the binding activities described above, minor variations have also been noted. For example, differences in binding activities with antigens presented in saliva vs. synthetic oligosaccharides have been observed for some strains.33,70 A recent study also showed that two recombinant VLPs that did not bind to Lewis epitopes recognize sialyl-related epitopes.71 These results suggested that the interaction between noroviruses and HBGAs is a typical protein/carbohydrate interaction

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which is highly diverse and complex, similar to lectin/carbohydrate interactions. Minor structural differences of carbohydrates between saliva and synthetic antigens could be responsible for this variation. The sialyl epitope is another common epitope in vivo which may represent a novel subtype of HBGA-related receptors to some strains. In conclusion, our understanding of the genetic and host range variation remains limited, and further expansion of this field is necessary.

4.1.3 The receptor-binding interface The P domain of noroviral capsid is directly involved in receptor binding, and this has been demonstrated by binding assays using recombinant P protein alone or chimeric capsids with heterologous S and P domains.70,72 Direct evidence of the P domain as the receptor-binding domain also came from the crystallographic study of VA387 (GII-4) P dimer complexed with HBGAs.10 The receptor-binding interface is in the P2 subdomain on the outermost surface of the capsid (Figure 3) between two P monomers. Thus, dimerization of the P domain is structurally critical for receptorbinding function. Extensive hydrogen-bond networks between the capsid P dimer and the saccharide ligands have been predicted based on the electron density map of the complex. One major side-chain of the carbohydrate ligands, the α-1,2-fucose (the H epitope, see Figure 3), interacts with the bottom (Thr344 and Arg345) and the wall (Ala346, Asp374, Cys 400 and Gly442) of an open cavity in the receptor-binding interface (Figure 3). The A- and B-epitopes (β-1,3-N-acetylgalactosamine and α-1,3-galactose, respectively) interact with the wall on another side (Ala346, Lys348 and Ser441) of the open cavity [Figure 3,10 reviewed in Ref. 73]. The involvement of these amino acids in the binding has been confirmed by mutagenesis studies.74 These studies also indicated that both the H- and the A/B-binding sites are important for norovirus binding. The H-epitope binding site may be shared by many strains which is sensitive to mutagenesis study; while the A/B-epitope binding site may be responsible for binding specificity of the virus thus may be more strain-specific because mutations at this site affected binding to A but not B antigen.74 Our understanding of the structural basis of receptor binding remains limited, because these crystallographic studies were performed with only

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one norovirus strain using type A- and B-trisaccharides.10 The remaining sugars of human HBGA may also participate in the interaction. In addition, it remains unknown whether the backbone of the HBGAs that carry the carbohydrate epitopes is also involved in the binding. Furthermore, other surface molecules such as the 105-kDa membrane protein and heparan sulphate also may be involved in the viral-host interaction.75,76 Therefore, further studies with additional strains and HBGAs to closely mimic the in vivo conditions are necessary.

4.2 Entry of Norovirus Studies of viral attachment and penetration were performed using recombinant VLPs as a probe. Norwalk virus VLPs could bind and internalize into differentiated Caco-2 cells, but the efficiency of penetration was low [∼5% of the bound VLPs77]. A similar conclusion was obtained from a study using another strain (UEV).76 After the discovery of norovirus recognizing HBGA receptors, Marionneau et al.78 demonstrated direct involvement of HBGAs in norovirus attachment and internalization on CHO cells following transfection with a FUT-2 cDNA. However, a great deal of efforts in many laboratories to cultivate noroviruses using variable human and animal cell lines including the Caco-2 cells failed,79 suggesting that the limit of norovirus replication in vitro may not be due to inability of attachment or penetration. Recently evidence of one cycle replication of Norwalk viral RNA in mammalian cells following transfection has been reported, although progeny virus particles were not infectious to secondary cells.80,81

4.3 Norovirus Replication Despite our inability to grow human noroviruses in cell culture, understanding of the viral replication has been significantly advanced through the elucidation of the genomic organization followed by characterization of individual viral proteins, particularly the viral non-structural proteins. The recent progress in the development of cell culture, in vitro replication systems, and animal models for human noroviruses have a significant impact on studies of norovirus replication. The roles of the six non-structural

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B

A

ß-Gal

D B

H

ß-Gal

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C

E Figure 3. Receptor-binding interface on the P domain dimer of a GII-4 norovirus (VA387). The side (A) and top (B) views of the crystal structure of the P dimer (ribbon diagram) with indication of the B trisaccharide (sphere model, orange) binding on the P2

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proteins have been well summarized in a review by Hardy.4 The VPg and RNA polymerase are two viral proteins whose roles in viral replication have been elucidated the most.

4.3.1 VPg and RNA polymerase in norovirus replication It is known from the studies of picornaviruses that the VPg and the RNAdependent RNA polymerase (3Dpol) play a critical role in viral RNA replication and translation. Recent studies on noroviruses have shown that an incubation of recombinant noroviral VPg with the 3Dpol produced VPgpoly(U) and the VPg-poly(U) could prime the replication of the genomic RNA through the 3Dpol after annealing to the poly(A)-tail of the genome RNA.82,83 These experiments also indicated that replication of the negative strand of the geonomic RNA was initiated in a primer-independent manner, starting at the very beginning of the 3′ terminus of the template RNA, which differs from the VPg-primed initiation of the genomic RNA replication. Addition of a poly(C) stretch to the 3′ terminus of the replicated product was carried out through the terminal transferase activity of the 3Dpol.82 This poly(C) stretch would allow de novo initiation to start a new replication on this template. Whether other non-structural viral proteins such as the NTPase also play a role in noroviral RNA replication remains to be determined. Figure 3. (Continued ) region of the P dimer. Green (P1) and red (P2) represent one P domain monomer; yellow (P1) and blue (P2) represent the other P monomer. (C) A closeup of the receptor-binding interface with a side view at the up-right corner (D). The open cavity of the binding interface is shown in surface representation with indication of the amino acid components but the water molecules are omitted. The B-trisaccharide is shown by ball-and-sticks with the oxygen and carbon atoms colored red and cyan, respectively. H, B, and β -Gal indicate the H epitope (α -1,2-fucose), B epitope (α -1,3 galactose), and β -1,3 galactose of the B-trisaccharide, respectively. The predicted hydrogen bonds are indicated by yellow dashed lines. (E) A close-up of hydrogen-bonding network between the α-1,2 fucose ring (H epitope) and the P dimer. The fucose ring and residues involved in interaction are shown by a ball-and-stick representation, with nitrogen, oxygen, and carbon atoms colored purple, red and yellow, respectively. The dotted lines indicate hydrogen bonds. Backbones of the two monomers of the P dimer are shown in blue and green ribbons, respectively. A–D were made by PyMOL (DeLano Scientific LLC) using the PDB file 2OBT (www.pdb.org). E was adapted with permission from Ref. 10.

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Involvement of noroviral VPg in translation of viral RNA has also been shown by the interaction of Norwalk virus VPg with translation initiation factors eIF3 and eIF4E, and with ribosomal subunits.84,85 Therefore, VPg interacts with the translational machinery, which may help to recruit ribosomes to the viral RNA. The observation that removal of VPg from FCV genomic RNA decreased viral protein synthesis in vitro86 supports the role of VPg in viral RNA translation. In addition, the loss of infectivity of isolated Norwalk virus RNA to mammalian cells after protease treatment also supports the critical role of a protein linked to RNA for infectivity.80

4.3.2 Replication of noroviruses in vitro and in animals After over 30 years of efforts by many laboratories, Straub and colleagues87 described the first cell culture system to grow human noroviruses using a 3-D organoid model of human small intestinal epithelium. Viral replication was indicated by cytopathic effect (CPE), detection of viral RNA, and increase of viral titer during subsequent passages (up to five cell passages). Recently, another in vitro cultivation system has been reported, in which an ex vivo model using fresh human duodenal biopsy cultures was used.88 Furthermore, a one-cycle norovirus replication has also been observed in human hepatoma Huh7 cells following a transfection of Norwalk virus RNA isolated from stool of volunteers.80 These are exciting advances which could eventually result in a useful cell culture system to study the replication and pathogenesis of noroviruses. Various in vitro replicon systems using reverse genetic techniques have also been reported. For example, a full-length genomic cDNA clone of Norwalk virus was able to replicate in mammalian cell lines (HEK293T and BHK-21),81 in which subgenomic RNA was transcribed and translated into VP1, and viral genomic RNA was packaged into virus particles. However, progeny viruses could not pass to secondary cells. Similar results were also obtained by others.89 These results indicated that mammalian cells are able to replicate norovirus genomic RNA. Attempts to develop a nonhuman primate challenge model for norovirus gastroenteritis, such as rhesus macaques and chimpanzee,

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reported some promising initial findings,90–92 however, possibly due to the high cost and other issues it has not been fully evaluated. Development of a gnotobiotic pig model for norovirus infection is also under progress. Pigs share several characteristics with humans in their gastrointestinal anatomy, physiology, immune responses and the presence of HBGAs, such as A- or H-antigens on mucosal surfaces.93,94 In a neonatal gnotobiotic pig model, human norovirus infection has been demonstrated by mild diarrhea, virus shedding, seroconversion, detection of intestinal noroviral antigen, and transient viremia,95,96 although the efficiency of viral replication is still low and the clinical manifestation remains undefined.

4.4 Pathogenesis of Norovirus Gastroenteritis Early biopsy studies of human volunteers indicated that norovirus replication occurs in the proximal small intestine, revealing typical histopathologic changes of the villi, including villus atrophy, disarray of epithelial cell, as well as cytoplasmic vacuolization.97–99 No histological alteration was noted in the stomach100 and large intestine. In the gnotobiotic pig model, noroviruses were found to infect mostly the villus cells, but only minimally in the crypt cells, which led to mild diarrhea with typical histopathologic lesions in the proximal small intestine.95,101 Immunohistochemical studies using specific antibodies against the capsid protein and nonstructural proteins detected patchy infection of duodenal and jejunal enterocytes (Figure 4). Similarly, in the 3-D organoid culture model of human small intestinal epithelium, norovirus infection was found in highly differentiated enterocytes.87,95 EM study revealed cytopathic alteration of the infected cells: shortening of the microvilli, internal membrane rearrangement, and cytosol vacuolization showing accumulation of suspected norovirus particles.87 Lysis of villus cells, resulting in the reduction of absorptive capacity of the intestine, could be a major cause of diarrhea. Enterocyte lysis could also induce inflammatory response and release of potential toxic proteins in the lumen, which are attributable to the syndrome of diarrhea.102 In addition, cell lysis helps the release of virus progeny to infect more enterocytes which can cause further functional damage to the intestine, contributing to diarrhea. It has been noted that there is a large quantity of a soluble

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Figure 4. Human norovirus (HS66, GII) infected intestinal villus cells of gnotobiotic pig. Confocal microscopy showing indirect immunofluorescent (IF) localization of the norovirus capsid protein in small intestinal tissues from virus-inoculated gnotobiotic pigs

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P protein in the stool of Norwalk virus-infected patients.103,104 This P protein may be derived from trypsinization of the capsid protein at two highly conserved trypsin cleavage sites104,105 and it may differ functionally in receptor binding from that of the intact capsids.105 The high conservation of the trypsin digestion sites and the fact that only the P domain survives through the intestinal tract suggest that future studies to elucidate the role of this protein in viral replication, immune response, and pathogenesis are of significance.

5. CONCLUSION Significant advancements have been made in the past decade in many aspects of norovirus research, including the fields of viral structure, disease burden, molecular diagnosis, transmission, host susceptibility and immune response, viral receptors, and pathogenesis. The novel data indicate that norovirus gastroenteritis is a globally important disease that has been paid increasing public attention worldwide. However, norovirus gastroenteritis remains difficult to control due to its widespread nature and the challenges arising from the lack of an efficient cell culture and/or animal model. In addition, the wide genetic and antigenic variation of noroviruses also pose serious problems for the diagnosis and development of vaccine and antivirals. Despite these challenges, the advances in molecular characterization of noroviruses have resulted in new understanding of this important viral disease and new hope for future studies. The finding of the viral receptors for noroviruses has provided a completely new concept on virus/host interaction which not only affect our understanding of Figure 4. (Continued) at 3 days postinfection. Primary antibody NS14 MAb was used to detect the capsid protein. Secondary antibodies: goat anti-mouse IgG Alexa488 (green) nuclear counterstain SYTOX orange (red) and actin stained with phallotoxin Alexa633 (blue). (A) Jejunum tissue showing scattered IF-positive cells on the villi tips or sides. (B) Jejunum of a mock-inoculated pig with no IF-positive cells evident. (C) Jejunum tissue showing individually infected enterocytes on the side of a villus. (D) Tip of a villus in the duodenum with several contiguous infected cells. (E) Tip of a villus on the jejunum with positive signal in the apical portion of the enterocyte cytoplasm. (F) Enterocytes from the duodenum showing nuclear displacement and positive signal throughout the cytoplasm of individual cells. Bars: A and B, 100 µm; C to F, 20 µm. Adapted with permission from Ref. 95.

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the epidemiology, immunology, host range and evolution of the viruses, but also will lead to new strategies to treat and prevent norovirus gastroenteritis. The successful cell culture and reverse genetic systems will significantly impact the research on the viral replication, immunology and pathogenesis and will also help in the evaluation of methods for treatment and prevention of norovirus disease. Advances in many other areas such as the identification of shared antigenic epitopes and the generation of monoclonal antibodies against the common epitopes will find application in diagnosis and vaccine development. In addition, the structure and functional analyses of a number of non-structural proteins and elucidation of their roles in viral replication, and the recent advances in the development of animal models will result in new strategies for the development of effective antivirals and vaccine against norovirus gastroenteritis.

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CHAPTER 25

Host Responses During Sindbis Virus Encephalomyelitis in Mice: New Implications for Understanding the Pathogenesis of Alphavirus Infections of the Central Nervous System David N. Irani & Natalie A. Prow

ABSTRACT Although most human alphavirus infections are either asymptomatic or result in transient febrile illnesses, a few can involve the central nervous system (CNS), causing acute encephalitis. Such encephalitic alphaviruses are usually transmitted to humans via infected mosquito vectors, but some can also be spread as aerosols, making them potential bioterrorism agents. This remains of concern because antiviral agents with activity against these pathogens are not available. The pathogenesis of alphavirus infection of the CNS has been studied in a number of experimental models, most notably Sindbis virus (SV) infection of mice. Here, both viral and host determinants influence disease outcome. Neurons are infected by SV and may undergo classical apoptosis. This is the primary mechanism by which newborn animals succumb to disease. In older mice, however, many neurons undergo a non-apoptotic death, including cells that are not infected but are damaged via bystander mechanisms. Recent studies have focused attention on glial cells that contribute to this bystander neuronal dysfunction and death. Finally, both innate and adaptive host immunity influence the outcome of SV encephalomyelitis (SVE), as certain immune responses clear virus from the CNS but others actually drive 601

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this bystander neuronal injury. Therapies that augment these beneficial host responses or that suppress the detrimental ones are effective in treating lethal SVE in mice, and these approaches deserve further investigation in models where the virus is a known human pathogen. This chapter will review host responses during SVE, with particular emphasis on how they present effective therapeutic targets in these diseases.

1. INTRODUCTION Although alphaviruses constitute a large genus of pathogens with similar structures and molecular characteristics, they vary widely in terms of their geographic distribution, host range, and disease manifestations. As a group, these pathogens are found worldwide, but no single alphavirus is represented in all populated regions of the world. They exhibit a broad host range, being capable of causing natural infections of arthropods (principally mosquitoes), birds, rodents, primates, horses, and humans. Human infections occur primarily via mosquito transmission; the main ecological maintenance strategy of alphaviruses in nature is to cycle from mosquito to vertebrate host and back to mosquito ad infinitum. In almost all cases, humans are not the principal amplifying target but are instead “dead-end” hosts that become accidentally infected when they come into proximity with burgeoning populations of infected mosquito vectors. Human alphavirus infections span a full range of disease, including asymptomatic infection, self-limited febrile illness, acute arthropathy, and rarely, invasion of the central nervous system (CNS) resulting in acute encephalomyelitis. It is this capacity for neuroinvasion and neurovirulence that fuels much of the ongoing research into the biology of these pathogens.

1.1 Alphavirus Encephalitis in Humans Three alphaviruses characteristically invade the human CNS to cause acute encephalitis: eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV). All can produce epidemic outbreaks of disease, and as their names imply, all may cause overt encephalitis in equine hosts. Indeed, cases observed in horses are often a harbinger of a localized outbreak that

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may appear in nearby human populations. Such transmissions invariably occur via the bite of an infected mosquito vector, after which local virus replication causes viremia that can lead to hematogenous dissemination to the CNS. The risk of neuroinvasion depends on both host and viral factors; in general it is more likely to occur in children compared to adults, and it happens more frequently with EEEV than with WEEV or VEEV.1 Neurological disease begins abruptly once the blood-brain barrier (BBB) is penetrated; patients develop fever, nuchal rigidity, and seizures that can progress to coma and death.2 Pathological features are confined to the CNS, and fatal cases usually show prominent perivascular and meningeal inflammation that accompanies neuronal destruction in regions such as the cortex, hippocampus, basal ganglia, and brainstem.2 Mortality is variable, as is the occurrence and severity of neurological deficits in patients who survive the acute infection. A diagnosis is typically confirmed by serological assays, although infectious virus can sometimes be directly recovered from the CNS at autopsy. Important features of the human encephalitic alphaviruses are summarized in Table 1.

1.2 Animal Models of Alphavirus Encephalitis Rodents have been used extensively in studies of alphavirus pathogenesis, both as a means to attempt virus isolation from clinical specimens and to investigate disease mechanisms in a vertebrate host. In terms of virus isolation, the inoculation of potentially infectious clinical material directly into the brains of neonatal mice has been a well-utilized strategy to screen for new pathogens for many years. This is because these hosts uniformly develop fatal encephalitis following such a challenge even when a small amount of virus is present, and there is little risk that a more subtle disease will occur and be overlooked. Once disease is observed in this setting, viral isolation and characterization efforts can be rapidly pursued. From the standpoint of disease pathogenesis, older mice (and to a lesser degree hamsters and guinea pigs) have been commonly used to investigate both viral and host factors that influence disease outcome. Weanling mice are useful because they resist more attenuated viral strains and therefore help to discriminate between different clinical isolates or different laboratory variants. Murine models of VEEV, WEEV, and EEEV

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Table 1. Clinical and epidemiological features of the human encephalitic alphaviruses. Feature

WEEV

EEEV

VEEV

Natural Cycle (Host-Vector)

Birds-Culiseta, Culex

Birds-Culiseta, Aedes

Rodents, Birds, Culex, Aedes, etc.

Geographic Distribution

Western U.S., Canada, Central America

Atlantic and Gulf coasts of the U.S., Caribbean

Central and South America

Ratio of Encephalitis Cases to Infections

Children 1/50 Adults 1/1000

Children, Elderly 1/17 Adults 1/40

Children 1/50 Adults 1/200

Age

Any (some predilection for infants)

Any (predilection for children)

Children

Clinical Features

Headache, fever; seizures are common

Malaise, disorientation, rapid progression to coma

Febrile prodrome and prostration

Mortality

5–10%

50–75%

< 10%

Neurological Sequelae

Common in infants

Common

Rare

Other

Equine vaccination has reduced human cases to fewer than 25 cases/year

0–5 confirmed cases/year

Transmission as aerosol confirmed in lab workers

infections in mice have been used to study the processes of neuroinvasion and neurovirulence, as well as in efforts to develop both antivirals and protective vaccines.3,4 Here, peripheral challenge with virus is usually sufficient to induce neurological disease, and the clinical endpoints of debilitating disease or death are readily apparent and occur with high frequency. Still, these pathogens do not cause universally fatal encephalitis in humans, and more subtle neurological morbidity cannot be reliably modeled in mice. Primates have also been used in alphavirus pathogenesis studies, most notably in the testing of candidate vaccines and to better understand mechanisms involved in aerosol transmission,5,6 but these

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models are more cumbersome and resource-intensive to work with. Nevertheless, successful disease protection or post-exposure treatment studies in rodents are usually extended into primates before they are considered for human application.

