ADVISORY BOARD DAVID BALTIMORE ROBERT M. CHANOCK PETER C. DOHERTY H. J. GROSS B. D. HARRISON BERNARD MOSS ERLING NORRBY J. J. SKEHEL M. H. V. VAN REGENMORTEL
Academic Press is an imprint of Elsevier 32 Jamestown Road, London, NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2010 Copyright # 2010 Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) (0) 1865 843830, fax: (þ44) (0) 1865 853333; e-mail:
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CHAPTER
1 Getting the Message: Direct Manipulation of Host mRNA Accumulation During Gammaherpesvirus Lytic Infection Karen Clyde and Britt A. Glaunsinger
Contents
I. Introduction II. Modulation of Host Transcription by the bZIP and RTA Proteins A. Introduction B. Manipulation of the cell cycle C. Modulation of the immune response D. Cell proliferation, differentiation, and tumorigenesis III. EBV SM Alters Splicing and Stability of Cellular mRNAs A. Introduction B. Induction of STAT1 and IFN-stimulated genes IV. Global Repression of Host Gene Expression by the Alkaline Exonucleases A. Introduction B. Degradation of cellular mRNAs C. Hyperadenylation and nuclear retention of nascent messages D. Relocalization of poly(A) binding protein E. Evasion of host shutoff
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Department of Plant and Microbial Biology, University of California, Berkeley, USA Advances in Virus Research, Volume 78 ISSN 0065-3527, DOI: 10.1016/S0065-3527(10)78001-5
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2010 Elsevier Inc. All rights reserved.
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V. Conclusions Acknowledgments References
Abstract
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The Gammaherpesvirinae subfamily of herpesviruses comprises lymphotropic viruses, including the oncogenic human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus. During lytic infection, gammaherpesviruses manipulate host gene expression to optimize the cellular environment for viral replication and to evade the immune response. Additionally, although a lytically infected cell will itself be killed in the process of viral replication, lytic infection can contribute to pathogenesis by inducing the secretion of paracrine factors with functions in cell survival and proliferation, and angiogenesis. The mechanisms by which these viruses manipulate host gene expression are varied and target the accumulation of cellular mRNAs and their translation, signaling pathways, and protein stability. Here, we discuss how gammaherpesviral proteins directly influence host mRNA biogenesis and stability, either selectively or globally, in order to fine-tune the cellular environment to the advantage of the virus. Appreciation of the mechanisms by which these viruses interface with and adapt normal cellular processes continues to inform our understanding of gammaherpesviral biology and the regulation of mRNA accumulation and turnover in our own cells.
I. INTRODUCTION All viruses must manipulate the environment of the host cell in order to optimize viral replication and evade cellular defenses. Mechanisms by which viruses regulate cellular processes include blocking or initiating signaling pathways, modulating protein stability and translation, and regulating levels of host RNA. Here we will highlight how gammaherpesviruses interfere with cellular transcription, mRNA processing, and mRNA stability to directly control gene expression during lytic infection. While these viruses also manipulate host mRNA levels during latency, for example via the activity of numerous virus-encoded miRNAs, this topic is the subject of several recent reviews (Ganem, 2007; Ganem and Ziegelbauer, 2008; Kieff and Rickinson, 2007; Pang et al., 2009; Rickinson and Kieff, 2007; Swaminathan, 2008) and thus will not be covered here. Gammaherpesviruses are the lymphotropic tumor viruses of the Herpesviridae family of large DNA viruses. The Gammaherpesvirinae subfamily includes the genera Lymphocryptovirus and Rhadinovirus (Fig. 1). The two human gammaherpesviruses, Epstein-Barr virus (EBV or HHV4)
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Cytomegalovirus Simplexvirus
Roseolovirus
Varicellovirus
Rhadinovirus
Lymphocryptovirus
FIGURE 1 Phylogenetic relationship between select primate and murine herpesviruses. Amino acid sequences of DNA polymerase catalytic subunit genes from reference strains were aligned with Clustal X. Alphaherpesvirinae subfamily is indicated in orange, Betaherpesvirinae in red and Gammaherpesvirinae in green.
and Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV8) are important causes of lymphomas and other neoplasms associated primarily, but not exclusively, with immunosuppressed populations. EBV is endemic worldwide and is most commonly regarded as the agent of infectious mononucleosis, although it is also associated with a number of neoplastic diseases, including Hodgkin’s and Burkitt’s lymphomas (BL), nasopharyngeal carcinoma (NPC), and gastric carcinoma (Kutok and Wang, 2006). While EBV-associated malignancy is generally a consequence of latent infection, one proliferative disease with a prominent lytic signature is oral hairy leukoplakia (OHL; Mendoza et al., 2008). The 184-kbp EBV genome encodes nearly 100 known genes, the majority of which are expressed exclusively during lytic infection. EBV latency is complex and occurs in four distinct forms depending on the number and complement of genes expressed, with all 11 latency-associated genes and viral miRNAs expressed in latency type III (Kieff and Rickinson, 2007; Rickinson and Kieff, 2007). Like all gammaherpesviruses, EBV establishes latency in lymphocytes, primarily B cells, in vivo; however, it also infects oropharyngeal epithelial cells and T cells (Swaminathan and Kenney, 2009). As with EBV, KSHV-induced diseases are primarily associated with immunocompromised individuals and include Kaposi’s sarcoma (KS),
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primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD; Laurent et al., 2008). KS was one of the AIDS-defining diseases in the 1980s, and KSHV remains the leading cause of cancers in untreated AIDS patients (Ganem, 2007). Its 170-kbp genome encodes approximately 100 known genes, most of which are strictly lytic. In vivo, KSHV infects B cells and endothelial cells, the latter making up the bulk of the histologically complex KS lesion. For both EBV and KSHV, latency is the default outcome of infection in culture, although lytic reactivation can be induced by a number of stressors, chemicals, or the overexpression of the viral lytic transactivators (RTA, ZTA). The rodent virus murine gammaherpesvirus-68 (MHV-68) has also been extensively studied as an important model of in vivo infection. Officially known as murid herpesvirus 4, MHV-68 is a naturally occurring Rhadinovirus that infects bank voles and other rodents (Simas and Efstathiou, 1998). MHV-68 causes a mononucleosis-like disease in laboratory rodents and long-term infection can also induce tumors in the lymphoid compartment as well as in other, nonlymphoid organs (Nash et al., 2001). Unlike KSHV and EBV, MHV-68 directly enters the lytic pathway in culture and replicates to relatively high titers. The ability of MHV-68 to infect mice, coupled with its tractable genetics, has made it a useful model to study the immune response and pathogenesis of gammaherpesvirus infection in vivo. Although latency is the form of the gammaherpesviral life cycle usually associated with immortalization and transformation of host cells (Damania, 2007; Ganem, 2007; Rickinson and Kieff, 2007), lytic replication likely also plays a vital role in disease, particularly for KSHV (Ganem, 2010). As is characteristic of herpesviruses, the lytic cycle features a temporal program of gene expression, beginning with viral immediate early (IE) genes, which are often transcriptional activators necessary to drive the subsequent viral gene expression cascade. Delayed early (DE) genes prime the infected cell for replication of the viral genome, counteract host antiviral responses, and activate the primarily structural late (L) genes involved in virion assembly. As will be discussed here, lytic viral gene expression is responsible for the downregulation of bulk host mRNA, as well as selective induction of a number of cellular factors that influence cell proliferation and migration, immune evasion, and angiogenesis.
II. MODULATION OF HOST TRANSCRIPTION BY THE BZIP AND RTA PROTEINS A. Introduction Through selective activation or repression of host genes, viral transcription factors facilitate the establishment of a cellular environment ideal for replication while dampening the host immune response. As described below,
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gammaherpesviral transcription factors direct a succession of host gene expression changes, often through interactions with cellular proteins.