1.3 Sindbis Virus Encephalitis in Mice One alphavirus, Sindbis virus (SV), was first isolated from a pool of infected mosquitoes in the Nile Delta in 1955 and has served as a prototype member of this genus for the purpose of experimental studies ever since.7 Neonatal mice are highly susceptible to SV, as a single plaque-forming unit (PFU) kills 100% of animals following either subcutaneous (s.c.) or intracerebral (i.c.) challenge.8 With peripheral inoculation into these hosts, local replication in s.c. tissue and muscle produces high-titer viremia within 24–48 hours.9 Hematogenous dissemination then leads to CNS spread via infection of capillary endothelial cells at the BBB.10 Viral titers in the brains of neonates increase over the course of disease, and death usually ensues 3–4 days after challenge.9,11 Disease outcome in older mice infected with the original wildtype strain of SV (AR339) is variable; most animals can survive s.c. virus challenge by 1–2 weeks of age, and survival is uniform in 3–4 week old mice even following an i.c. route of inoculation.11 In weanling mice, CNS viral titers peak somewhat later and at levels 100- to 1000-fold lower than those found in newborn animals, and titers decline thereafter as recovery ensues.11 Since these original pathogenesis studies were undertaken, a neuroadapted strain of SV (NSV) that is lethal for adult animals has been generated by serial passage of the original AR339 stock through mouse brain.12 Furthermore, the molecular basis for the virulence of this virus has been characterized.13,14 Construction of a full-length complementary DNA (cDNA) clone of SV now allows for the experimental introduction of even single nucleotide mutations into the virus genome and the production of recombinant viruses whose pathogenicity can be easily tested in vivo.15

2. OVERVIEW OF SINDBIS VIRUS PATHOGENESIS Many studies have investigated various aspects of SV pathogenesis in mice, and as a group, this large body of data makes the model a prototype

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for understanding the biology of alphavirus infections as they occur in other vertebrate hosts, including humans. Important issues related to CNS virus tropism as well as both viral and host determinants of outcome will be reviewed here.

2.1 Cellular Tropism Following direct i.c. challenge in mice, all strains of SV target neurons of the CNS with minimal productive infection of supporting glial cell populations.10,11 Interestingly, while the virus spreads directly from cell to cell in these animals, not all populations of neurons are equally susceptible and immunohistochemical studies reveal that the infection can be quite patchy throughout the CNS.9–12 Infected neurons are most easily identified in regions such as the hippocampus of the brain and the ventral gray matter of the lumbar spinal cord. This selective tropism presumably reflects differential cell surface expression of viral receptor(s), although the identity of such molecule(s) remains unknown. For more virulent viral strains such as NSV, cellular tropism does not change compared to more attenuated isolates or the wild-type strain, but they do replicate to somewhat higher peak titers within the CNS, and the cellular responses to infection are clearly different (discussed below).12 Viral infection of mammalian cell lines or even of primary neurons in vitro are generally less helpful in understanding in vivo tropism issues, as most cultured cells are broadly permissive for infection and virus replication. Still, such models have served to identify putative viral receptor proteins on mouse neural cells.16 This strong tropism for neurons is different from what is found in some murine alphavirus encephalitis models such as VEEV where glial cells can also be infected.3,4

2.2 Determinants of Outcome 2.2.1 Viral determinants The production of a SV cDNA has been an important tool in the study of virus-host interactions, and it is now clear that even single nucleotide mutations resulting in coding changes to the viral genome can alter pathogenicity

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in mice. While many such changes in the three main viral structural proteins (the capsid protein and the two surface glycoproteins, E1 and E2) contribute to the virulence of SV in vivo, for a virus such as NSV that remains virulent in older animals, the most important change involves a substitution of histidine for glutamine at residue 55 of the E2 glycoprotein (Table 2).14,17 This change increases the efficiency of neuronal infection, thus resulting in better replication and higher peak CNS viral titers.16,18 It is also one that is rapidly selected during persistent SV infection of the CNS.19 As a result, animals infected with NSV show more neuronal cell death in CNS tissue sections,12 and emerging evidence suggests that multiple neuronal cell death pathways are activated in response to this infection.20–22 The various neuronal responses following SV and NSV infections will be discussed in Table 2. Viral determinants of age-dependent virulence observed with different SV strains. Amino Acid Residues at Individual Positions in the E1 and E2 Glycoproteins E2

E1

Viral Strain

3

23

55

172

209

72

237

313

NSV AR339 HRSP*

T T I

E E V

H Q Q

G G R

G R G

A V A

A A S

D D G

Viral Strain Host Age (Days) 1 7 21

NSV

AR339

HRSP*

% Mortality

Virus Titer†

% Mortality

Virus Titer†

% Mortality

Virus Titer†

100 100 100

8.9 9.1 7.2

100 18 0

8.5 8.0 6.6

52 0 0

8.4 7.9 6.0

* HRSP, heat-resistant small plaque laboratory strain of SV (multiply passaged in tissue culture). † Log10 PFU/gram of brain tissue taken 24 hours after i.c. challenge.

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detail below. Other viral mutations also influence pathogenicity in vivo, although none to the same degree as the change at E2, position 55.13–18

2.2.2 Host determinants Age and genetic background are the two main host factors that influence the outcome of SV encephalitis (SVE) in mice. It has been known for some time that neonatal mice die rapidly following challenge with most strains of SV; there is a sharp decline in the susceptibility to fatal infection that develops some time during the first two weeks of life (Table 2).8,9,11 Infected neurons in neonates show evidence of apoptosis in histological preparations of CNS tissues, and the magnitude of these changes correlates with neurovirulence.23 The neurons of older mice, however, are much more resistant to virus-induced programmed cell death, and it is proposed that downregulation of the endogenous cell death machinery in neurons as part of normal maturation and differentiation of the CNS accounts for this change.24 In terms of genetic background, while all inbred strains of mice tested to date can be infected with NSV, a few such as the BALB/cBy sub-strain of BALB/c mice have shown a remarkable resistance to its lethal effects.25 Such a resistance cannot be attributed to either altered virus tropism or impaired virus replication within the CNS,25,26 but instead it is related to an intrinsic resistance of the neurons of these animals to virus-induced cell death.26 The genetic basis for this resistance remains unknown, but it has been linked to several loci on chromosome 2.27 The identity of these genes and their potential roles in disease pathogenesis remain of significant interest to this field.

3. NEURONAL RESPONSES DURING SINDBIS VIRUS ENCEPHALOMYELITIS Neurons are the primary cellular targets of all SV strains within the CNS; infection, dysfunction, and death of these cells underlie the various clinical manifestations of disease. While earlier studies focused on the destruction of virus-infected cells via apoptosis, it is now apparent that non-infected neurons are also susceptible to injury via non-apoptotic mechanism(s). Furthermore, as part of the hind limb paralysis that accompanies

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NSV infection of weanling mice, retraction of the synaptic inputs onto lower motor neurons of the spinal cord cause these cells to lose function even without undergoing cell death. This process, referred to as “synaptic stripping”, remains poorly understood but may underlie some of the reversible neurological deficits that can occur with this disease.

3.1 Apoptosis of Virus-Infected Neurons Direct infection of neurons by SV, both in vitro and in vivo, results in the target cells undergoing classical apoptosis as assessed by morphological and biochemical criteria.23,28 The importance of this cell death pathway to SV pathogenesis in vivo has been elegantly demonstrated in newborn mice where overexpression of different members of the Bcl-2 family, proteins known to regulate apoptosis induced by a variety of stimuli, can potentially protect animals from fatal encephalitis.29,30 Indeed, the degree of neuronal apoptosis found in the brains of newborn mice correlates directly with the neurovirulence of different viral strains.23 The subsequent unraveling of molecular pathways involved in neuronal apoptosis has presented new targets to be exploited in the treatment of neonatal mice with SV infection, and indeed, SV has itself become a powerful tool to dissect the intracellular workings of the various apoptotic cascades in neurons.24 Largely as a result of studies undertaken in the SV model, neuronal apoptosis has now been identified in the brains of newborn mice infected with other neurotropic viruses including flaviviruses, reoviruses, and bunyaviruses.31–33 These findings suggest that neuronal apoptosis is an important general mechanism in the pathogenesis of viral encephalitis in young hosts.

3.2 Non-Apoptotic Neuronal Cell Death Despite these significant advances in clarifying the pathogenesis of SVE, not every piece of experimental evidence fully supports the importance of neuronal apoptosis in the development and evolution of clinical disease. In weanling mice infected with NSV, spread of virus from the brain to motor neurons of the lumbar spinal cord causes the hind limb paralysis that animals consistently develop before death.34 When the mechanisms

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underlying the destruction of these neurons were more thoroughly investigated, no morphological or biochemical evidence of apoptosis was seen.20 Furthermore, multiple Bcl-2 family members previously shown to protect neonatal mice from lethal SVE failed to prevent NSV-induced paralysis or motor neuron destruction in older animals.21 In histological sections of spinal cord, infected motor neurons exhibited evidence of cellular swelling, which is uncharacteristic of apoptosis and much more consistent with glutamate-mediated excitotoxic damage (Figure 1).20 Indeed, pharmacological blockade of a non-N-methyl-D-aspartate (non-NMDA) glutamate receptor subtype successfully prevented paralysis and death in these animals without having any effect on CNS virus tropism, replication, or clearance.35,36 Consistent with in vitro findings,22 these data strongly support a role for glutamate-mediated excitotoxicity in the pathogenesis

Figure 1. Motor neurons in the lumbar spinal cord of NSV-infected mice five days after viral challenge show morphological features not typical for apoptosis. Degenerating cells appear swollen, hypochromatic, with dissolution of nuclear and cytoplasmic membranes. Apoptotic neurons (not shown) would appear shrunken, with densely stained chromatin and evidence of fragmentation and blebbing. Toluidine blue, 100×.

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of lethal alphavirus encephalomyelitis in older animals. They also show for the first time that significant clinical protection of these hosts can be achieved without having any direct antiviral effect.

3.3 Bystander Injury of Non-Infected Neurons Upon further consideration, that neurons could be protected in NSVinfected animals without altering CNS virus tropism, replication, or clearance raised the intriguing possibility that many cells are being damaged via some bystander mechanism. Intrinsic to such a possibility is that neurons not directly infected with virus are susceptible to injury. Since completion of the glutamate receptor blocking studies, bystander damage to non-infected neurons in both the brain and spinal cord of NSV-infected animals has been confirmed.36,37 Furthermore, other neuroprotective interventions beyond glutamate receptor antagonists that protect mice from paralysis and death without affecting the replication or clearance of virus from the CNS have been identified.37,38 Some (and perhaps all) of these bystander pathways still involve glutamate-mediated excitotoxicity,35–37 suggesting that these other neuroprotective drugs act upstream to block a cascade that triggers this process.38 Emerging evidence implicates innate immune responses as some of these important triggering events (discussed below). If confirmed in related models, the fact that bystander neuronal injury may be an important cellular substrate of fatal disease in older animals with alphavirus encephalomyelitis may force some new thinking about potential treatment strategies for these diseases. Anti-inflammatory and/or neuroprotective interventions may become realistic options along with various antiviral approaches.

4. GLIAL CELL RESPONSES DURING SINDBIS VIRUS ENCEPHALOMYELITIS Although glial cells (astrocytes, oligodendrocytes, microglial cells) are not directly infected by SV in vivo, these cells still undergo a variety of changes in response to SV infection that are increasingly being recognized as important events in disease pathogenesis. For astrocytes, such changes

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involve their capacity to buffer levels of the excitatory neurotransmitter, glutamate, via reuptake transporters. For microglia, infection causes cellular activation and the production of a variety of inflammatory mediators that contribute to neurodegeneration. Changes to both cell types present novel therapeutic targets during infection and will be reviewed here.

4.1 Astrocyte Responses Astrocytes serve many supportive roles within the CNS. One important function is the capacity to buffer extracellular levels of the excitatory neurotransmitter, glutamate, by means of specific surface transporters that carry this amino acid out of the synaptic cleft and away from neurons once synaptic transmission has occurred. Indeed, experimental knockdown of the gene encoding glutamate transporter-1 (GLT-1) revealed a central role for this protein in glutamate reuptake by astrocytes (it provides more than 90% of total CNS glutamate reuptake capacity) and in the prevention of excitotoxic injury to neurons.39 When GLT-1 expression was examined in the spinal cords of NSV-infected animals, focal loss in the ventral horn around inflammatory cells and degenerating motor neurons was seen (Figure 2).36 This change proved to be reversible in the few animals that survived disease, and there was no evidence that the astrocytes themselves were damaged or destroyed during infection.36 Furthermore, BALB/cBy mice that are resistant to NSV-induced paralysis did not show altered spinal cord expression of GLT-1, and various drug treatments that prevented downregulation of this protein during the acute stages of infection in susceptible animals also effectively blocked motor neuron degeneration and the development of hind limb paralysis.36,38 Together, these findings strongly implicate astrocytic glutamate reuptake proteins in NSV pathogenesis, and the researchers are fueling efforts to understand the factors that contribute to their altered expression. As mentioned above and discussed further below, innate immune responses are strongly implicated in this process.

4.2 Microglial Responses Microglia are the main endogenous immune cell of the CNS and they become activated in response to a wide variety of injuries, infections,

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Figure 2. Immunohistochemical staining shows altered expression of the astrocytic glutamate transporter, GLT-1, in the lumbar spinal cords of mice during acute NSV infection. Left: a low power view of the ventral horn of an uninfected animal shows diffuse GLT-1 staining of the neuropil (brown) as well as multiple large angulated cells with the appearance of normal motor neurons (purple). Right: a similar region from a paralyzed animal six days after infection with NSV shows focal loss of GLT-1 expression in the ventral horn (brown), infiltration of mononuclear cells (purple), and loss of motor neuron cell bodies. Immunoperoxidase and hematoxylin, 20×.

and inflammatory stimuli.40 Activated microglial cells can proliferate, migrate, change morphology, and assume many macrophage-like functions including phagocytosis and production of inflammatory mediators.40,41 These cells in such an activated state may have either beneficial or injurious effects on surrounding neural elements.42 During SVE, there is histochemical evidence of widespread microglial activation within the CNS (Figure 3),43 although the functional significance of this change has until recently been poorly understood. Since drugs that inhibit this activation process have proven to be beneficial in other animal models of neurological diseases where it has been observed, their effects were examined in the NSV system. Two unrelated agents both provided notable clinical benefit in terms of paralysis and survival, and each was linked to reduced bystander injury of uninfected neurons without having any impact on CNS virus replication or spread.37,38 Thus, these cells, too, are important contributors to NSV pathogenesis and are therapeutic targets in these diseases.

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Figure 3. Lycopersicon esculentum (tomato) lectin immunostaining identifies numerous microglial cells (brown) in the spinal cords of NSV-infected animals. Compared to uninfected controls (not shown), the number of lectin-positive cells detected in tissue sections increases more than 5-fold over the first 72 hours of infection. Immunoperoxidase and hematoxylin, 60×.

5. HOST IMMUNE RESPONSES DURING SINDBIS VIRUS ENCEPHALOMYELITIS Although not so apparent in newborn animals where direct interactions between the virus and infected host cells are the main determinant of outcome, the immune system is recognized to make important contributions to disease pathogenesis in weanling mice during SVE. On one hand, elements of the adaptive immune system become activated to promote viral clearance from the CNS. On the other hand, innate and adaptive immune responses both contribute to the lethal outcome of mice infected with NSV. The beneficial and detrimental aspects of host immunity will be reviewed here.

5.1 Mechanisms of Viral Clearance From the CNS As in other tissues, successful viral clearance from the CNS involves both the eradication of extracellular virus and the elimination (or at least the permanent suppression) of virus-infected cells. Indeed, if virus-infected neurons are to recover normal cellular function, then clearance mechanisms

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must inhibit the intracellular synthesis of viral proteins and nucleic acids without significantly altering cellular structure and physiology. Short of fully cleansing all viral genomes from each cell, host responses must at least be able to prevent the resumption of viral replication over the longterm in order to avoid persistent or recurrent disease.44 Antiviral antibodies play a central role in clearing SV from the neurons of infected mice.45,46 Many of the antibodies that mediate CNS viral clearance are directed against viral surface proteins, and most also can inhibit virus replication in primary neurons in vitro.46 The precise mechanisms by which such large molecules act to suppress an intracellular process such as viral replication have only been partially clarified. Bivalent antibodies against viral envelope glycoproteins bind the infected cell surface to inhibit replication; monovalent fragments are ineffective, implying that crosslinking of surface viral proteins is required.47 Antibody treatment of infected cells restores normal cellular processes such as the maintenance of membrane polarity and host cell protein synthesis that are shut off by infection,48 and it also directly inhibits the synthesis of viral proteins.46,47 Type-I interferons (IFNs) can synergize with antiviral antibodies to control SV replication in vitro,49 and this combination of host factors is optimal for CNS clearance of SV in vivo.46,50 Still, “clearance” in this model is an incomplete process, as viral RNA can be amplified from the brains of mice many months after clinical recovery has occurred.51 Indeed, long-term production of antiviral antibodies within the brains of these recovered animals suggests the continued need for local immunological control over the lifespan of the host.44 The role of T cells in the clearance of SV from the CNS is somewhat less clear, although recent data suggest that the prototype, T cell-derived cytokine, IFN-γ , can clear SV from neurons.52 Here, different neuronal populations vary in their responsiveness to the antiviral effects of IFN-γ ; spinal motor neurons are highly responsive and clear virus in the complete absence of antibody, while cortical neurons require antiviral antibodies for full clearance to be achieved.52 It is not known whether this difference relates to the ability of these different neuronal populations to bind IFN-γ or to respond to this mediator. Neurons, to a much greater extent than glial cells, widely express the IFN-γ receptor.53

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5.2 Role of Immune Responses in the Generation of Neuropathology 5.2.1 T cell responses A contribution of T cells to the fatal outcome seen in mice with NSV encephalomyelitis was first suggested in studies showing that disease mortality was reduced in animals lacking CD8+ T cells, as a result of disruption of the β 2-microglobulin gene.54 Here, messenger RNAs (mRNAs) encoding various components of major histocompatibility complex (MHC) class I molecules were induced in neurons during infection, despite a lack of detectable surface expression of MHC class I proteins.54 On the other hand, both perforin and Fas, important effectors of direct CD8+ T cell-mediated cytotoxicity, were seemingly not involved as both genetically deficient animals were equally susceptible to NSV infection compared to strain-matched controls.55 In this setting, a role for CD8+ T cell-derived cytokines was proposed, though specific effectors of damage have not been identified. Subsequent experiments showed that CD4+ T cell-deficient mice were also partially protected against NSV infection, and that IFN-γ -deficient animals survived the disease at very high rates.55 While none of these defects in T cell subsets or in T cell cytokine production altered peak virus titers in the CNS, the mechanisms underlying their pathogenic effects during NSV infection remain unexplained.

5.2.2 Innate immune responses Once it was observed that drugs inhibiting microglial activation could protect mice from NSV-induced paralysis and death, a search for the molecular mechanisms underlying their beneficial effects was pursued. An important clue came from earlier studies showing that mice deficient in the proinflammatory cytokine, interleukin (IL)-1β , were highly resistant to NSV;56 this led to the discovery that both agents blocking microglial activation also suppressed IL-1β production within the CNS,37,38 and that replacement of IL-1β to the experimental system overcame their protective effects.37 Furthermore, pathogenic IL-1β production was clearly implicated in the loss of GLT-1 expression, since downregulation of transporter expression was markedly reduced in IL-1β -deficient animals.38,57

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This finding puts these pathogenic microglial responses upstream of the effects that NSV exerts on astrocytes. Although the molecular signals that lead to both the induction of these pathogenic innate immune responses and the cytokine-driven effects on glutamate transport remain unknown, taken together, the data suggest a model where microglial activation leads to specific changes on astrocytes that, in turn, promote conditions leading to neurodegeneration. It must also be appreciated that not all innate host responses are detrimental in the setting of NSV encephalomyelitis. Animals unable to respond to type-I IFN develop higher CNS viral titers and are more susceptible to lethal disease than age-matched controls.58 Likewise, pharmacological inhibition of inducible nitric oxide synthase, was found to increase mortality in NSV infection, suggesting that production of nitric oxide is an important protective host defense mechanism.25 Tumor necrosis factor-alpha (TNF-α )-deficient animals developed synaptic abnormalities around motor neuron cell bodies in the spinal cord during NSV infection, suggesting that this mediator acts to maintain synaptic connectivity in the motor pathway. Accordingly, any therapy designed to target innate immune responses must avoid suppressing those with protective or beneficial effects.

6. IMMUNOTHERAPY FOR LETHAL SINDBIS VIRUS INFECTION As can be perceived from the data already presented, therapies designed to augment beneficial host immunity or to inhibit detrimental host responses provide novel approaches to counteract the lethal effects of NSV infection in weanling mice. By extension, some of these strategies may also eventually be useful in treating humans with related forms of alphavirus encephalitis. Two of the most promising immunotherapeutic interventions identified to date involve the administration of exogenous antiviral antibodies to augment CNS viral clearance and the use of drugs designed to block pathogenic innate immune responses.

6.1 Antibody-Mediated Clearance of Virus From the CNS Since adoptive transfer experiments showed that antiviral antibodies could effectively cross the BBB of persistently infected severe combined

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immunodeficient (SCID) mice and clear an avirulent strain of SV from the CNS,46 a similar approach was used to treat immunocompetent mice given a lethal challenge of NSV. These studies showed that various monoclonal anti-SV antibodies against either the E1 or E2 surface glycoproteins (but not C, the capsid protein) could literally “cure” animals of disease, even when treatment was delayed for some time after viral challenge (Table 3).59 Most of these antibodies neutralized NSV infectivity and/or lysed NSV-infected cells in the presence of complement in vitro, but neither biological function was necessary or sufficient to guarantee recovery. Even though the effective therapeutic window of these curative antibodies was relatively brief (∼48 hours), the data provide some of the first evidence that an antiviral approach can be used in vertebrates with ongoing alphavirus encephalitis. Similar findings have since been shown in mice infected with the flavivirus, West Nile Virus (WNV),60,61 and these encouraging findings in animals have prompted a clinical trial of immune serum in humans with neurological involvement as a result of WNV infection.62 While this clinical study has yet to report any beneficial effects, the data do show how experimental results in animal models can be translated to humans in a rapid manner.

6.2 Inhibition of Detrimental Innate Immune Responses As IL-1β -deficient animals are known to resist NSV-induced paralysis and death,56 a more therapeutically relevant strategy to confirm a specific role for the IL-1 pathway in this model was sought. This led to the discovery that treatment of mice with a recombinant form of the naturally occurring IL-1 receptor antagonist (IL-1ra) could provide significant protection against both severe paralysis and death following NSV challenge (Figure 4).37 As might be predicted, treated animals maintained astroglial GLT-1 expression and showed less histological evidence of neuronal cell death compared to untreated controls. Recombinant IL-1ra is already in clinical use in humans with rheumatoid arthritis and diabetes mellitus, where the drug seems to be well tolerated and efficacious as either an adjunctive or a stand-alone form of treatment.63,64 The availability of this safety and efficacy data makes its application in human alphavirus encephalitis a realistic option at some point in the future. Further study of its effects in animal models of alphavirus encephalitis where the virus is

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Table 3. Effects of different monoclonal antibodies used as immunotherapy of lethal NSV encephalomyelitis in mice.

Antibody Clone

Biological Activities†

Target Viral Antigen

Antibody Isotype

Mortality (%)*

N

C

HI

E1 E1 E1 E1 E1 E2 E2 E2 E2 E2 E2 E2 C C

IgG2a IgG2a IgG2a IgG1 IgG2a IgG3 IgG3 IgM IgA IgG3 IgG3 IgG1 IgG2a IgM

0 30 0 42 0 0 0 100 100 4 17 85 100 100

− − + + − + + − + + + − − −

+ − + − + + + − − + − + − −

+ − − − − + + − − + + − − −

101 102 103 108 109 201 202 204 208 209 210 212 1 3

* 0.2 ml of purified ascites fluid given intravenously 24 hours after lethal NSV challenge. † N, neutralizes infectivity in vitro; C, mediates antibody-dependent, complement-mediated cytotoxicity of infected cells; HI, inhibits hemagglutination.

Clone 202 202 202 202 202 202

Dilution

Time of Transfer After Challenge (h)

Mortality (%)

None 1:10 1:100 1:1000 None None

24 24 24 24 48 72

0 0 0 0 14 87

also an important human pathogen (WEEV, VEEV, EEEV) are in progress. It would also seem prudent to consider its use in conjunction with other agents (i.e., antiviral antibodies) to combat the effects of the pathogen at multiple steps in its pathogenic cascade.

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Figure 4. Exogenous administration of a specific IL-1 receptor antagonist (IL-1ra) protects mice from otherwise lethal NSV encephalomyelitis. Parallel groups of mice (n = 6/group) were treated with IL-1ra (10 µg intranasally/animal/day) or a vehicle control starting at the time of viral challenge. Hind limb paralysis (A) and death (B) were monitored on a daily basis over a 14-day study interval, and the significance of paralysis and death between the two groups was calculated by Kaplan-Meier analysis.

7. CONCLUSIONS Although most alphaviruses infect humans via the bite of infected mosquito vectors, some can be transmitted as aerosols, making them potential biological terrorism threats. Since conventional antiviral therapies have proven ineffective against these pathogens in vivo, most efforts have focused on the development of vaccines to prevent these diseases. Still, there remains a significant need to understand the biology of these infections in order to develop therapeutic approaches that are applicable following virus exposure. Studies in the SV model summarized here reveal multiple mechanisms of neuronal injury in infected animals, show that host responses play an important role in the pathogenesis of these diseases, and demonstrate that these responses and their immediate downstream effects are novel potential therapeutic targets. Indeed, since many human patients with these infections no longer have detectable virus present within the CNS at the time that precipitous clinical deterioration occurs, it may be that blockade of these detrimental host responses are the best and perhaps the only treatment option in this clinical setting.

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REFERENCES 1. Johnston RE, Peters CJ (1996). In Fields Virology, 3rd edn (eds.) Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, Straus SE. Lippincott-Raven pp. 843–898. 2. Whitley RJ (1997). In Infections of the Central Nervous System (eds.) Scheld WM, Whitley RJ, Durack DT. Lippincott-Raven pp. 147–168. 3. Nalca A, Fellows PF, Whitehouse CA (2003). Antiviral Res 60, 153–174. 4. Holbrook MR, Gowen BG (2007). Antiviral Res 77, 112–121. 5. Reed DS, Larsen T, Sullivan LJ, Lind CM, Lackemeyer MG, Pratt WD, Parker MD (2005). J Infect Dis 192, 1173–1182. 6. Reed DS, Lind CM, Lackemeyer MG, Sullivan LJ, Pratt WD, Parker MD (2005). Vaccine 23, 3139–3147. 7. Taylor RM, Hurlbut HS, Work TH, Kingstin JR, Frothingham TE (1955). Am J Trop Med Hyg 4, 844–862. 8. Reinarz ABG, Broome MG, Sagik BP (1971). Infect Immun 3, 268–273. 9. Griffin DE (1976). J Infect Dis 133, 456–464. 10. Johnson RT (1965). Am J Pathol 46, 929–943. 11. Johnson RT, McFarland HF, Levy SE (1972). J Infect Dis 125, 257–262. 12. Jackson AC, Moench TR, Trapp BD, Griffin DE (1988). Lab Invest 58, 503–509. 13. Lustig S, Jackson AC, Hahn CS, Griffin DE, Strauss EG, Strauss JH (1988). J Virol 62, 2329–2336. 14. Tucker PC, Griffin DE (1991). J Virol 65, 1551–1557. 15. Rice CM, Levis R, Strauss JH, Huang HV (1987). J Virol 61, 3809–3819. 16. Ubol S, Griffin DE (1991). J Virol 65, 6913–6921. 17. Tucker PC, Strauss EG, Kuhn RJ, Strauss JH, Griffin DE (1993). J Virol 67, 4605–4610. 18. Tucker PC, Lee SH, Bui N, Martinie D, Griffin DE (1997). J Virol 71, 6106–6112. 19. Levine B, Griffin DE (1993). J Virol 67, 6872–6875. 20. Havert MB, Schofield B, Griffin DE, Irani DN (2000). J Virol 74, 5352–5356. 21. Kerr DA, Larsen T, Cook SH, Fannjiang YR, Choi E, Griffin DE, Hardwick JM, Irani DN (2002). J Virol 76, 10393–10400. 22. Nargi-Aizenman JL, Griffin DE (2001). J Virol 75, 7114–7121.

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23. Lewis J, Wesselingh SL, Griffin DE, Hardwick JM (1996). J Virol 70, 1828–1835. 24. Griffin DE (2005). Curr Top Microbiol Immunol 289, 57–78. 25. Tucker PC, Griffin DE, Choi S, Bui N, Wesselingh SL (1996). J Virol 70, 3972–3977. 26. Thach DC, Kimura T, Griffin DE (2000). J Virol 74, 6156–6161. 27. Thach DC, Kleeberger SR, Tucker PC, Griffin DE (2001). J Virol 75, 8674–8681. 28. Levine B, Huang Q, Isaacs JT, Reed JC, Griffin DE, Hardwick JM (1993). Nature 361, 739–742. 29. Levine B, Goldman JE, Jiang HH, Griffin DE, Hardwick JM (1996). Proc Natl Acad Sci USA 93, 4810–4815. 30. Lewis J, Oyler GA, Ueno K, Fannjiang YR, Chau BN, Vornov J, Korsmeyer SJ, Zhou S, Hardwick JM (1996). Nat Med 5, 832–835. 31. Despres P, Frrenkiel MP, Ceccaldi PE, dos Santos CD, Deubel V (1998). J Virol 72, 823–829. 32. Oberhaus SM, Smith RL, Clayton GH, Dermody TS, Tyler KL (1997). J Virol 71, 2100–2106. 33. Pekosz A, Phillips J, Pleasure D, Merry D, Gonzalez-Scarano F (1996). J Virol 70, 5329–5335. 34. Jackson AC, Moench TR, Griffin DE, Johnson RT (1987). Lab Invest 56, 418–423. 35. Nargi-Aizenman JL, Havert MB, Zhang M, Irani DN, Rothstein JD, Griffin DE (2004). Ann Neurol 55, 541–549. 36. Darman J, Backovic S, Dike S, Maragakis NJ, Krishnan C, Rothstein JD, Irani DN, Kerr DA (2004). J Neurosci 24, 7566–7575. 37. Irani DN, Prow NA (2007). J Neuropathol Exp Neurol 66, 533–544. 38. Prow NA, Irani DN (2007). Exp Neurol 205, 461–470. 39. Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, et al. (1996). Neuron 16, 675–686. 40. Lawson LJ, Perry VH, Dri P, Gordon S (1990). Neuroscience 39, 151–170. 41. Gehrmann J, Matsumoto Y, Kreutzberg GW (1995). Brain Res Brain Res Rev 49, 569–575. 42. Schwartz M (2003). J Cereb Blood Flow Metab 23, 385–394. 43. Tyor WR, Stoll G, Griffin DE (1990). J Neuropathol Exp Neurol 49, 21–30.

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44. Tyor WR, Wesselingh S, Levine B, Griffin DE (1992). J Immunol 149, 4016–4020. 45. Griffin DE (2003). Nat Rev Immunol 3, 493–502. 46. Levine B, Hardwick JM, Trapp BD, Crawford TO, Bollinger RC, Griffin DE (1991). Science 254, 856–860. 47. Ubol S, Levine B, Lee S-H, Greenspan NS, Griffin DE (1995). J Virol 69, 1990–1993. 48. Depres P, Griffin JW, Griffin DE (1995). J Virol 69, 7006–7014. 49. Depres P, Griffin JW, Griffin DE (1995). J Virol 69, 7345–7348. 50. Byrnes AP, Durbin JE, Griffin DE (2000). J Virol 74, 3905–3908. 51. Levine B, Griffin DE (1992). J Virol 66, 6429–6435. 52. Binder GK, Griffin DE (2001). Science 293, 303–306. 53. Robertson B, Kong G, Peng Z, Bentivoglio M, Kristensson K (2000). Brain Res Bull 52, 61–74. 54. Kimura T, Griffin DE (2000). J Virol 74, 6117–6125. 55. Rowell JF, Griffin DE (2002). J Neuroimmunol 127, 106–114. 56. Liang XH, Goldman JE, Jiang HH, Levine, B (1999). J Virol 73, 2563–2567. 57. Prow NA, Irani DN (2008). J Neurochem 105, 1276–1286. 58. Byrnes AP, Durbin JE, Griffin DE (2000). J Virol 74, 3905–3908. 59. Stanley J, Cooper SJ, Griffin DE (1986). J Virol 58, 107–115. 60. Diamond MS, Shrestha B, Marri A, Mahan D, Engle M (2003). J Virol 77, 2578–2586. 61. Engle MJ, Diamond MS (2003). J Virol 77, 12941–12949. 62. Jester PM, Tilden SJ, Li Y, Whitley RJ, Sullender WM (2006). Contemp Clin Trials 27, 254–259. 63. Waugh J, Perry CM (2005). BioDrugs 19, 189–202. 64. Larsen CM, Faulenbach M, Vaag A, Volund A, Ehses JA, Seifert B, Donath MY (2007). N Engl J Med 356, 1517–1526.

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SECTION IV

Double-Stranded RNA Virus

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CHAPTER 26

Host Signaling Responses to Reovirus Infection Da Pan, Paola Marcato, Maya Shmulevitz & Patrick W. Lee

ABSTRACT Mammalian reovirus is a non-enveloped double-stranded RNA virus. Although isolated from respiratory and enteric tracts, reovirus is rarely linked to human disease and is thus regarded as benign. Recently, reovirus was found to preferentially kill many types of cancer cells, raising the prospect of using reovirus as a cancer therapy reagent. Studies in reovirus biology reveal that Ras signaling in host cells provides advantages for reovirus replication in transformed cells over normal cells. Further studies of reovirus oncolysis show that three distinct steps of oncolytic reovirus replication are enhanced by Ras activation: virion uncoating, progeny virus infectivity and reovirus-triggered apoptosis. Identifying the role of downstream effectors in reovirus oncolysis will further reveal how reovirus takes advantages of host cell signaling and will better define the differences between normal and cancer cells. Fully understanding reovirus and host cell interactions will also enable us to better apply reovirus as an anti-cancer reagent.

1. INTRODUCTION Reoviridae, currently consisting of eleven virus genera, are non-enveloped double-stranded RNA (dsRNA) viruses that ubiquitously infect vertebrates, invertebrates, and plants (for the most updated list of viruses belonging to Reoviridae, see http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm). 627

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Mammalian reovirus (respiratory, enteric, orphan virus) belongs to the Reoviridae family and was so named because of its isolation from human respiratory and gastrointestinal tracts and its lack of connection to any severe disease state.1 The only case of acute respiratory disease in humans caused by a reovirus occurred recently in 20072 by a previously unknown, bat-origin reovirus that is serologically unrelated to the commonly isolated mammalian reovirus strains which infect human asymptomatically. The mammalian reovirus isolates that have been studied in the laboratory for decades are serotype 1 strain Lang (T1L), serotype 2 strain Johns (T2J), serotype 3 strain Dearing (T3D) and strain Abney (T3A).3–5 Extensive studies on reovirus over the years have defined the general process of its replication. However, since the recent discovery that T3D reovirus preferentially replicates in cancer cells, the focus of much research has shifted to defining the cellular characteristics of cancer cells which make them intrinsically permissive to reovirus infection. This chapter will focus on reovirus infection in the context of cancer, or transformed cells and will highlight the signaling pathways of cancer cells that make them highly infectible by reovirus. Henceforth, unless otherwise listed, we will refer to T3D reovirus as reovirus in this chapter.

2. BIOLOGY OF REOVIRUS: STRUCTURE AND REPLICATION Reovirus has a segmented genome consisting of ten double-stranded RNA segments which are grouped into three size classes: small, medium or large (S, M and L). Each segment of the genome encodes one viral protein, with the exception of the S1 segment that encodes two proteins. Eight of the eleven viral proteins are structural proteins that appear in the viral particles while the remaining non-structural proteins are critical players in replication of reovirus. The dsRNA reovirus genome is surrounded by two concentric protein shells: inner and outer capsids. The inner capsid consists of λ1, λ 3, µ 2, σ 2, and the outer capsid consists of µ 1, σ 1, σ 3, and λ 2. Protein µ 1 (76 kDa) undergoes autocleavage, yielding a small 4.2 kDa fragment µ 1N and the 71 kDa fragment µ 1C, both of which appear to be components of the mature virions.6 Recently, Nibert et al.7 showed that using certain conditions (high pH, absence of reducing

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agent and low temperature) to disrupt reovirus for protein separation on polyacrylamide gel, a high percentage of intact µ 1 protein could be recovered, leading the authors to propose that µ 1 autocleavage might be a process important for virus disassembly during reovirus entry. When infecting cells, reovirus virions undergo two distinct stages of sequential disassembly, yielding the intermediate subviral particles (ISVPs),8 and cores.9 Both of these subviral particles are isolatable and can also be generated in vitro with specific proteases.10 As illustrated in Figure 1, reovirus infection begins with virus binding to cell surface receptors, such as sialic acid and/or in combination with the junctional adhesion molecule (JAM).11,12 Following binding, reovirus enters cells through receptor-mediated endocytosis. Beta-integrin plays a role in mediating reovirus internalization following attachment, possibly through interaction with λ 2 protein.13 Once in acidified endosomes or lysosomes, reovirus uncoating/disassembly (digestion of σ 3 coat protein and cleavage of µ 1/µ 1C into δ ) occurs in a protease-dependent manner, and ISVPs are formed.14 It has been postulated that these subviral particles can penetrate membranes such as plasma membrane,15 as well as lysosomal membranes and as a result viral particles enter into the cytosol16 where the remaining reovirus replication cycle occurs until virus release. During the process of penetration, σ 1 proteins are released from the particles and transcriptionally active virus cores are formed. Reovirus mRNAs (i.e. plus strand RNAs) are transcribed in the cores by reovirus RNA-dependent RNA polymerase λ 3 independently of host cell machinery and released into the cytoplasm (primary transcription). Like all other viruses, reovirus mRNAs are translated into proteins using host protein translation machinery. Concordantly, newly transcribed mRNAs and certain viral proteins form cytoskeleton-associated structures, “viral factories”,17 where new core particles will assemble. Non-structural proteins µ NS, σ NS, and the structural protein µ 2 form the framework for the viral factories and are crucial in recruiting viral transcripts and proteins, and initiating assembly of virus particles.18 Following progeny virus cores formation, secondary transcription (i.e. transcription from de novo assembled cores) begins. Minus strand RNAs are synthesized within the assembled cores, using the plus strand RNAs as templates.19 At a late stage of the reovirus life cycle, outer capsid proteins are added to the cores, halting transcription and

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Figure 1. Overview of reovirus replication cycle. Reovirus infection includes: σ 1 protein interaction with receptors such as sialic acid and/or JAM, receptor-mediated endocytosis, proteolytic enzymes causing virus uncoating/disassembly, penetration of subviral particles (ISVPs) through endosomal membrane, formation of core particles, primary transcription of viral mRNAs by viral RNA-dependent RNA polymerase (λ 3) within reovirus cores, viral protein synthesis in the cytoplasm, assembly of virus cores in viral factories containing viral transcripts and proteins, minus strand synthesis to form the viral genomic RNAs within the newly assembled reovirus cores, secondary transcription of viral mRNA, virion maturation through complete assembly of outer capsid, and virus release. Refer to text for more details.

completing the assembly of virus particles.20 These now mature virion particles are released from the cells, which can be mediated by apoptosis.21 During the reovirus replication cycle, viral mRNA and proteins are exposed to the intracellular environment and can potentially trigger host signaling pathways. Studies in recent years revealed that reovirus exploits deregulated Ras-signaling pathways in transformed and cancer cells for its own enhanced replication.

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3. CELL SIGNALING EVENTS IN CANCER THAT ASSIST REOVIRUS INFECTION In 1977, Hashiro et al.,22 by screening cancer and transformed cells and normal cells, found that reovirus T2J can infect tumor cells and certain spontaneously transformed cells, but normal cells are resistant to infection. Later, Duncan et al.23 discovered that WI-38 cells (human embryonic lung cells) transformed by SV40 displayed drastically enhanced susceptibility to T3D reovirus infection. However, it was not until the 1990s that a distinct connection between reovirus and oncolysis was made, when it was shown that preferential replication of reovirus in transformed cells was dependent on having an activated Ras-signaling pathway. Further studies showed that a single injection of reovirus could induce tumor regression in a mouse xenograft model.24 It is estimated that Ras mutations occurs in more than 30% of human cancers.25 However, when mutations upstream and downstream of Ras are taken into consideration, a much higher percentage of cancers would have mutations in Ras-signaling pathways, making them potentially susceptible to reovirus. Since this discovery, studies using reovirus as a potential anti-cancer therapy have been aggressively pursued and reovirus is currently being tested in phase I/II clinical studies.

3.1 The Ras Signaling Pathway Determines Reovirus Oncolysis 3.1.1 The Ras signaling pathway is usurped by reovirus In 1993, the importance of epidermal growth factor receptor (EGFR) for increased susceptibility to reovirus was demonstrated.26 Following this observation, it was found that the v-erbB oncogene, which encodes a mutated EGFR homolog that lacks the extracellular ligand domain but has a constitutively active intracellular effector domain, confers enhanced cellular susceptibility to reovirus infection.27 This finding indicated that reovirus requires the intrinsic signaling events initiated by EGFR. Further, cells that were transformed by activated son of sevenless (sos) or ras, both of which are key factors in Ras signaling pathways downstream of EGFR,

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are significantly more susceptible to reovirus compared to normal cells.28 These findings link the Ras signaling pathway to preferential replication of reovirus in transformed and cancer cells.

3.1.2 Ras signaling Ras proteins are small GTP-binding proteins at the hub of convergent signaling pathways activated by extracellular stimuli.29 Ras activation is tightly regulated by a group of guanine nucleotide exchange factors, such as sos, and GTPase activating proteins, such as necrosis factor 1 (NF1). These factors control the cycling of Ras between its active GTP-bound state and its inactive GDP-bound state. In its GTP-bound state, Ras initiates signaling of numerous downstream cascades, which as shown in Figure 2, control a plethora of cellular functions, such as trafficking, transcription, translation, cell cycle progression, differentiation and apoptosis.29–31 Constitutively activated Ras is insensitive to GAP regulation and induces deregulated signaling pathways. These mutated Ras are found in different human tumors.25,32 Three of the main downstream signaling pathways regulated by Ras are the Raf/MEK/Erk pathway, the PI3K/Akt pathway and the RalGEFs/Ral pathway. Among the sub-Ras effectors, Raf is the most studied and is extensively targeted in cancer therapies that employ Raf-specific pharmaceutical inhibitors.33–36 Activated Ras interacts with Raf through a Ras-binding domain (RBD) and recruits Raf onto the plasma membrane where Raf is activated. Activated Raf then phosphorylates and activates MEK1/2, which in turn phosphorylates and activates p42 and p44 Erk1/2. Activated Erk1/2 can phosphorylate a group of substrates including p90 ribosomal S6 kinase (RSK) and TSC2 (tuberous sclerosis complex, also known as hamartin).37 It also controls cell proliferation, transcription, translation and apoptosis aspects of transformation.30 As will be mentioned later in this chapter, lysosomal proteases like cathepsin L,38,39 cathepsin B38 and neutrophil elastase40 are responsible for reovirus uncoating. Interestingly, the production and activity of cathepsin L is upregulated by the activated Raf pathway in murine fibroblast cells. However, in epithelial cells, all three of the main sub-Ras pathways are needed,41 indicating that the downstream pathways activated by Raf might be cell type-dependent.

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Figure 2. Activation of Ras and its effector pathways. Upon binding of the epidermal growth factor (EGF) to the receptor, EGFR, the intrinsic tyrosine kinase activity of the receptor is activated. This causes the phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, which are then recognized by the Grb2 adaptor protein. The association of the Grb2:Sos complex with the activated EGFR allows Sos to activate Ras. Activated Ras then interacts with its effectors and activates downstream signaling pathways. The three main sub-Ras effector pathways are shown: Raf/MEK/ERK, RalGEFs/Ral, PI3K/Akt pathways.

The next most extensively studied sub-Ras pathway is PI3K/Akt. PI3K or phosphatidylinositol 2-kinase catalyzes the conversion of phosphatidylinositol (4,5)-biphosphate (PtdIns[4,5]P2) to phosphatidylinositol (3,4,5)-triphosphate (PtdIns[3,4,5]P3) in response to growth factors and cytokines. PI3K and its lipid second messenger products PtdIns[3,4,5]P3 and PtdIns[4,5]P2 control cell proliferation, cell survival, cell cycle regulation and cell metabolism.31 The most important downstream effector of PI3K is Akt (also known as protein kinase B, PKB). PtdIns[3,4,5]P3 can directly interact with Akt and causes the translocation of Akt to the plasma membrane followed by activation of this kinase. Activated Akt regulates a plethora of downstream effectors such as glycogen synthase kinase-3 (GSK-3) and TSC2. As functional GSK-3 is often linked to impaired cell cycle regulation and Akt phosphorylation of GSK-3 can inhibit its function, activation of Akt is believed to cause abnormal cell growth and has been repeatedly shown to be important for Ras-mediated transformation.42,43 Moreover, Akt can activate NF-κ B by phosphorylating

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and activating IKK. NF-κ B will be released and translocate into the nucleus, where it mediates cell survival by turning on genes that encode anti-apoptotic proteins such as IAPs.44 However, it was also shown that blocking the activation of NF-κ B abrogated cell cycle arrest effect caused by Ras transformation, indicating that NF-κ B can promote events other than anti-apoptosis.45 As will be described below, NF-κ B is critical in reovirus-triggered apoptosis and is playing a proapoptotic role in this process. Ras may be able to activate NF-κ B and benefit reovirusmediated apoptosis. The third so called “orphan” sub-Ras effector pathway is RalGEFs/ Ral. RalGEFs represent a group of guanine exchange factors that stimulate GDP/GTP exchange of Ral and activate the protein in a Rasdependent manner.46 Activated Ral binds to its downstream effectors such as Cdc42/Rac, phospholipase D and Ral binding protein (RalBP1, also known as RIP1 or RLIP76)47,48 and is closely associated with cell functions such as vesicle formation and Golgi trafficking. RalGEFs have been shown to be an essential component of Ras-mediated transformation.49,50 There are two isoforms of human Ral proteins, RalA and RalB. These two isoforms were shown to collaborate to both enhance proliferation and limit cell death.51 Moreover, RalB was able to directly recruit and activate the atypical Iκ B kinase family member Tank Binding Kinase (TBK1) to support survival in cancer cells.52 Although RalB-dependent activation of TBK1 is dispensable for normal cells, it helps to respond to dsRNA virus infection.52 It was also shown that the RalGEFs/Ral pathway plays a role in the metastasis of prostate cancer to bone,53 further indicating the importance of this sub-Ras pathway in tumorigenesis. Other Ras effector pathways, such as the Tiam and the Rho/Rac pathways, control many aspects of cell transformation mediated by Ras, and cross-talk between different sub-Ras pathways is common.30 However, there are still many missing links among these pathways. As described earlier, reovirus takes advantage of an activated Ras-pathway to replicate and spread in transformed and cancer cells. In addition, different sub-Ras pathways may affect various steps of reovirus replication. Experiments involving the use of Ras mutants that activate different sub-Ras pathways54 have revealed that the Ras/RalGEFs pathway and the p38 pathway both enhance reovirus infection in NIH3T3 cells, suggesting that RalGEFs

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and p38 pathways might be linked. Reovirus can therefore be used as a tool to reveal the cross-talks between sub-Ras pathways and provide a better understanding of the molecular mechanisms of Ras-mediated cell transformation. As will be mentioned below, various steps of reovirus replications are affected by Ras-transformation. It is possible that different sub-Ras pathways have distinct effects on individual steps of reovirus lifecycle. Pathways initiated by Ras-transformation may cooperate with each other to enable reovirus preferential replication and cell killing in transformed cells.

3.2 Activated Ras Signaling Enhances Three Steps of Reovirus Replication To effectively apply reovirus as a cancer therapy, we need to fully understand the mechanisms of its preferential replication in transformed and cancer cells. By comparing each step of reovirus replication in Rastransformed and non-transformed murine NIH3T3 cells, it was observed that Ras transformation can augment three distinct steps of reovirus replication.55 Firstly, uncoating of the otherwise equally bound virus particles occurs with greater efficiency in Ras-transformed cells. As mentioned above, activities of lysosomal proteases such as cathepsins are required for reovirus uncoating. Ras transformation in murine fibroblast cells can enhance production and activity of cathepsins through the activation of Raf pathway.41 In this case, Raf/MEK pathway might upregulate cathepsins and cause higher level of reovirus uncoating. However, RalGEFs/Ral pathway is implicated indirectly in the process of endocytosis.48 As a result, an augmented level of uncoating could be the result of a faster endocytosis process caused by activation of RalGEFs/Ral pathway as well. Using reovirus as a tool, we may be able to reveal unresolved relationship between sub-Ras pathways and either endocytosis or proteolytic enzyme activity/expression. Previous studies on mechanisms of reovirus oncolysis showed differences in viral protein synthesis between Rastransformed and non-transformed cells by metabolically labeling viral protein between 12 and 48 hour postinfection (hpi).27,28 In this way, the radiolabeled proteins represented accumulation of proteins from almost

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two reovirus replication cycles. Subsequent analysis the levels of viral transcripts using highly quantitative real-time reverse transcriptase polymerase-chain reaction (qRT-PCR) in both Ras-transformed and nontransformed cells that were infected by reovirus, coupled with shorter pulse-labeling (1 hour) of viral proteins,55 revealed that the translation rate of the viral transcripts are similar in Ras-transformed and nontransformed cells. Enhanced efficiency of uncoating of incoming virions results in the generation of more transcriptionally active viral core particles, which in turn leads to the production of more viral transcripts and viral proteins at a later time of virus replication.55,56 The double-stranded RNA activated protein kinase (PKR) is also believed to be important for reovirus oncolysis28: activation of PKR in normal cells prohibited reovirus propagation while PKR activation was inhibited in Ras-transformed cells.28 Furthermore, Inhibition of PKR phosphorylation by 2-aminopurine, or deletion of the Pkr gene, led to drastic accumulation of reovirus proteins from 12 to 48hpi.28 Since PKR activation can result in inhibition of protein translation,57 it was originally proposed that activated Ras promotes reovirus protein synthesis by suppressing PKR activation.28 More recently, it was found that the reovirus protein translation rate was not drastically different between infected Ras-transformed and nontransformed cells,55 suggesting that PKR affected reovirus oncolysis at levels other than protein translation. PKR involvement has also been shown in the activation of many signaling transduction factors such as NF-κ B58,59 and mitogen-activated protein kinase (MAPK).60 Pathways activated by these factors are also regulated by Ras. The possibility that Ras mediates the reovirus-induced interferon pathway via PKR is particularly intriguing as reovirus mutants that were shown to be more sensitive to interferon (which induces PKR activation)61 differentiated Ras-transformed and nontransformed cells better than their interferon insensitive counterparts.62 Secondly, progeny virus particles produced by Ras-transformed cells have a four-fold higher infectious-to-noninfectious particle ratio than those from non-transformed cells. Virus particles from Ras- and non-transformed cells, however, are identical by transmission electron microscopy and the virus particles show similar protein profiles following gel electrophoresis. This suggests that subtle modifications on virus structural proteins may be responsible for the enhanced infectivity of reovirus generated from

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Ras-transformed cells. Notably, reovirus is the first oncolytic virus shown to be regulated at the level of infectivity by host cells. It is noteworthy that reovirus proteins have been shown to undergo extensive modifications.63 For instance, the reovirus outer capsid protein µ 1 is modified by a myristoyl group and this modification is implicated in the process of penetration through membrane.64 Other proposed modifications on µ 1 have included phosphorylation and polyadenylation.20 It is possible that Ras activation in transformed cells promotes modifications or conformational changes of certain reovirus proteins which can positively affect virion infectivity, most likely at the early stages of virus infection (i.e., binding, entry, and/or uncoating). Identification of the enzymes that are responsible for modifications on viral proteins in Ras-transformed cells may provide important information on potential targets for anti-cancer drugs. The third step of reovirus replication caused by Ras-transformation is enhanced reovirus-mediated apoptosis, which results in a higher level of reovirus release. Reovirus-induced apoptosis is a major factor in determining virus spread. Decades of research have delineated much of the pathways and effectors involved in reovirus-mediated apoptosis. Before we discuss the role of Ras in promoting apoptosis in reovirus-infected cells, we will first summarize our current understanding of how reovirus infection triggers apoptosis in a cell.

4. REOVIRUS-TRIGGERED APOPTOSIS IN HOST CELL By analyzing the morphological changes and fragmentation of cellular DNA of reovirus-infected cells, it was found that reovirus causes the host cell to undergo apoptosis.65 One of the important tools used in reovirus studies is reovirus reassortment. When host cells are infected with two different strains of reovirus, the two genomes might be reassorted in a virion. As a result, newly assembled viruses will contain a mixture of the ten double-stranded RNA segments from the two genomes. By using reovirus reassortants, strain-specific signaling pathways induced by reovirus infection can be mapped to certain genes. First identified using reovirus reassortment, the reovirus S1 and M2 gene segments, which encode σ 1 and µ 1 proteins, respectively, were found to be critical for reovirus-mediated apoptosis.64,65 However, it is still not clear how these proteins interact with

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the host cell to trigger apoptosis. Reovirus infection also triggers host cell signaling events such as the activation of the stress-activated c-jun N-terminal kinase (SAPK/JNK) pathways, and activation of the Nuclear Transcription Factor κ B (NF-κ B) pathway. These pathways are critical players in reovirus-triggered apoptosis. Meanwhile, S1 and M2 genes are implicated to be important in triggering these host pathways.66,67 The exact nature of involvement of these proteins in activating the host signaling pathways is still not clear. Genome-wise analysis of gene expression changes upon reovirus infection68 revealed that the transcription of genes most altered are highly clustered in cellular apoptosis and DNA repair pathways. Recently, the long-awaited plasmid-based reverse genetics system for double-stranded RNA viruses has been successfully produced,69 enabling reovirus researchers to mutate individual genes and incorporate them into infectious viral particles. This technology will help us to better understand the roles which individual reoviral proteins play in triggering host signaling pathways, including the apoptotic pathways.

4.1 Reovirus-Triggered Apoptosis There are two main apoptosis pathways in cells (Figure 3): the extrinsic and the intrinsic pathways. The extrinsic or cytoplasmic pathway is activated by extrinsic stimuli and is initiated by the association of tumor necrosis factor (TNF) superfamily proteins to their receptors. TNF family consists of more than 20 proteins including TNF-related apoptosis-inducing ligand (TRAIL) and CD95-ligand. This event triggers oligomerization of the receptors and the recruitment of the adaptor protein Fas-associated Death Domain (FADD). This recruitment in turn causes procaspase-8 accumulation and autocleavage, followed by activation of effector caspases which often feeds into the intrinsic apoptosis pathway. Protein cFLIP can interfere with the generation of active caspase-8 and as a result inhibit the extrinsic apoptosis pathway. The intrinsic or mitochondrial apoptotic pathway depends on loss of mitochondrial membrane integrity and the release of cytochrome c, Smac (second mitochondria-derived activator of caspase)/DIABLO (direct binding of inhibitor of apoptosis protein (IAP) with low isoelectric point, PI), or apoptosis inducing factor (AIF). Release of cytochrome c can trigger activation of the key effector

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Figure 3. Apoptotic pathways triggered by reovirus infection. Reovirus triggers apoptosis through both cytoplasmic and mitochondria pathways. Cytoplasmic pathway functions in a caspase-8-dependent manner that can be interfered by cFLIP. Activation of mitochondrial pathway depends on the selective release of Smac/DIABLO, which selectively neutralizes apoptosis inhibitor proteins (AIPs) including XIAP, survivin and c-AIP1. Cleavage of Bid by caspase-8 mediates the connection between cytoplasmic and mitochondrial apoptosis pathways. Reovirus infection also triggers activation of JNK as well as activation of NF-κ B. RIG-1 and IPS-1 are important for reovirus-mediated apoptosis but are dispensable for activating NF-κ B. Bold lines showed pathways that are activated by reovirus or are required in reovirus-triggered apoptosis. Dashed lines represent pathways that are dispensable for reovirus-mediated apoptosis.

caspase, caspase-3, through formation of the cytochrome c/Apaf-1/caspase-9 containing apoptosome complex. Smac/DIABLO facilitates activation of downstream caspases by neutralizing the inhibitor of apoptosis proteins (IAPs). At the same time, AIF can translocate into the nucleus and cause direct DNA condensation. Extrinsic and intrinsic apoptosis pathways are interconnected by the cleavage of Bid, a proapoptotic Bcl-family protein.

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Caspase-8-dependent cleavage of Bid allows the protein to translocate into the mitochondria where it facilitates pore formation of the mitochondrial membrane and cytochrome c release, and initiates mitochondrial apoptosis pathway. This process can be blocked by anti-apoptotic proteins such as Bcl-2.70 It was first observed that apoptosis triggered by reovirus infection in human embryonic kidney cell line HEK293 could be drastically decreased by interfering with TRAIL-receptor interaction, including pretreatment using anti-TRAIL (TRAIL1/2) antibodies, overexpression of decoy death receptors DcR1/2, or incubation with soluble TRAIL receptors such as DR5.71,72 Antibodies against Fas or soluble TNF receptor had no effect on reovirus-triggered apoptosis, indicating that Fas and TNF were not likely to be involved in reovirus-triggered apoptosis. Downstream adapter protein FADD mediates the downstream activation of caspases following ligand-receptor interactions. When a dominant negative (DN) FADD mutant was expressed, reovirus-triggered apoptosis was reduced significantly.71,73 Treatment with caspase-8 inhibitor can abrogate reovirus-triggered apoptosis. These findings suggest that the extrinsic apoptosis pathway is triggered by reovirus through TRAIL/death receptor/FADD and caspase-8 (Figure 3). It was later shown that the mitochondrial pathway also plays an important role in reovirus-triggered apoptosis.74 Release of cytochrome c and Smac/DIABLO but not AIF, and cleavage proapoptotic protein Bid were observed following reovirus infection.73–75 Overexpression of DN-FADD could block the release of cytochrome c and cleavage of Bid, indicating that activation of caspase-8 is critical to initiate the mitochondria-dependent pathway after reovirus infection.73,74 Overexpression of Bcl-2 could inhibit the release of cytochrome c or Smac/DIABLO, and activation of effector caspases-3 and -7,73,74 implicating the mitochondrial apoptosis pathway involvement in reovirus-mediated apoptosis. Meanwhile, expression of a dominant negative form of caspase-9 did not prevent the activation of caspase-3 and the resulting cleavage of Poly (ADP-ribose) polymerase (PARP), suggesting that activation of caspase-9, which is mediated by the release of cytochrome c, is dispensable to reovirus-mediated apoptosis.75 As the result of Smac/DIABLO release induced by reovirus infection, antiapoptotic IAPs including XIAP, c-IAP1 and survivin were downregulated

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while c-IAP2 level remained relatively the same throughout the reovirus replication cycle,75 indicating that reovirus infection triggers selective downregulation of cellular IAPs. These results suggest that reovirustriggered apoptosis involves both extrinsic and intrinsic apoptosis pathways (Figure 3). It was also observed that reovirus infection caused the release of TRAIL from host cells.71 In addition, reovirus could sensitize cells to TRAIL-induced apoptosis.67,71,72 Several cancer cell lines were tested for TRAIL sensitivity and it was shown that reovirus and TRAIL could synergistically kill cancer cells and this effect is TRAIL-dependent and caspase8 mediated.67,72 Reovirus infection of ovarian cancer cells showed that expression of cFLIP, a negative mediator of apoptosis initiated by TRAIL, decreased after infection. Downregulation of cFLIP by RNA interference could render cancer cells more sensitive to TRAIL-induced apoptosis after reovirus infection.67 The combination of reovirus and TRAIL may represent a novel cancer therapy for TRAIL-resistant cancer.

4.2 Activation of NF-κ B by Reovirus Infection One of the early signaling events following reovirus infection is the activation of NF-κ B, which is important to reovirus-mediated apoptosis.76,77 NF-κ B is a transcription factor that regulates cellular functions such as immune response, inflammation and cell death in response to different signals. Deregulated NF-κ B has been implicated in human disease including cancer.78–83 NF-κ B transcription factors are assembled through dimerization of the five Rel/NF-κ B proteins: RelA (p65), c-Rel, RelB, NF-κ B1(p50/p105) and NF-κ B2(p52/p100), with p50 and p52 proteolytic products of p105 and p100 respectively.84 Latent NF-κ B is retained in the cytoplasm by the bindings of inhibitors of NF-κ B (Iκ Bs) for RelA and cRel, or NF-κ B2p100 for RelB. NF-κ B activation involves its translocation from the cytoplasm to the nucleus. In a canonical NF-κ B activation pathway, site specific phosphorylation of Iκ Bs by the Iκ B kinase (IKK) complex results in ubiquitination and proteosomal degradation of these NF-κ B inhibitor proteins,84–89 and this allows the prototypical NF-κ B, which contains p50/RelA dimers, to translocate into the nucleus. The IKK complex contains two catalytic subunits, IKKα and IKKβ , and two

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regulatory subunits IKKγ /NEMO and ELKS.90–92 IKKβ is essential for NF-κ B activation in the canonical pathway in the presence of IKKγ /NEMO. In an alternative NF-κ B activation, IKKα is phosphorylated by NF-κ B-inducing kinase (NIK) and in turn phosphorylates NF-κ B2p100,93,94 targeting NF-κ B2p100 for ubiquitination and degradation. NF-κ B dimers containing RelB or p52 are then released and translocated into the nucleus. A third pathway of NF-κ B activation involves Iκ Bα phosphorylation by CK2, followed by ubiquitination and degradation.93,94 It was determined in electrophoretic mobility shift assays (EMSA) that NF-κ B was activated (bound to its DNA consensus sequence) following reovirus infection in a number of cell lines including HeLa, HEK293, L and Madin-Darby canine kidney (MDCK) cells.76,77 In HeLa cells, activation of NF-κ B starts as early as 4 hours post infection, peaks at 10 hpi and starts to decline at 12 hpi.77 Activation of NF-κ B by reovirus infection results in the translocation of p65 and p50 into the nucleus. Experiments suggest that reovirus-triggered apoptosis requires NF-κ B activation because reovirus-induced apoptosis can be significantly blocked by: a proteasome inhibitor which blocks degradation of Iκ Bs; or transient expression of a dominant-negative form of Iκ Bα which lacks the sites for ubiquitination and degradation, and quenches activation of NF-κ B.77 Regulation of inhibitor protein Iκ Bα, but not Iκ Bβ nor Iκ Bε, is essential for reovirus-mediated NF-κ B regulation. Blocking the activity of IKKα instead of IKKβ diminishes translocation of RelA protein and as a result blocks the activation of NF-κ B. In addition, reovirusmediated apoptosis and NF-κ B regulation both depend on IKKγ /NEMO, since cells lacking IKKγ /NEMO undergo a lower level of apoptosis after reovirus infection compared to normal cells. These results suggest that reovirus infection triggers activation of NF-κ B through a novel IKKαand IKKγ /NEMO-dependent pathway (Figure 3).95 In HEK293 cells, however, NF-κ B activation was observed at an early time postinfection but was blocked at later times through inhibition of Iκ Bα degradation.76 Reovirus infection was shown to be able to block NF-κ B activation by stopping Iκ Bα from being degraded at later stage of infection.76 This downregulation of NF-κ B sensitized HEK293 cells to TRAIL- or TNF-induced apoptosis. This study indicates that reovirus can both activate and inhibit NF-κ B activation and this regulation can be

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cell-type dependent. It is also possible that this cell-type dependent activation of NF-κ B is a result of various signaling pathways activated in different cell lines. In order to identify downstream events that are activated by NF-κB in reovirus-infected cells, human HeLa cells were engineered to express a degradation-resistant mutant of Iκ Bα in an inducible manner.96 Expression of 112 genes under the activation of NF-κ B was altered upon reovirus infection. Most of these genes play a role in the innate immune response and cell death programs. It will be interesting to identify how certain genes that are activated by NF-κ B upon reovirus infection can contribute to reovirus-triggered apoptosis and which specific pathways are involved. It was shown recently that reovirus activates interferon regulatory factor-3 (IRF-3), an important factor in host immune response97 and IRF3 is activated at a similar time when NF-κ B is activated. Activation of IRF-3 requires retinoic acid-inducible gene-1 (RIG-1) and interferon-β promoter stimulator-1 (IPS-1). Moreover, IPS-1 and IRF-3 are also required for reovirus-triggered apoptosis, indicating the involvement of apoptosis in antiviral responses following reovirus infection (Figure 3). However, IPS-1 and IRF-3 are dispensable for activation of NF-κ B, indicating that other apoptosis pathways are triggered.

4.3 JNK is Activated During Reovirus Infection An important kinase in pathways that transduces extracellular signals, SAPK/JNK, was found to be activated after reovirus infection.66 Strain differences in inducing JNK activation were mapped to S1 and M2 viral genes which are also critical for reovirus-triggered apoptosis.66 Activity of JNK closely correlated with reovirus-induced apoptosis and c-jun was phosphorylated after reovirus infection.66 This was further confirmed by using an inhibitor of JNK which blocked reovirus-induced apoptosis but not virus yield.98 Furthermore, inhibition of JNK delayed release of cytochrome c and Smac/DIABLO, with the latter an essential effector for reovirus-mediated apoptosis.98 Using mouse embryonic fibroblast cells harboring either wild-type or no MEKK, an upstream kinase that activates JNK,99 it was shown that reovirus-induced caspase-3 activation was diminished in MEKK null cells, an effect similar to that caused by the

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JNK inhibitor.98 These results suggest that JNK plays an important role in reovirus-induced apoptosis (Figure 3). Whether the activation of JNK and NF-κ B in reovirus-mediated apoptosis are interconnected, is still unclear at present.

4.4 Reovirus-Triggered Host Signaling Requires Reovirus Uncoating/Disassembly It was shown that reovirus-mediated apoptosis requires virus uncoating, but not virus replication. Uncoating inhibitors such as ammonium chloride and E64, both of which stop reovirus uncoating by inactivating proteases, can abolish reovirus-triggered apoptosis. On the other hand, in vitro generated ISVPs can infect cells and trigger apoptosis and are unaffected by these inhibitors.100 Activation of NF-κ B and JNK are correlated to both reovirus uncoating and reovirus-triggered apoptosis.66,77 The importance of virus disassembly might indicate that the uncoating process of reovirus replication can release signals necessary to be recognized by host sensors. It will be of great interest to determine which host proteins or processes are able to recognize and respond to reovirus disassembly.

4.5 Activated Ras-Signaling Enhances Reovirus-Induced Apoptosis As described extensively above, reovirus triggers apoptosis in host cells and activates NF-κ B and JNK. Ras signaling is closely associated with cellular signaling events such as activation of NF-κ B.101 Activated Rassignaling pathways can also regulate apoptosis at different levels. For example, Raf activation may lead to ERK phosphorylation of p90 ribosomal S6 kinase (RSK) and other translation factors, leading to either apoptosis or survival; PI3K/Akt activation can promote anti-apoptotic signaling by phosphorylation of proapoptotic protein Bad, preventing it from binding and inhibiting anti-apoptotic proteins Bcl-2 or Bcl-XL101; the RalGEFs pathway, as mentioned above, can regulate apoptosis with the collaboration of the two homolog Ral proteins.51 Reovirus is likely able to differentiate between the conflicting signals caused by Ras-activation and selectively synergize the ones that trigger cell death. In normal cells, on

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the other hand, reovirus-triggered apoptotic pathways might be counteracted by anti-apoptotic signals, resulting in reduced apoptosis than that observed in Ras-transformed cells. It is important to identify both the antiand pro-apoptotic signals in Ras-transformed and non-transformed cells to better understand the differences between normal and transformed cells, and to better use reovirus as a cancer therapy.

5. REOVIRUS IN CANCER THERAPY The linkage of reovirus infection to activated Ras signaling has led to various attempts to test the cancer therapeutic potential of reovirus. So far, a variety of human cancers, including brain, colon, ovarian, breast, pancreatic and bladder cancer cell lines or ex vivo human tumor surgical specimens have been found to be susceptible to reovirus killing.103–106 Animal models using tumor cell implants have also been tested and significant tumor regression occurred after intratumoral injections of reovirus.103,105,107 Generally, tumors implanted in immunocompromised animals need only a single injection of reovirus, while immunocompetent animals require multiple injections of reovirus or in combination with immunosuppressive drugs.108 Reovirus has also been shown to have the potential to treat metastatic tumors.109,110 Systemic i.v. therapy with reovirus inhibited metastatic tumor growth and improved the survival rate of the animals, raising the possibility of using reovirus to attack metastatic tumors in inaccessible locations. Although Ras mutations are rare in lymphoid malignancies, when lymphoid cell lines were tested for efficacy of reovirus therapy, several cell lines were found to be susceptible to infection.111 Lymphoma cells implanted subcutaneously in mice regressed after a single injection of live reovirus. Of the 27 human lymphoma specimens, 21 of them were susceptible to reovirus, suggesting that reovirus can be used in the treatment of some lymphoma.111 Persistent infection (PI) of reovirus occurs during long term culture of survived infected cells.112 Concerns were raised that long term of exposure to reovirus infection may cause the tumor to be resistant to reovirus. Raji lymphoid cells were infected with reovirus and the surviving cells were cultured and reinfected to create persistently infected cell lines. PI Raji cells could be treated by anti-reovirus antibodies so that the cured cells

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would no longer release reovirus. It was found that PI Raji cells were nontumorigenic, while cured Raji cells which are resistant to reovirus infection in vitro can form tumors but are susceptible to reovirus infection in vivo. Further study showed that the proteases present in the microenvironment of the tumor augmented the uncoating efficiency of reovirus and thus enhanced reovirus oncolysis. Following this study, the same group further compared a range of cell lines susceptible or resistant to reovirus infection and discovered that the proteolytic stripping of the outer capsid protein of reovirus virions (i.e. the uncoating step) is critical for reovirus oncolysis.56 Although Ras activation was not found in every susceptible cell line, a pathway downstream of Ras might be activated. As discussed before, Ras-transformation can enhance the uncoating step of reovirus infection.55 On the other hand, the uncoating process is essential for reovirus-induced apoptosis100 and Ras activation can enhance reovirusinduced apoptosis,55 indicating that apoptosis caused by reovirus infection might be affected by upregulation of proteases caused by Ras activation. In order to use reovirus as an effective cancer therapy, it will be necessary to determine which component of signaling pathway upregulates protease production and activity. Interestingly, a fibrosarcoma cell line (HT1080) develops resistance (HTR1) to reovirus infection in vitro while the cured HTR1 cells are more highly tumorigenic.113 Lower level of cathepsin B activity was shown in the HTR1-resistant cells. However, whether the highly tumorigenic cured cell could be killed by rechallenge of reovirus in vivo was not addressed in this study. Co-implantation of persistently infected HTR1 and cured cells did not show tumor formation, indicating that residual reovirus released by the HTR1 cells might be able to infect and kill the tumor cells in vivo. Meanwhile, the cured cells could be triggered to undergo apoptosis by other apoptosis-inducing reagents such as camptothecin, indicating that the reovirus-resistant tumors might be susceptible to a combined therapy. Most of the current animal models of reovirus oncolysis were tested in tumors formed by established tumor cell lines or transformed cells. Colon cancer induced by azoxymethane, which resembles natural tumor development, was recently tested for reovirus oncolysis.114 Reovirus-treated animals showed a significantly lower level of tumor formation and metastases.

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Reovirus cancer therapy is undergoing clinical trials in Canada, the United States and the United Kingdom. By delineating the molecular mechanisms of reovirus oncolysis, we may be able to better apply reovirus to target cancer cells that are from different origins but share similar signaling activation.

6. SUMMARY Reovirus has been used as a tool to understand virus-host interactions. Comprehensive results on reovirus-induced apoptosis suggest that a network of host signaling pathways is involved in reovirus infection. The Ras signaling pathway has been implicated in reovirus preferential replication in cancer cells and different steps of reovirus replication can take advantage of activated Ras-signaling. Characterizing the effects of sub-Ras pathways on reovirus oncolysis can provide us with more information on the basic differences between transformed cells and normal cells. Knowledge of reovirus oncolysis will help us further identify targets for cancer therapy.

ACKNOWLEDGMENTS We thank Dr. Don Stoltz for his critical reading of this chapter. Da Pan and Maya Shmulevitz are supported by Cancer Research Training Program (CRTP) at Dalhousie University. Paola Marcato is funded by Alberta Heritage Foundation for Medical Research (AHFMR).

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93. Schomer-Miller B, Higashimoto T, Lee YK, Zandi E (2006). J Biol Chem 281, 15268–15276. 94. Yamaoka S, Courtois G, Bessia C, Whiteside ST, Weil R, Agou F, Kirk HE, Kay RJ, Israel A (1998). Cell 93, 1231–1240. 95. Hansberger MW, Campbell JA, Danthi P, Arrate P, Pennington KN, Marcu KB, Ballard DW, Dermody TS (2007). J Virol 81, 1360–1371. 96. O’Donnell SM, Holm GH, Pierce JM, Tian B, Watson MJ, Chari RS, Ballard DW, Brasier AR, Dermody TS (2006). J Virol 80, 1077–1086. 97. Holm GH, Zurney J, Tumilasci V, Leveille S, Danthi P, Hiscott J, Sherry B, Dermody TS (2007). J Biol Chem 282, 21953–21961. 98. Clarke P, Meintzer SM, Wang Y, Moffitt LA, Richardson-Burns SM, Johnson GL, Tyler KL (2004). J Virol 78, 13132–13138. 99. Yujiri T, Ware M, Widmann C, Oyer R, Russell D, Chan E, Zaitsu Y, Clarke P, Tyler K, Oka Y, et al. (2000). Proc Natl Acad Sci USA 97, 7272–7277. 100. Connolly JL, Dermody TS (2002). J Virol 76, 1632–1641. 101. Cox AD, Der CJ (2003). Oncogene 22, 8999–9006. 102. Norman KL, Lee PW (2005). Drug Discov Today 10, 847–855. 103. Hirasawa K, Nishikawa SG, Norman KL, Alain T, Kossakowska A, Lee PW (2002). Cancer Res 62, 1696–1701. 104. Norman KL, Coffey MC, Hirasawa K, Demetrick DJ, Nishikawa SG, DiFrancesco LM, Strong JE, Lee PW (2002). Hum Gene Ther 13, 641–652. 105. Etoh T, Himeno Y, Matsumoto T, Aramaki M, Kawano K, Nishizono A, Kitano S (2003). Clin Cancer Res 9, 1218–1223. 106. Hanel EG, Xiao Z, Wong KK, Lee PW, Britten RA, Moore RB (2004). J Urol 172, 2018–2022. 107. Yang WQ, Lun X, Palmer CA, Wilcox ME, Muzik H, Shi ZQ, Dyck R, Coffey M, Thompson B, Hamilton M, et al. (2004). Clin Cancer Res 10, 8561–8576. 108. Smakman N, van der Bilt JD, van den Wollenberg DJ, Hoeben RC, Borel Rinkes IH, Kranenburg O (2006). Cancer Gene Ther 13, 815–818. 109. Hirasawa K, Nishikawa SG, Norman KL, Coffey MC, Thompson BG, Yoon CS, Waisman DM, Lee PW (2003). Cancer Res 63, 348–353. 110. Yang WQ, Senger D, Muzik H, Shi ZQ, Johnson D, Brasher PM, Rewcastle NB, Hamilton M, Rutka J, Wolff J, et al. (2003). Cancer Res 63, 3162–3172.

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111. Alain T, Hirasawa K, Pon KJ, Nishikawa SG, Urbanski SJ, Auer Y, Luider J, Martin A, Johnston RN, Janowska-Wieczorek A, et al. (2002). Blood 100, 4146–4153. 112. Alain T, Kim M, Johnston RN, Urbanski S, Kossakowska AE, Forsyth PA, Lee PW (2006). Br J Cancer 95, 1020–1027. 113. Kim M, Egan C, Alain T, Urbanski SJ, Lee PW, Forsyth PA, Johnston RN (2007). Oncogene 26, 4124–4134. 114. Alain T, Wong JF, Endersby R, Urbanski SJ, Lee PW, Muruve DA, Johnston RN, Forsyth PA, Beck PL (2007). Cancer Gene Ther 14, 867–872.

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CHAPTER 27

Rotavirus Antagonism of the Host Innate Immune Response John T. Patton & Mario Barro

ABSTRACT Rotaviruses, members of the Reoviridae family of segmented doublestranded RNA viruses, are the primary cause of life-threatening diarrhea in young children. Rotavirus replication is sensitive to the antiviral activities triggered by interferon (IFN)-signaling pathways. Recent studies have revealed that rotaviruses subvert the effects of IFN through the actions of NSP1, a viral protein with a putative N-terminal RING-finger motif and a hypervariable C-terminal region. NSP1 interacts with several members of the interferon regulatory factor (IRF) family of proteins, including IRF-3 and IRF-7, transcription factors that are critical to the expression of type I IFNs (IFN-α and -β ). This interaction requires the C-terminal region of NSP1 and results in the proteasomal degradation of IRF proteins. Rotaviruses incapable of producing fully functional NSP1 are characterized by deficiencies in growth and spread. The presence of a RING-finger motif in NSP1 is reminiscent of E3 ubiquitin-protein ligases, suggesting that NSP1mediated ubiquitination may drive IRF degradation. By inducing the degradation of multiple members of the IRF family, rotavirus NSP1 serves as a highly effective broad-spectrum antagonist of the innate immune response.

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1. INTRODUCTION An array of innate immune pathways have evolved to hinder virus replication. The best defined of these pathways involve interferon (IFN) signaling,1,2 RNA interference (RNAi),3 apoptosis,4 and protein kinase R (PKR)-mediated phosphorylation.5 Induction of the IFN-signaling pathway is of particular importance, as it culminates not only in the expression of proteins with non-specific antiviral activity, but also in the expression of immunoregulatory proteins that stimulate virus-specific adaptive immune responses.6 Activation of the pathway for type-I IFN (IFN-α and -β ) is triggered by host sensors that recognize pathogen components or infection products. These sensors include the Toll-like receptors (TLR), a family of membrane proteins located on the plasma membrane or in endosomes where they screen for pathogen-associated molecular patterns (PAMP).7–9 The endosomal TLR3 is a general sensor of viral infection, since it can recognize the dsRNA by-products of virus replication.2 The cytoplasm also contains other sensors of viral dsRNAs, such as the RNA helicases retinoic acid-inducible gene (RIG-I) and melanoma differentiationassociated gene 5 (mda5).2 Detection of pathogen markers by the sensors results in the activation of cellular kinases that direct the phosphorylation of constitutivelyexpressed transcription factors. One such factor is IFN regulatory factor-3 (IRF-3), a member of the IRF family of immunomodulatory DNA-binding proteins.6,10 Once phosphorylated, IRF-3 undergoes structural changes that lead to its dimerization and nuclear translocation.11 The binding of activated IRF-3 with the large cellular coactivators CBP/p300 to IFNstimulated response elements (ISRE) upregulates genes expressing IFN-β and other products.12 Secreted IFN-β interacts with receptors for type-I IFN (IFN-α /β ) displayed on infected cells and neighboring uninfected cells. This interaction transmits signals through the Jak-Stat pathway that stimulates IRF-7 expression.1 This transcription factor is defined as the master regulator of type-I IFN, due to its capacity to enhance the expression of IFN-β beyond that achieved by IRF-3 alone and to trigger the expression of multiple classes of IFN-α.13 The mechanism by which

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IRF-7 functions is reminiscent of IRF-3, involving phosphorylation, dimerization (with itself or IRF-3), and nuclear translocation where it binds to and activates ISREs. Unlike constitutively-expressed IRF-3, most cell types (e.g. fibroblasts) have little or no detectable IRF-7 in the non-activated state and require exposure to IFN-β to trigger IRF-7 expression.11 In contrast, some trafficking cells (plasmacytoid dendritic cells and macrophages) produce IRF-7 constitutively, negating the need for IRF-3 activation and IFN-β signaling to mount a strong and broad type-I IFN response.14 The production of type I IFN triggers the expression of antiviral products that undermine virus replication, creating an antiviral state. The products include the 2′,5′-oligoadenylate-dependent RNase L, myxovirusresistance (Mx)-GTPases, the dsRNA-activated protein kinase R (PKR), and RNA-specific adenosine deaminase (ADAR).2,5,15 The effectiveness of the IFN-signaling pathway in limiting virus replication is perhaps best revealed by the recognition that many, if not most, viruses produce proteins that subvert activation of the pathway.2,6 Examples of such antagonistic viral proteins include the lymphocytic choriomeningitis virus (LCMV) nucleoprotein NP,16,17 Hantavirus (NY-1) glycoprotein G1,18 rabies virus phosphoprotein P,19 and human papilloma virus (HPV16) E6 protein,20 all of which prevent IRF-3 activation, but through distinct mechanisms. Other virus-encoded antagonists include: Ebola virus VP35 protein, which impedes activation of both IRF-3 and PKR;21 hepatitis C virus NS3/4A protease, which cleaves intermediates in RIG-I activation;22 paramyxovirus SV5 V protein, which targets an RNA sensor of the RIG-I recognition system;23 and influenza A NS1 protein, which interferes with PKR activation.24 As reviewed in this chapter, recent studies reveal that rotaviruses express a protein that subverts IFN signaling. This viral protein, nonstructural protein 1 (NSP1), induces the proteasomal degradation of multiple members of the IRF family in a manner suggestive of an E3 ubiquitin ligase activity. Rotaviruses join the pestiviruses25,26 and Kaposi’s sarcomaassociated herpesvirus (KSHV)27 as viruses known to interfere with innate immune responses by degrading IRF proteins needed for type-I IFN expression.

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2. ROTAVIRUS BIOLOGY 2.1 Pathogenesis Rotaviruses, members of the Reoviridae, are recognized as one of the major causes of severe gastroenteritis in many animal species.28 In humans, rotaviruses are responsible for most cases of acute dehydrating diarrhea in infants and children under the age of five.29 Globally, such infections account for ∼500 000 deaths each year, the vast majority occurring in developing countries.30 The primary site of rotavirus replication in humans are the mature enterocytes at the tips of villi in the small intestine.28,29 Infection of these cells leads to a characteristic blunting and denuding of villi, an outcome likely to account for the loss of fluid homeostasis in the infected child. Recent studies suggested that in some animal species, rotavirus infection may spread beyond the gastrointestinal (GI) tract to include deep blood organs.31,32 Evidence that some rotavirus strains may infect dendritic cells raises the possibility that these trafficking cells provide an avenue through which the virus could escape from the GI tract.33,34

2.2 Replication Rotavirions are icosahedral particles formed from three concentric layers of protein that enclose eleven segments of dsRNA (Figure 1).35 The components of the outer, intermediate, and inner (core) layers are the protease-activated attachment protein VP4 and the glycoprotein VP7, the trimeric protein VP6, and the core lattice protein VP2, respectively. Inside the core are the viral RNA-dependent RNA polymerase (RdRP) VP1, and the RNA-capping enzyme VP3.36,37 During viral entry, the outer protein layer of the virion is lost, yielding a double-layered particle (DLP). Subsequently, VP1 and VP3 within the core direct the synthesis of eleven species of viral mRNAs, products that are extruded from channels extending through the DLP.38 The mRNAs contain typical 5′-cap structures but lack 3′-polyA tails. Instead, rotavirus mRNAs end with the 3′-consensus sequence (3′CS), 5′-UGUGACC-3′.39 This 3′CS serves as a dual-functioning cis-acting signal, promoting both

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Figure 1. Rotaviruses with gene 5 rearrangements produce C-Truncated NSP1. (A) Electrophoretic analysis of the genomes of a wild-type rotavirus (SA11-4F) and an NSP1defective rotavirus (SA11-5S). A sequence duplication increases the size of the gene 5 RNA of SA11-5S, causing the RNA to migrate with the gene 2 and 3 RNAs. (B) Diagram of the gene 5 RNAs (g5-wt and g5-re) and NSP1 proteins (wtNSP1 and NSP1∆C) of SA11-4F and SA11-5S. The ∼550-nucleotide duplicated sequence in the g5-re RNA is labeled DUP-2, and derives from duplication of the DUP-1 region. The location of the RING finger domain (RFD) and the IRF binding domain (IRF-BD) in NSP1 are indicated. The duplication sequence in the g5-re RNA interrupts the ORF, causing it to encode a form of NSP1 with a 17-amino acid C-terminal truncation. The truncation removes part of the IRF-BD. As a result, NSP1∆C does not induce degradation of IRF proteins.

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translation and replication of rotavirus mRNAs.40,41 Translation of the mRNAs gives rise to six structural proteins (VP1-VP4, VP6, VP7) and six non-structural proteins (NSP1-NSP6). Replication of the rotavirus genome is an asymmetrical process with viral mRNAs acting as templates for synthesis of the dsRNA segments.42 This replication process is coordinated with the packaging of newly-made dsRNAs into cores.36 Genome replication and packaging take place in electron-dense inclusions bodies, termed viroplasms, that form in the cytoplasm.43 Formation of viroplasms is driven by two non-structural proteins, NSP2 and NSP5.44 Following their formation, cores undergo maturation into DLPs, a process that is also viroplasm associated. DLPs are recruited to the rough endoplasmic reticulum (ER) through the interaction of VP6 with the ER-transmembrane protein NSP4.45,46 During budding into the ER, DLPs associate with VP4 and VP7 complexes, forming the outer protein layer of the virion.47

2.3 Classification Rotavirus are classified into groups based on antigenic properties (or sequences) of their VP6 component. Presently, seven groups have been defined (A–G), with rotaviruses in group A recognized as the most important cause of human mortality and morbidity.48 The genome segments of rotaviruses contained within the same group can be exchanged (reassort) with one another during co-infections to form progeny with mixed genotypes. In contrast, rotaviruses belonging to different groups are not known to undergo reassortment.49 Rotaviruses within the same group are further classified into P-types (P = protease-sensitive) and G-types (G = glycoprotein) based on antigenic properties or sequences of their outer capsid components VP4 and VP7, respectively.50,51

2.4 Properties of NSP1 Although rotaviruses typically only cause disease in their natural host, vis-à-vis, homologous infections, these viruses can frequently infect heterologous hosts. However, the virus yields of heterologous infections are typically magnitudes less than those of homologous infections, suggesting

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that rotaviruses encode one or more proteins that operate in a species specific manner to promote virus replication. Some obvious candidate proteins that promote species specific virus growth include the outer capsid proteins, VP4 and VP7, due to their roles in virus attachment and entry. Another intriguing candidate is NSP4, a protein known not only to have a role in virus morphogenesis, but also to function as a viral enterotoxin.46 Finally, NSP1, the viral antagonist of IFN-signaling pathways in rotavirus-infected cells is also likely to act in a species specific manner to promote virus growth. NSP1, the 55 kDa product of the gene 5 RNA, shows the most sequence variation of any rotavirus protein, with identities extending well below 50% between some group A strains.52–54 However, for strains isolated from the same host species, sequence identities can be quite high. This observation, combined with phylogenetic analyses, suggests that NSP1 has co-evolved with its natural host.53,55 Such co-speciation may explain why NSP1 proteins of different virus strains differ in their capacity to promote virus spread in any one type of animal. For example, while murine rotavirus (e.g. EDIM), expressing its natural NSP1, is strongly virulent and spreads efficiently in the mouse, equivalent murine reassortant viruses expressing instead a rhesus rotavirus (RRV) NSP1 are deficient in these characteristics.56 Co-speciation may also account for the selective pressure that leads to the non-random introduction of gene 5 RNAs into progeny viruses formed upon co-infection of animals or cell lines.56,57 Despite the marked overall sequence variation among group A NSP1 proteins, two shared features suggest that their structures are highly conserved. First, all NSP1 proteins possess an N-terminal domain that consists of an identical arrangement of cysteine and histidine residues, generally similar to those that constitute RING fingers (Figure 2).58–61 The putative RING-finger domain is present not only within NSP1 proteins of all group A rotaviruses, but also those of the evolutionarily distant group C rotaviruses.62 The prediction that NSP1 contains a RING-finger domain is supported by the fact that the protein binds to zinc.63 The second shared feature of NSP1 proteins is that they all exhibit a similar distribution of proline residues throughout their primary amino acid sequences.54 This implies that a shared set of forced turns in the α-carbon backbone of the

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Figure 2. RING-finger domain of rotavirus NSP1. (A) Schematic representation indicating the general location of structural and functional domains in NSP1: RING-finger domain, RFD; cytoplasm localization domain, CytoLS; RNA-binding domain, RNA-BD; IRF-binding domain, IRF-BD; and the recognition epitope of the C19 antibody used in detecting wtNSP1, α C19. (B) Alignment of the putative RNA-finger domain of group A and C rotaviruses, demonstrating conservation of cysteine and histidine residues (boxed). Included are domains of human (Wa, DS1, AU-1, and Bristol), porcine (OSU), bovine (RF), and simian (SA11) viruses, as well as viruses belonging to group A (Wa, DS1, AU-1, OSU, RF, and SA11) and group C (Bristol). The conserved cysteine and histidine residues of the NSP1 RING domain are boxed. (C) Predicted organization of the RING-finger domain of NSP1, including possible cross-bracing between the two zinc fingers. The signature cysteine-histidine motif shared by NSP1 proteins is shown in brackets.

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proteins, thus supporting the prediction that all NSP1 proteins have similar architectures. Immunofluorescence and cell fractionation studies indicate that wildtype (wt) NSP1 is distributed throughout the cytoplasm of infected cells in association with cytoskeletal components.64 Deletion mutagenesis has identified a cytoplasmic localization signal in NSP1 of ∼100 amino acids that is positioned immediately downstream of the RING-finger domain (Figure 2). Mutant forms of NSP1 lacking the localization signal accumulate in the nucleus, a characteristic of undefined significance.64 Separate studies have determined that NSP1 has an affinity for single-stranded RNA.63,64 One analysis of its RNA-binding an activity suggested that it specifically recognizes a common element near the 5′-end of rotavirus mRNAs.64 The function of the RNA-binding activity is not known, but hints at additional roles for NSP1.

3. NSP1-MEDIATED EVASION OF IFN SIGNALING Early studies indicated that the growth of rotaviruses is restricted in IFNtreated cells,65 albeit to a lesser extent than that observed for vesicular stomatitis virus (VSV) and reovirus, which are noted for their increased susceptibility to the antiviral effects of IFN.66 In related studies, investigators determined that pretreatment of calves or piglets with IFN suppresses rotavirus replication and/or ameliorates disease severity.67,68 More importantly, Vanden Broecke et al.69 found that IFN production was delayed in newborn calves infected with low doses of rotavirus, even though this initial lag period was characterized by severe but transient diarrhea. In contrast, calves infected with high doses of virus produced IFN without delay and remained free of severe diarrhea. These studies were the first to raise the possibility that, although rotaviruses are susceptible to IFN, these viruses may have a mechanism to suppress its signaling activity during early stages of infection.

3.1 Contribution of NSP1 to Virus Spread Initial evidence that the rotavirus product NSP1 is an antagonist of IFN signaling came from the analysis of rotavirus variants with genomes that

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included dsRNA segments of atypical size (Figure 1).70,71 Such variants have been recovered from rotavirus-infected animals and immunocompetent and immunocompromised children.72,73 Rotaviruses with similar atypical segments have also been generated by serial passage of the virus at high multiplicity of infection (MOI) in highly-permissive cell lines.74 Molecular analysis has shown that the atypical segments result from an intragenic sequence rearrangement, most frequently taking the form of a head-to-tail sequence duplication, and less frequently, a sequence deletion.71 Such rearrangements have been detected for genome segments encoding NSP1 (gene 5), VP6 (6), NSP3 (7), NSP4 (10), and NSP5 (11).75,76 In the case of rotavirus variants with gene 5 rearrangements, the sequence alteration typically interrupts the ∼500 amino-acid open reading frame (ORF) of NSP1 (Figure 1). Thus, such viruses only produce C-truncated forms of the protein (NSP1∆C).70,77 The most extreme gene 5 rearrangements described so far encode forms of NSP1∆C that are only 40 or 50 amino acids in length.78 The fact that rotaviruses with gene 5 rearrangements (NSP1-defective rotaviruses) are viable in cell culture indicates that NSP1 has a non-essential role in virus replication.70,78,79 Recent experiments performed with small interfering (si)RNAs which knockdown the expression of NSP1 in rotavirus-infected cells, confirmed that NSP1 is a non-essential viral protein in cell culture.80 Although NSP1 is not required for virus replication, the inability of the virus to produce wtNSP1 is associated with changes in growth characteristics. Most notably, NSP1-defective rotaviruses have small-plaque phenotypes in comparison to their wild-type counterparts.64,70 Knockdown of NSP1 expression using siRNAs similarly converts the large-plaque phenotypes of wild-type rotaviruses into the small-plaque phenotypes seen for NSP1-defective strains.81 In some highly-permissive cell lines (e.g. monkey kidney MA104 epithelial cells), NSP1-defective rotaviruses and their wild-type counterparts grow equally well.70 However, in other cell lines (e.g., rhesus fetal lung diploid FRhL2 and human colon Caco-2 epithelial cells), rotaviruses expressing wtNSP1 can grow to titers ten-fold greater than viruses expressing C-truncated NSP1 (NSP1∆C).82 The importance of NSP1 in cell-to-cell spread of the virus was indicated by studies examining the growth characteristics of a virus inoculum prepared by mixing equal amounts of an NSP1-defective rotavirus with its

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wild-type counterpart.74 Infection of cells with the mixed inoculum at low MOI produced a progeny virus population with gene 5 RNAs encoding wtNSP1. In contrast, infection at high MOI produced a progeny virus population with rearranged gene 5 RNAs encoding NSP1∆C. These results indicated that under conditions in which the virus must spread from cellto-cell to achieve complete infection of a cell population, vis-à-vis, a low MOI infection, a strong selection exists favoring the multiplication of viruses encoding wtNSP1. On the other hand, when the virus is not required to spread from cell-to-cell to fully infect a culture (high MOI), the selective pressure to maintain the expression of wtNSP1 is lost. Additional information on the importance of NSP1 to rotavirus spread was gained from sequencing the gene 5 RNAs of rhesus rotavirus (RRV) passaged serially at high or low MOI in highly permissive cell lines.83 Unlike many strains of rotavirus (e.g. SA11-4F), serial passage of RRV at high MOI does not lead to gene 5 rearrangements.83 Instead, such passage conditions introduce stable mutations into the 3′CS of the gene 5 RNA. These mutations decrease the activity of the translational enhancer in the gene 5 3′CS,40 leading to reductions in the expression of wtNSP1 in RRV-infected cells. In contrast, the gene 5 3′CS of RRV passaged at low MOI was unchanged, therefore retaining the translational enhancer activity of wild-type virus.83 Collectively, studies with rotaviruses that contain rearranged gene 5 RNAs or a defective gene 5 translation enhancer reveal a link between NSP1 and the capacity of rotaviruses to spread efficiently from cell-to-cell and, in some cell types, to replicate to high titers.

3.2 Suppression of IFN Expression by NSP1-Producing Virus Direct evidence that NSP1 interferes with IFN signaling was gained from studies examining whether media recovered from cells infected with wildtype rotavirus or NSP1-defective rotavirus contain factors that interfere with replication of VSV-GFP, an IFN-sensitive virus engineered to express green fluorescent protein (GFP).82 The analysis demonstrated that media of cells infected with wild-type rotavirus did not inhibit VSV-GFP replication, revealing that it lacked IFN. On the other hand, media recovered from cells infected with NSP1-defective rotavirus prevented VSV-GFP

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replication, suggesting that the defective virus had induced the expression of IFN. This assumption was confirmed using a neutralizing IFN-β antibody, which abrogated the inhibitory effect of the media on VSV-GFP replication.82 Together, these findings show that the expression of wtNSP1 by rotavirus mediates a process by which the virus avoids activating the IFN-signaling pathway of the host.

3.3 NSP1-Induced Degradation of IRF Proteins An important discovery in the elucidation of NSP1 function came from Graff et al.,84 who determined by using a yeast two-hybrid assay system, that bovine and murine rotavirus NSP1 proteins interact with IRF-3. Additional support of this interaction was provided by assays showing that recombinant GST-tagged IRF-3 could pull-down NSP1 from rotavirus-infected cell lysates.84 Subsequent co-immunoprecipitation experiments by Barro and Patton81 directly demonstrated the existence of endogenous NSP1-IRF-3 complexes within infected cells. The impact of the NSP1-IRF-3 interaction was revealed by Western blot assays, which compared the endogenous levels of IRF-3 in cells infected with wild-type rotaviruses versus NSP1-defective rotaviruses.81 The examination showed that the expression of wtNSP1 correlated with a near complete loss of IRF-3 from infected cells, while expression of NSP1∆C stimulated little decrease in IRF-3 levels. Indeed, infection of cells with virus encoding NSP1∆C led to the activation of IRF-3, as judged from assays showing that IRF-3 in these cells underwent dimerization and nuclear translocation.81 Evidence that NSP1 was directly responsible for the loss of IRF-3 was provided by transient expression experiments in which wtNSP1 or NSP1∆C was expressed with IRF-3.81 The results showed that wtNSP1, but not NSP1∆C, induced IRF-3 degradation. Treatment of cells coexpressing wtNSP1 and IRF-3 with the inhibitor MG132 reversed IRF-3 degradation, indicating that its turnover was mediated by proteasomes.81 Members of the IRF family share certain structural elements, including a common DNA-binding domain (Figure 3).10 The presence of such shared features raised the possibility that, in addition to IRF-3, NSP1 might target other members of the family. Initial experiments addressing

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Figure 3. IRF family members targeted by NSP1. Full-length NSP1 mediates the proteasomal proteolysis of IRF-3, IRF-5, and IRF-7 in rotavirus-infected cells. The general location of functional and structural domains in IRF targets are indicated: tryptophan-pentad repeat DNA-binding domain, DBD; proline-rich region, PRO; IRF interactive (dimerization) domain, IAD; autoinhibitory domain, ID; proline-glutamate-serine-threonine-rich domain, PEST; constitutive activation domain, AD; region similar to domain B in dwarfin family proteins, DWB; nuclear localization signal, NLS; nuclear export signal, NES; and phosphorylation-mediated response domain, PRD.11,12,14,97–100 The DBD is the single domain shared by all IRF proteins, making it the potential common target of the broadspectrum activity of NSP1. Many IRF proteins including IRF-3, IRF-5, and IRF-7 not only form homodimers, but also heterodimers (e.g., IRF-3-IRF-7 dimers). This characteristic indicates the presence of shared interactive elements among some IRF proteins, which may also serve as recognition targets for NSP1.

this possibility showed that cells infected with wild-type rotavirus contained little or no IRF-7, the essential transcription factor required for type I IFN expression.82 What little IRF-7 that was present in cells could be coprecipitated with wtNSP1, indicating a physical interaction. In comparison to the relative lack of IRF-7 in cells infected with wild-type rotavirus, cells infected with rotavirus encoding NSP1∆C showed high levels of IRF-7 accumulation.82 As cells infected with wild-type rotavirus also lack IRF-3, a transcription factor that upregulates IRF-7 expression, it was not clear whether the absence of IRF-7 in infected cells expressing wtNSP1 stemmed from decreased levels of IRF-7 synthesis or from the degradation of newly-made IRF-7, or from a combination of the two. To resolve this issue, wtNSP1 or NSP1∆C was transiently expressed along with IRF7, and the level of IRF-7 in the transfected cells was examined by Western blot assay. The analysis showed that wtNSP1, but not NSP1∆C, induced degradation of IRF-7.82 Furthermore, the degradation was reversed using

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the proteosome inhibitor MG132. Thus, NSP1 directs the proteolysis of IRF-3 and IRF-7 through mechanisms that appear indistinguishable. The capacity of NSP1 to degrade IRF-7 may be critical for rotavirus replication in some trafficking cells (macrophages and plasmacytoid dendritic cells) in which IRF-7 is typically expressed in a constitutive manner. In fibroblasts, where IRF-7 expression is stimulated in an IRF-3-dependent manner, the combined capacity of NSP1 to degrade an upstream activator of IRF-7 expression (i.e., IRF-3) as well as the IRF-7 product itself defines NSP1 as a remarkably strong antagonist of the IFN-signaling pathway. The effect of NSP1 on a third IRF family member, IRF-5, has also been examined using transient expression assays.82 IRF-5 is constitutively expressed in B lymphocytes and dendritic cells and it plays a role not only in the expression of type I IFN but also in cell cycle regulation and apoptosis (Figure 3).6,85 Co-expression of wtNSP1 with IRF-5 resulted in the degradation of the latter, which as determined using MG132, was mediated by proteasomes. Replacement of wtNSP1 with NSP1∆C reversed the degradative effects on IRF-5. Thus, NSP1 brings about the degradation of multiple members of the IRF family through a similar, if not identical mechanism. The characteristics of NSP1 define the protein as a broadspectrum antagonist of IRF function and suggest that the protein can recognize an element shared by many IRF family members. Several rotaviruses encoding wtNSP1 have been shown to induce the degradation of one or more of the IRF proteins, including simian SA11related (SA11-4F and SA11-30-19), rhesus RRV, and bovine UK, NCDV and B641 strains.81,82,86,87 The porcine OSU strain is an exception in that, despite encoding a full-length NSP1 with an intact RING-finger domain, infection with OSU does not induce IRF-3 degradation in either human or monkey cell lines.86 Transient co-expression studies have confirmed that the expression of recombinant OSU NSP1, even at high levels, does not cause the loss of IRF-3. Additional experiments have indicated that OSU NSP1 fails to interact with human or monkey IRF-3, or does so relatively weakly.86 These results raise the possibility that human and monkey IRF3 proteins are not appropriate substrates for analyzing the function of NSP1 encoded by the porcine OSU strain. It is also possible that OSU NSP1 is functional as an IFN antagonist, but does so not by targeting IRF-3 for degradation, but by targeting other members of the IRF family

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(e.g., IRF-7). A final possibility is that NSP1 encoded by the laboratoryadapted strain of rotavirus OSU used in earlier studies86 has acquired a mutation, disabling its function.

3.4 NSP1 Domains Linked to IRF Degradation Initial attempts to locate determinants within NSP1 which are important for its interaction with IRF-3 were performed using a yeast two-hybrid system.84 These experiments showed that a C-terminal 163-amino acid (aa) fragment of NSP1 interacts with IRF-3, although not as strongly as wtNSP1. In contrast, a 200-aa N-terminal fragment, which includes the RING domain (residues 42-79), did not interact with IRF-3. Surprisingly, an NSP1 mutant with only an N-terminal 63-aa truncation failed to interact with IRF-3, despite the fact that the protein retained its C-terminal 163 amino acids. These results suggested that the IRF-3 interactive domain is located at the C-terminus of NSP1, but that the function of this domain is influenced by upstream residues. This latter point has been reaffirmed by pull-down assays performed with mutant forms of NSP1 that contained site-specific changes to the conserved cysteine and histidine residues of the RING domain.86 Thus, the structural integrity of the RING domain of NSP1 appears to be crucial to the function of the protein, including the interaction of NSP1 with its IRF target proteins. As might be anticipated from the importance of the RING domain on NSP1-IRF-3 interactions, mutations of the conserved cysteine and histidine residues of the RING domain also prevent NSP1 from inducing the degradation of IRF-3.86 Studies of NSP1∆C made by the rotavirus variant SA11-5S have shown that this truncated protein is defective as an IFN antagonist even though it lacks only the last 17 amino acids of wtNSP1.81,82 Specifically, transient expression assays have demonstrated that this small C-terminal deletion renders NSP1 incapable of (a) preventing IFN expression, (b) inducing IRF-3 or IRF-7 degradation, or (c) suppressing the activation of IFN-α or -β promoter elements. These findings suggest that the IRF binding domain is located at the C-terminus of NSP1. The results also reveal that although the RING-finger domain may be critical to NSP1 activity, but in the absence of an intact C-terminal region, it cannot subvert IFN signaling.

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3.5 NSP1 Ubiquitin-Dependent Turnover Many factors may affect the capacity of NSP1 to function as an IFN antagonist, other than those directly tied to its primary sequence. Perhaps chief among these is the level of NSP1 that accumulates in infected cells, a parameter influenced by the rate of gene 5 transcription, the stability and translational efficiency of the gene 5 mRNA, and the stability of NSP1. Although the extent to which variations in these parameters account for strain-to-strain differences in the ability of rotavirus to grow and to suppress innate immune responses remains unresolved, it is clear that steadystate levels of NSP1 do vary among rotavirus-infected cells.86,88 The fact that gene 5 RNAs produced by some rotavirus strains contain mutations in their 3′-terminal translation enhancer element provides one explanation for the variations seen in the intracellular levels of NSP1.70,83 In comparison to other rotavirus proteins, the half-life of NSP1 is relatively short (∼90 min).88 Experiments using the proteasome inhibitors MG132 and clasto-lactacystin-β -lactone (CCL) indicate that the proteasomes mediate turnover of NSP1, a situation analogous to the IRF targets.86,88 Ubiquitin groups, ligated to the lysine residues of proteins, serve as signals for recognition and degradation by proteasomes.89 The degree to which the extensive sequence variation noted for NSP1 influences the susceptibility of the protein to ubiquitination, and thus turnover rates, is unknown. However, this degradation provides another possible explanation for differences seen in intracellular levels of NSP1. Interestingly, proteasomal degradation of NSP1 is slowed by the presence of other viral proteins in the cell.88 Perhaps this is a nonspecific competitive effect resulting from an increased number of ubiquitination/proteasome targets in cells containing other viral proteins, since they too are subjected to proteasomal degradation.90

3.6 NSP1 as a Possible Homolog of E3 Ubiquitin Ligases Ubiquitination places signals on a protein that promote its recognition and degradation by proteasomes.89 Ligation of the 76-aa ubiquitin polypeptide onto a target protein involves three enzymes.91 The first of these, the E1 ubiquitin-activating enzyme forms a covalent linkage with ubiquitin in an

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ATP-dependent manner. The ubiquitin moiety is subsequently transferred from E1 onto the E2 ubiquitin-conjugating enzyme. The E3 ubiquitin-protein ligase E3 forms a bridge between a charged E2 enzyme and a target substrate, thereby mediating the transfer of the ubiquitin moiety onto lysine residues of the target. Iterative rounds of this process generates unique polyubiquitin chains on the target that act as signals to trigger proteasomal proteolysis. A common feature of a large number of E3 ligases is the presence of a RING-finger or closely related zinc-binding domain (e.g., PHD).89,92 The function of the RING domain appears connected to protein-protein interactions that drive the dimerization of the E3 ligase with itself or with other co-activator proteins needed in forming active E3 ligase complexes. The presence of a RING-finger-like domain in NSP1 combined with the capacity of NSP1 to bind and induce the degradation of IRFs in a proteasome-dependent manner suggests that NSP1 has E3 ligase activity. The ability of the C-terminus of NSP1 to interact with IRF proteins would provide specificity to this activity. It remains unclear whether NSP1 has the capacity to interact with E2, which would be an expected feature of the protein if it truly functions as a viral E3 ligase. A limited number of other viral proteins with RING-finger like domains have been identified to have activities similar to E3 ligases. These include KSHV K3 and K593 and poxvirus p2894 proteins. Although many viruses utilize E3 protein ligases to manipulate the host machinery in favor of their own replication, typically the E3 enzymes are of host origin.95

4. SUMMARY AND PERSPECTIVES Existing studies have comfortably established rotavirus NSP1 as an antagonist of IFN signaling. This property is consistent with earlier reports indicating that NSP1 affects virus growth, spread, and virulence. NSP1 subverts IFN expression by inducing the proteasomal proteolysis of multiple members of the IRF family. The presence of a RING-finger domain in NSP1, combined with its manipulation of the proteasomal pathway to degrade the IRF proteins, suggests that NSP1 is an E3 ubiquitin ligase. Proof of such an activity awaits evidence that NSP1 not

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only interacts with IRF proteins, but also with an ubiquitin-activated E2 enzyme, and that these interactions result in the ubiquitination of IRF targets. Yet to be defined is the nature of the determinant in IRF proteins that is recognized by NSP1. Elucidating whether this determinant is possessed by all IRF family members, or only a subset, would prove useful in defining the full potential of NSP1 in subverting innate and adaptive immune responses. Equally important is establishing whether variations in the Cterminal sequences of the NSP1 proteins alters their spectrum of IRF targets and influences the host range of the virus. Although much remains unclear about the function of NSP1, its capacity to induce the degradation of multiple IRF targets makes it a remarkably potent broad-action IFN antagonist. There is no evidence that the RNA-binding activity described for NSP1 is directly linked to IRF degradation. Nonetheless, this activity may play a role in undermining innate immune responses, possibly by sequestering viral RNAs that might otherwise act as triggers for the activation of RNA-dependent antiviral pathways. For example, the interaction of NSP1 with viral RNAs may prevent the RNA sensors of TLR3, RIG-I and PKR from serving as effective sentinels of rotavirus infection. Of the twelve rotavirus proteins, eight have RNA-binding activity, leaving open the possibility that viral proteins other than NSP1 may impede innate immunity by sequestering viral RNAs.96 On average, rotavirus infections cause approximately 1000 to 2000 deaths each day. The impact of these viruses on human health has stimulated extensive efforts towards the development of rotavirus vaccines. Insights gained on the function of NSP1 suggest that through the mutation of this protein, it may be possible to generate attenuated human rotavirus strains that would be ideal vaccine candidates.

ACKNOWLEDGMENT We thank Sarah McDonald for her assistance in the preparation of this review. The authors’ work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

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Index

Apolipoprotein E and B (Apo E; Apo B), 480, 482 Apoptosis inducing factor (AIF), 47, 638–640 Astrocyte response, 612 Atelectasis, 159 Atg5, 219–222, 531, 532 Atg8, 219, 531, 532 Atg12, 219, 221, 222, 531, 532 ATF-2/c-Jun, 116, 350 Autocrine or paracrine type I IFN signaling, 140 Autophagosomes, 219, 222, 223, 532 Autophagy, 209, 211, 218–223, 531, 532, 535 Autophagy-related genes, 218

Activating transcription factor 3 (ATF3), 508 Acute encephalomyelitis, 602 Acute viral infection, 60, 433 ADAR, 10, 286, 447, 448, 454, 657 Aggresomes, 227 Alb-urokinase plasminogen activator, 482 Alphavirus, 601–606, 611, 617, 618, 620 Alveolar fluid, 134 Alveolar macrophages, 135, 136, 162, 165, 176, 189, 309, 377 Akt, 119, 120, 124, 125, 277, 321, 333–337, 509, 540, 632, 633, 644 Anaphylatoxin complement fragments, 233 Anosmia, 232 Antigenic drift, 133, 146, 582 Antigenic-presenting cells, 404 Antigenic shift, 133 Antisense, 91, 94, 330 Antiviral immunity, 131, 132, 177, 178, 215, 218, 223, 419, 504 Apaf-1, 47, 639

Bag-1, 508, 509 Bayesian modeling, 4 Bax, 48, 307, 405 β-catenin, 508, 532, 533 Bcl-2, 35, 36, 405, 507, 509, 527, 609, 610, 640, 644 Bcl-family proteins, 507, 609 Bcl-xL, 337, 527, 644

679

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680

Index

BH3 (Bcl-2 homologous domain 3), 509 Bid, 405, 513, 639, 640 Bidirectional spread, 417 Biomarkers, 4, 5, 12, 20, 21 Blood-brain barrier, 214, 232, 415, 417, 603 Bronchiolitis, 157–159, 177, 185, 186, 188, 194, 195, 296 Bronchus associate lymphoid tissue (BALT), 142 Bunyaviridae, 248, 257, 263 Bystander injury, 611, 613 Bystander killing, 71 cAMP, 35, 37, 325, 328, 329 Cannabinoids, 235, 236 Caspase-3, 36, 37, 47, 327, 332, 333, 405, 514, 540, 639, 640, 643 Caspase-7, 332 Caspase-8, 36, 47, 48, 405, 638–641 Caspase-9, 47, 48, 335, 405, 639, 640 Caspase-10, 36 Caspase-recruiting domains (CARDs), 348 Cathepsin L, 304, 632 CCL2, 13, 37, 38, 194, 195, 428, 558, 561 CCL3, 38, 194, 195, 427, 428 CCL5, 13, 38, 166, 190–195, 203, 400, 427, 428, 561 CCL8, 554, 559, 560 Cell-mediated immune response, 71, 76 Cell surface marker, 38, 145 Cell surface receptors, 210, 476, 629

Cellular tropism, 401, 606 c-FLIP, 36, 554, 560 Chemokine receptor, 12, 68, 175, 398, 402, 404, 415, 427, 477 CCR5, 9, 11, 13, 34, 60, 61, 65, 67, 74, 194, 398, 400, 404, 415, 427, 428, 433, 477 CCR5-∆32 deletion, 65 CXCR3, 22, 402, 415, 427, 501 CXCR4, 9, 11, 34, 60, 65 Childhood morbidity, 159 Cholesterol trafficking, 38 Chronic hepatitis, 471, 473, 474, 486–489 Cilia, 134 Cirrhosis carcinoma, 446, 471, 473, 474, 485 Clades, 417 Classical pathway, 169, 480, 559 Coagulation disorders, 371, 380, 382 Collectin, 135, 136, 210 Complement system, 428–430, 480, 549, 550, 559 Congenital heart disease (CHD), 157, 160, 161 Connective tissue growth factor (CTGF), 510 COPD, 186, 187, 201–203 Conventional DCs (cDCs), 132, 133, 139–141 Coronavirus, 18, 219, 295–303, 321, 322, 531, 532. See also SARS-CoV Coxsackie and adenovirus receptor (CAR), 498, 499, 506, 526, 538

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Coxsackievirus B (CVB), 496, 498, 499, 502–508, 512, 513, 515, 516, 525, 526, 541 CVB3, 495–510, 512–515, 526, 527, 530, 532–536, 538–541 CVB4, 496, 498, 503, 505, 511 3A protein, 311, 312 2B protein, 514, 515 VP1 capsid protein, 497, 499, 526, 577 CpG motif, 211, 213 CTL response, 62, 73, 403 CXCL1, 194, 558, 561 CXCL8, 38, 190, 192, 194, 195, 501 CXCL9, 427, 501 CXCL10/IP10, 22, 38, 166, 190–194, 402, 403, 415, 423, 425, 427, 428, 501–503, 509, 554 Cyclooxygenase, 235, 333 Cyr 61, 508, 510 Cytochrome c, 47, 336, 405, 638–640, 643 Cytochrome c oxidase (COX5B/COX6B), 405 Cytokine and receptor IL-1α, 500, 504 IL-1β , 34, 43, 176, 178, 351, 377, 500, 506, 511, 512, 559, 616, 618 IL-1 receptor A (IL-1RA), 212, 377, 618, 620 IL-1 receptor associated kinase (IRAK), 212

681

IL-4, 48, 144, 200, 272, 275, 277, 459, 479, 500 IL-6, 34, 43, 44, 49, 62, 169, 176, 178, 283, 284, 309, 325, 330, 352, 377, 421, 425, 500, 501, 511, 557, 558, 560, 563, 583 IL-8, 34, 49, 136, 161, 166, 169, 176, 305, 309, 311, 333, 479, 501, 511, 539, 540, 557, 561 IL-10, 13, 35, 39, 43, 48, 49, 176, 178, 275, 277, 283, 330, 377, 457–459, 500, 501, 505, 583 IL-12, 13, 35, 39, 43, 48, 49, 176, 178, 275, 277, 283, 330, 377, 456–459, 500, 501, 505, 583 IL-12 Rβ 1, 500 IL-13, 166, 186, 194, 200–203, 511 IL-18, 43, 235, 500 Cytokine regulation, 34, 193, 272 Cytopathic effect (CPE), 295, 304, 313, 326, 327, 332, 404, 508, 527, 533, 539, 590 Cytoskeleton, 36, 310–312, 485, 533, 557, 629 DAP5/p97/NAT1, 513 Decay accelerating factor (DAF), 498, 499, 526, 538, 540 Decoy death receptor DcR1/2, 640 Defensins, 131, 133, 135, 136, 188

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682

Index

Dengue virus, 306, 309, 547–550, 552–554, 556–559, 561–564, 566 Dicer, 10, 85–87, 89–96, 232 DNA binding domain, 420, 666, 667 DNA fragmentation, 327, 333, 335 Dominant negative, 122, 123, 168, 305, 330, 362, 422, 540, 640, 642 3-D organoid model of human, 590 Drosha, 85, 86, 89, 90, 92 Double-layered particles (DLP), 658 dsRNA-dependent protein kinase (PKR), 22, 91, 112, 117, 118, 139, 193, 210, 219, 220, 236, 256, 286, 325, 349, 352, 355–357, 402, 421–423, 435, 447, 448, 480, 503, 554, 636, 656, 657, 672 Eastern equine encephalitis virus (EEV), 602 EBNA1, 222 Ebola virus (EBOV), 94, 345–354, 356–359, 361–363, 371–373, 375, 377–380, 382–385, 657 Ectodomain, 146, 215 Endocytosis, 33, 40, 41, 113, 114, 322, 393, 424, 473, 539, 629, 630, 635 Endoplasmic reticulum (ER), 42, 173, 277, 301, 311, 326, 393, 504, 514, 515, 660 Endosomes, 113, 190, 191, 209, 212, 214, 222, 629, 656 Eicosinoids, 234, 235 eIF4GI, 504, 513

eIF4GII, 513 Encephalomyocarditis virus, 221 Endothelial damage, 403, 547, 552, 553 Engulfed cargo, 218 Envelope glycoproteins, 450, 472, 473, 476, 477, 615 Epidermal growth factors (EGFR), 631, 633 Epithelial apoptosis, 160 Epizootic, 229 Epstein Barr virus (EBV), 87, 95, 222, 223, 457 E1-ubiquitin-activating enzyme, 528, 529, 670 E2-ubiquitin-conjugated enzyme, 528, 529, 671 E3-ubiquitin ligases, 670 Execution, 91, 219 Extra cellular signal regulated kinase 1 and 2 (ERK 1/2), 284, 328 Ex vivo model, 553, 590 Fas, 36, 40, 42, 43, 61, 143, 217, 220, 278, 306, 415, 433, 507, 554, 616, 638, 640 Fas apoptotic inhibitory molecule (FAIM), 306 Fas-associated death domain (FADD), 217, 220, 554, 560, 561, 638, 640 Filoviruses, 345, 346, 350, 353, 356, 363, 372, 375, 377, 379, 381, 383, 385 Flaccid paralysis, 392, 418

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Flaviviridae, 217, 391, 416, 445, 471, 472, 549 Focal adhesion kinase (FAK), 509 Formalin inactivated vaccine, 177, 204 Fulminant hemorrhagic disease, 371 Gamma delta T cells (γδ T cells), 198 Gastroenteritis, 575, 576, 579–584, 590, 591, 593, 594, 658 Gene expression omnibus (GEO) database, 3 Glial cell response, 611 Glycogen synthase kinase 3β (GSK 3β ), 333, 335, 336, 508, 633 Glycosylation, 71, 133, 215, 302, 394 Guanosine nucleotide analogue (loxoribine), 214 H539E, 215 Hantaviruses, 247–260, 262–264 G1 cytoplasmic tail, 258–260 pulmonary syndrome, 247, 249, 250 structure and genome, 248 Heat shock proteins, 295, 308, 537 Helper-T cell, 427 Hemagglutinin protein, 216 Hemorrhagic fevers (VHFs), 375 Hemorrhagic fever with renal syndromes (HFRS), 247, 249–251, 253, 255 Hepatitis C virus (HCV), 91, 94, 239, 261, 296, 227, 325, 445–462, 471–489, 657 HCV-like particles, 450, 476

683

Hepatocellular carcinoma (HCC), 445, 446, 471, 473, 474, 485 Herpes viruses, 88, 477, 532 Heterosubtypic immunity, 131, 146, 147 High-affinity immunoglobulin G Fc receptor, 476 High-density lipoprotein (HDL), 450 Highly active antiretroviral therapy (HAART), 36 HIV-1, 3, 4, 6–11, 15, 18, 21–23, 34, 38, 40, 42, 43, 45, 65, 66, 69–72, 83, 84, 89–96, 326, 356, 530, 537, 538, 540 gp120, 48, 70, 71, 76, 84, 326, 477 Nef, 31, 32, 38–43, 49, 74, 84, 89, 90, 92, 94 specific cellular immunity, 31 Tat, 9, 31–38, 42, 48, 49, 84, 90, 93–96, 356, 530 Virulence, 64, 66, 231, 273, 361, 362, 393, 394, 398, 401, 402, 420, 423, 426, 435, 498, 548, 582, 605, 607, 671 Host cell signaling pathway, 31, 349 Host immune responses, 59, 60, 64, 66, 253, 273, 275, 280, 407, 416, 424, 427, 434, 436, 445, 481, 577, 614 Host pathogen interaction, 296, 311 Host signature, 3, 4, 24 Human alpha defensin 1 (HNP-1), 136

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684

Index

Human beta defensin 1 (HBD1), 136 Human histo-blood group antigens (HBGAs), 577, 584–587, 591 Human leukocyte antigen (HLA), 13, 41, 42, 65, 67, 68, 73, 74, 404, 406, 407, 445, 449, 453–455, 460, 461, 484, 505, 506 Humoral immune response, 62, 69, 303, 371, 382, 449, 450, 484 Hyperinflation, 159 Hypermutation, 23, 286 Hypervariable region 1 (HVR-1), 450 ICAM1, 558 IFIT1, 563 IFITM1, 512, 563 IFN-α/β, 21, 22, 480 IFN-γ, 13, 22, 34, 65, 68, 71, 74, 75, 137, 144, 147, 234–236, 277, 282, 287, 309, 377, 400–402, 404, 419, 420, 423, 433, 435, 451, 453, 455, 483–485, 500–506, 511, 512, 565, 615, 616 IFN-β promoter stimulator (LPS-1), 217 IFN regulatory factors (IRFs), 22, 171, 172, 408, 420, 436, 671 IFN signaling pathway, 262, 281, 349, 427, 563, 655–657, 661, 666, 668 IFN-stimulated genes (ISGs), 117, 253, 255, 256, 258, 263, 406–408, 425, 426, 447, 448, 480, 481, 483, 485, 488, 502, 503, 507

IGTPase, 503 IκB-kinase β (IKKβ ), 121, 122, 170, 217, 641, 642 Immune antagonism, 132 Immunocompromise, 132, 231, 239, 392, 396, 579, 582, 645, 664 Immunodominant epitopes, 201 Immunotherapy, 617, 619 Indoleamine-pyrrole 2,3-dioxygenase (IDO), 404, 548, 551, 558, 564–566, 568 Inducible nitric oxide synthase (iNOS), 161, 504, 617 Inflammatory cytokines, 36, 43, 48, 62, 122, 132, 140, 143, 169, 176, 211, 212, 234, 273, 283, 302, 324, 347, 363, 378, 379, 396, 404, 422, 425, 433, 436, 501, 504, 505, 537, 539, 540, 552, 557, 563, 564 Inflammatory mediators, 160, 232, 233, 404, 612, 613 Influenza virus, 94, 109, 111, 115–125, 131–133, 135–149, 220, 326, 353, 355, 406 Inhibitor of apoptosis proteins (IAPs), 48, 634, 639–641 Inhibitory protein κ B kinase (IKK) ε, 116, 118–121, 167, 169, 170, 213, 634, 641 Innate and adaptive immune responses, 59, 229, 269, 270, 313, 345, 415, 436, 614, 672 Innate antiviral immune response, 222, 476

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Index

Innate cellular effectors, 137 Innate immune cells, 185, 186, 273, 379, 420, 446 Innate immune system, 89, 132, 133, 188, 209, 210, 223, 377, 428, 449 Interferon (IFN)-inducible protein, 210, 396 Intermediate subviral particles (ISVPs), 629, 630, 644 Internal ribosomal entry site (IRES), 497, 513 In vitro replicon systems, 590 IPS-1, 116, 118, 217, 222, 233, 253, 348, 354, 355, 448, 639, 643 IP10/CXCL-10, 501. See also chemokine IRAK1, 164, 564 IRAK4, 213, 564 ISRE, 172, 259, 260, 281, 282, 349, 350, 420, 423, 425, 426, 656, 657 JAK-STAT pathway, 22, 115, 174, 178, 656 Junctional adhesion molecules, 629 Jun-N-terminal kinase (JNK), 36, 37, 43, 44, 47, 123, 323, 324, 327, 333, 334, 337, 537, 540, 638, 639, 643, 644 Kruppel-like factor (KLF), 305 KSHV, 87, 88, 95, 657, 671 Kynureninase (KYNU), 558, 560, 566

685

La autoantigen, 499 LCMV, 75, 221, 282, 285, 286, 456, 458, 459, 657 Light chain 3 (LC3), 219, 531, 532 Lipoxygenase, 195, 235 Liver activation regulated chemokine (LARC), 478, 479 Long-term non-progressors (LTNPs), 64, 76 Low-density lipoprotein, 37, 480 Low molecular weight polypeptide-2 (LMP-2), 407, 484 Lysosome, 41, 213, 218, 220–222, 531, 535, 629 Macrophage derived chemokine (MDC), 501 Macrophage inflammatory protein-1 α (MIP-1α), 43, 48, 500 MAP kinase/ERK kinase (MEK), 122, 123, 125, 331, 537–539, 632, 633, 635 Marburg virus (MARV), 345, 346, 371–373, 375, 378, 382–384 Matrix metalloproteinase (MMP), 34, 310, 510 Matrix protein 1 (MP1), 222 mBNip 21, 508 MCP-1, 13, 22, 37, 501, 511 MCP-2, 501, 559, 563 MCP-3, 13, 501 MCP-5, 427, 428, 501 MEKK, 643 Melanoma differentiation associated gene-5 (MDA-5), 116, 138, 166,

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686

193, 348, 354, 422, 425, 448, 559 MHC-related polypeptide, 484 Microarray, 20, 21, 96, 255, 258, 295, 304, 352, 357, 391, 392, 398, 405, 407, 408, 420, 430, 471, 474, 478, 481, 482, 485, 489, 495, 496, 500, 501, 509, 510, 515, 516, 553, 580 Microautophagy, 218 Microglia responses, 38, 612, 617 MicroRNA (miRNA), 10, 83, 85–98, 232, 516 Mimic poly I:C, 215 Mitochondria antiviral signaling (MAVS), 141, 167, 169, 217, 253, 348, 448 Mitochondria outer membrane permeabilization (MOMP), 405 Mitogen-activated protein (MAP), 35, 123, 212, 284, 305, 321, 323, 325, 345, 362, 363, 513, 534, 536, 537, 636 Mitogen-activated protein kinase (MAPK), 35, 37, 40, 44, 46, 47, 111, 122, 123, 284, 305, 306, 321, 323–333, 337, 338, 534, 536, 537, 540, 564, 636 mRNA metabolism, 15, 16 Measles virus (MV), 211, 216, 269, 270, 272 MV-induced immunosuppression, 275, 286, 287 Myeloid dendritic cells, 296, 233 Myeloid differentiation factor (MyD 88), 212, 506

Index

Myxovirus resistance-1 (Mx1), 22, 119, 132, 139, 309, 349, 399, 406, 554 Mx2, 406, 554 MxA proteins, 256, 257, 349 Mx GTPase, 503, 504, 657 Native immunity, 252 Nature killer cells, 66, 131, 160, 197, 233, 379, 446, 501 Necrosis factor 1, 632 Neurodegeneration, 405, 612, 617 Neurokinin-1, 274 Neurotoxicity, 33, 36, 37, 48, 49 Neurovirulence, 219, 239, 391, 532, 602, 604, 608, 609 Neutralizing antibodies, 5, 63, 70, 145–147, 238, 248, 253, 383, 407, 427, 431, 432, 449–451, 457 Neutrophils, 135, 136, 233, 236, 238, 371, 404 NF-κB, 6, 7, 121, 165, 168, 170, 171, 306, 333, 337, 363, 425, 509, 633, 636, 643 NIK and IKK α, 170, 171 Nitric Oxide Synthase-1 (NOS-1), 234–236 NKG2D, 67, 137, 478 NKp44, 137 NKp46, 137 Norovirus, 575–594 virus-like particles (VLPs), 301, 363, 379, 385, 402, 577, 580, 583–585, 587

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Nucleotide-binding oligomerization domain (NOD) proteins, 210, 421 2′,5′ oligoadenylate synthetase (2-5 OAS), 447 OAS1, 554 OAS3, 554 OASL, 422, 554, 559 Oncolysis, 229, 230, 239, 627, 631, 635, 636, 646, 647 Orthomyxoviridae, 111 Osteopontin, 478, 479 Otitis media, 158, 159 OX40L, 196 P38 MAPK, 35, 40, 323–333, 337, 338, 537, 540 P53, 42, 43, 306–308, 502, 508, 509, 529, 530, 532, 533, 535 P90 ribosomal S6 kinase (RSK), 321, 329, 632, 644 PAMP, 262, 656 Paramyxoviridae, 157, 158, 217, 274 PARP, 513, 640 Passive antibody therapy, 186, 199 Pathogen associated molecular patterns (PAMPs), 138, 189, 204, 209–211, 233, 348, 421, 656 Pattern recognition receptors (PRRs), 133, 137, 157, 161, 163, 167, 168, 172, 185, 186, 189, 191, 209–212, 221, 223, 252, 253, 262, 274, 348, 421, 423, 425 PD-1 (programmed cell death 1), 75, 399, 456, 458, 459, 551

687

Pegylated interferon-alpha, 446 Perforin, 74, 75, 143, 197, 201, 287, 396, 417, 433–435, 507, 616 Perforin-Fas-mediated cytolytic mechanisms, 433 Peripheral blood mononuclear cells (PBMCs), 15, 18, 34, 192, 352, 458, 477, 487, 488 Perivascular, 160, 185, 186, 234, 603 Phagocytosis, 39, 136, 176, 210, 211, 613 Phosphatidylethanolamine, 219, 531 Phosphatidylinositide-dependent kinase 1 (PDK1), 329 Phosphatidylinositol-3-kinase (PI3K), 41–43, 45, 119, 120, 124, 125, 219, 277, 305, 333–336, 509, 515, 540, 632, 633, 644 PI3K/Akt, 119, 124, 125, 333, 334, 336, 509, 540, 632, 633, 644. See also Akt Picornaviridae, 217, 219, 496 Plasmacytoid, 116, 133, 165, 178, 190, 191, 196, 209, 211, 220, 233, 236, 283, 348, 352, 657, 668 Poly-A binding protein (PABP), 499, 513, 514 Polypyrimidine-tract binding protein (PTP), 499 Predictive gene set, 14, 19, 20 Primary infection and secondary infection, 5, 60–63, 66, 69, 144, 145, 147, 148, 198, 200–202, 285, 429, 549, 564, 565 Prophylaxis, 303, 383, 385

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688

Index

Protease 2A , 497, 513 Protease 3C, 497, 513 Proteasomal degradation, 258, 261, 655, 657, 670 Proteasome inhibitor, 530, 532, 534, 535, 642, 670 Proteasomes, 535, 666, 668, 670 Protein degradation, 118, 399, 525, 527–529, 532–534, 536 Protein kinase B (PKB), 335, 633 Protein modification, 38, 525, 530, 531, 534, 536 Protein ubiquitination and degradation, 247 Pseudo-particles, 474 PtdIns (3, 4, 5) P3, 335, 633 PtdIns (4, 5) P2, 633 Quasispecies, 394, 450, 460, 484 Raf/MEK/ERK, 122, 123, 125, 537, 632, 633 RANTES, 13, 37, 48, 65, 177, 357, 423, 425, 427, 511, 557 Ras signaling, 627, 630–632, 635, 644, 645, 647 Ras/Ral GEFs pathways, 634 Rhesus rotavirus (RRV), 661, 665, 668 Receptor DR4 and DR5, 562 Receptor interacting protein 1 (RIP1), 217, 554, 634 Recombinant norovirus, 577, 580, 584. See also norovirus Recombinant virus, 173, 605 Reovirus, 211, 215, 221, 627–632, 634–647, 663

Respiratory syncytial virus (RSV), 157–162, 164–166, 168, 170, 172–179, 185–195, 197–204, 211, 216, 221, 576, 580 asthma, 159, 185–187, 195, 201–204, 564 fusion protein, 211, 276, 362, 564 vaccination, 110, 149, 158, 187, 200, 204, 240, 384, 446, 604 Reticuloendothelial system, 232 Retinoid acid inducible gene-like receptor (RLR), 137, 138 Retrocyclin, 136 Retroviruses, 6, 10, 32, 87, 88, 91, 93, 111, 530 Rhabdoviridae, 217 Rho/Rac pathways, 634 Ribavirin, 188, 382, 446, 471, 474, 486–488 Ribonucleoprotein complex, 112 RNA-dependent RNA polymerase (RDRP), 248, 298, 353, 393, 394, 459, 526, 577, 578, 629, 630, 658 RNA helicases, 116, 138, 157, 161, 163, 166, 167, 209, 216, 217, 233, 253, 348, 354, 447, 448, 559, 656 RNAi suppressors, 88, 89 RNase L, 118, 236, 256, 396, 398, 402, 408, 422, 435, 503, 559, 657 RNA-specific adenosine deaminase 1 (ADAR 1), 447, 448 Rotavirus, 655, 658–672 RSAD2, 554

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Index

SAPK/JNK, 638, 643 Sarcoma (src) family of kinases (p56 lck), 506 SARS Coronavirus, 18, 297–299, 302, 303, 321 Scavenger receptor BI (SR-BI), 472 Sendai virus, 204, 214, 220, 260, 261, 354 Sialic-acid (neuraminic acid), 113 Signal transducer and activator of transcription 4 (STAT 4), 502 STAT1, 22, 69, 173, 174, 201, 236, 261, 270, 280–282, 332, 349–351, 358–361, 377, 399, 400, 406, 407, 426, 563 STAT2, 175, 270, 280, 281, 349, 350, 359, 360, 406, 426 Sindbis virus (SV), 601, 605, 608, 611, 614, 617 Single nucleotide polymorphism (SNP), 12, 13, 65, 397, 398, 422 Single-stranded RNA (ssRNA), 107, 111, 116, 118, 138, 163, 190, 214, 220, 293, 416, 526, 549, 663 Siva, 508 Smac, 638–640, 643 Small interferon RNAs (siRNA), 83, 85, 86, 88–92, 94, 95, 122, 168, 192, 193, 217, 257, 303, 309, 313, 358, 362, 533, 535, 559, 664

689

Spermidine/spermie N1-acetyltranferase (SAT), 405 Src, 39, 259, 537, 538 Stem cells, 232, 234, 280 Stress activated c-jun-N-terminal kinase (SAPK/JNK), 638, 643 Subacute sclerosing panencephalitis (SSPE), 269–271, 285, 286 Surfactant protein A (SP-A), 135, 136, 189 Surfactant protein D (SP-D), 135, 136, 311 Tank binding kinase (TBK1), 165, 167, 218, 247, 259–262, 264, 447, 634 TAR binding proteins (TRBP), 86, 91, 95, 96 Target of rapamycin (TOR), 219 T cell activation, 40, 196, 275, 278, 280, 284, 395, 456 T cell receptors (TCR), 39, 40, 43, 144, 198, 237, 259, 277, 432, 461 TFPI, 558 Th1 cytokine, 43, 192, 194, 272, 277, 503 Th2 cytokine, 43, 186, 192, 194, 200, 203, 272, 277 Tissue inhibitors of MMPs (TIMPs), 510 Tissue tropism, 395, 401, 498, 499, 502, 548 Toll-like receptors (TLRs), 21, 22, 116, 137, 138, 163, 164, 166, 168, 178, 186, 190, 193, 196,

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690

Index

197, 209, 211–213, 216, 223, 233, 252, 262, 283, 348, 403, 421, 422, 656 TNF receptor-associated factor (TRAF), 169, 212, 217, 405, 506 TRAF-1, 47, 405 TRAF-2, 47, 217 TRAF-6, 164, 212, 213, 217, 506, 564 TNF-related apoptosis-inducing ligand (TRAIL), 36, 122, 220, 379, 478, 547, 551, 554, 556, 561–563, 566, 638, 640–642 Toll-receptor-domain-containing adaptor inducing IFN-β (TRIF), 22, 141, 164, 165, 191, 192, 212, 213, 217, 447 Transcriptomics, 303 Transforming growth factor-β (TGF-β ), 87, 88, 324, 457, 500, 502, 507, 510 Translational machinery, 308, 514, 590 TRIM22, 22, 554 Tumor necrosis factor superfamily (TNFSF14), 405 Two-phase model of secretion of type I IFN, 172 Tyrosine kinase 2 (Tyk2), 173, 330, 350, 394, 425, 426 Ubiquitination, 170, 247, 259, 261, 262, 264, 307, 528–531, 533, 534, 536, 641, 642, 655, 670, 672 Ubiquitin-like molecules, 118

Ubiquitin-proteasome system, 525, 527, 529 Ubiquitin-protein ligase, 528, 529, 655, 671 UDP-glucose ceramide glucosyltransferase (UGCG), 482, 560 3′-Untranslated regions (3′UTRs), 10, 85, 472, 497, 499 5′UTR, 91, 497, 499 Vaccination, 110, 149, 158, 187, 200, 204, 240, 384, 446, 604 VCAM1, 558 VEGF receptor, 33 Venezuelan equine encephalitis virus (VEEV), 384, 602–604, 606, 619 Vesicles, 41, 113, 210, 212, 218, 233, 302, 550 Vesicular stomatitis virus (VSV), 209, 214, 229, 286, 384, 477, 663 Very low density lipoprotein, 483 Viral encephalitis, 232, 239, 609 Viral escape, 455, 459–462, 475 Viral nucleocapsid, 309, 357, 393, 472, 478, 479 Virion infectivity, 38, 637 Virulence, 64, 66, 231, 273, 361, 362, 393, 394, 398, 401, 402, 420, 423, 426, 435, 498, 548, 582, 605, 607, 671 Virus Hemagglutinin (HA), 112–115, 121, 123, 145–147, 137, 146, 271, 272, 276–278, 284

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Index

Virus host interactions, 124, 471, 472, 476, 477, 485, 489, 606, 647 Virus-induced signaling adaptor (VISA), 217, 253, 348, 448 Virus-like particles (VLPs), 301, 363, 379, 385, 402, 577, 580, 583–585, 587 VP1, 497–499, 526, 577, 578, 590, 658, 660 VP2, 497, 508, 577, 578, 658 VP24, 345, 350, 353, 357–362, 377

691

VP35, 345, 350, 353–358, 361, 657 VP40, 357, 363 VPg, 497, 577, 578, 589, 590 VPg-poly(U), 589 VSV encephalitis, 232–234, 238 Western equine encephalitis virus (WEEV), 602–604, 619 West Nile virus (WNV), 214, 391–409, 415–437, 433, 550, 556, 559, 618