1. EBV and KSHV bZIP Proteins Basic leucine zipper (bZIP) proteins are a class of transcription factors characterized by a basic leucine zipper motif, which allows for both dimerization and sequence-specific DNA-binding interactions (Ellenberger, 1994). Transcription factors containing bZIP domains are found across eukaryotes, from budding yeast to humans. Both KSHV and EBV encode bZIP proteins that function as transcriptional modulators during lytic infection (Table I). The EBV bZIP protein, known as EBV ZTA, is the product of the BZLF1 gene. ZTA contains an N-terminal transactivation domain, followed by a bZIP domain comprising a basic DNA-binding (DBD) and leucine zipper homodimerization domain. ZTA exhibits homology to cellular AP-1 transcription factors such as the c-Fos oncogene (Farrell et al., 1989) and transactivates both viral and host genes via interactions with high-affinity AP-1 elements or the related ZTA-response elements (ZREs; Swaminathan and Kenney, 2009). During lytic infection, ZTA activates a cascade of viral gene transcription and facilitates the replication of the viral genome by binding to the lytic origin of replication (Orilyt; Kieff and Rickinson, 2007; Sinclair, 2003). EBV ZTA thus serves as the main lytic switch protein, though EBV RTA (BRLF1) can also trigger reactivation (Ragoczy et al., 1998; Zalani et al., 1996). Importantly, ZTA also targets a multitude of cellular genes that directly influence the cell cycle, proliferation, and immune responses. Unique among known Rhadinoviruses, KSHV also encodes a bZIP protein (K-bZIP, also known as RAP), one of the two alternatively spliced products of the KSHV-specific K8 locus. Complete splicing generates the
TABLE I
Synonyms for viral homologs discussed in this chapter
Lymphocryptovirus (g-1)
Rhadinovirus (g-2)
EBV
KSHV
BZLF1, ZTA, EB1, Z, ZEBRA BRLF1, RTA, R BMLF1, SM, BSMLF1, EB2, M, MTA BGLF5
K8a, K-bZIP, RAP None ORF50, RTA, ART, Lyta ORF50, RTA ORF57, MTA, KS-SM ORF57, MTA ORF37, SOX
MHV-68
ORF37, muSOX
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K8a transcript, which encodes the full-length K-bZIP protein. Retention of an intron results in premature termination of translation and the production of a truncated product (K8b) (Tang and Zheng, 2002), which has been implicated in the negative regulation of full-length K-bZIP (Yamanegi et al., 2005). While ZTA is a major contributor to EBV lytic replication, the effects of K-bZIP are more subtle. Like ZTA, K-bZIP binds to the Orilyt, but does so indirectly, in complex with the cellular factor CCAAT/ enhancer-binding protein alpha (C/EBPa, CEBPA). While there is little sequence similarity between EBV ZTA and KSHV K-bZIP, the two are structurally similar, with an N-terminal transactivation domain and a Cterminal bZIP domain. Unlike ZTA, K-bZIP has been primarily associated with transrepression activity upon coexpression with other transcription factors, although in some instances it can function as an activator (Hwang et al., 2001, 2003; Park et al., 2000).
2. RTA proteins Whereas only the human gammaherpesviruses, EBV and KSHV, encode known bZIP proteins, the RTA transactivator is conserved across the entire subfamily (Table I). RTA is encoded by the ORF50 genes of KSHV and MHV68 and by the BRLF1 gene of EBV. The various RTA homologs are relatively well conserved, consisting of an N-terminal DBD and dimerization domain and a C-terminal activation domain. KSHV RTA transactivates viral early genes, thereby functioning as the lytic switch protein to drive the virus out of latency. Like ZTA, EBV RTA expression can also induce reactivation, and expression of the full complement of early genes requires both ZTA and RTA. Both homologs can activate transcription directly by binding to RTA response elements (RREs) in viral promoters and indirectly by distinct mechanisms. Transactivation of some EBV lytic genes requires the combined effort of RTA and ZTA, and KSHV RTA can interact with the cellular factor RBP-Jk to activate a subset of downstream targets of the Notch signaling pathway (Miller et al., 2007; Section II.D). Microarray studies have revealed that a host of cellular proteins are up- and downregulated in the presence of the RTA proteins of KSHV, EBV, and MHV68 (Brown et al., 2010; Chang et al., 2005a; Hair et al., 2007; Li et al., 2004a,b), although the functional significance of the majority of these changes remains to be determined.
B. Manipulation of the cell cycle Control of the cell cycle is a common strategy employed by the majority of DNA viruses. Small dsDNA viruses, such as human papillomavirus, often return quiescent cells to the cell cycle in order to facilitate viral replication (Doorbar, 2005). Large dsDNA viruses, such as herpesviruses, encode their own lytic replication machinery, but might benefit from the
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te
S
La
Early
A
G2
G1 M
B
C M H C
IFN-stimulated genes
Latency
Proliferation, Angiogenesis
IFNγRα
Inflammation
Angiogenesis
Apoptosis Immune supression
TNFR1
Proliferation Differentiation Immune supression
Differentiation Cell survival, Proliferation
FIGURE 2 Gammaherpesvirus bZIP homologs manipulate cellular gene expression during lytic infection. Effects of EBV BLZF1 and KSHV K-bZIP on (A) cell cycle, (B) immune system, and (C) cell proliferation and tumorigenesis are summarized. Downstream effects are hypothesized or confirmed.
generation of raw materials that occurs prior to DNA synthesis. Not surprisingly, a number of studies have demonstrated cell cycle arrest in lytically infected cells. As detailed below, there is some disagreement as to the mechanisms and downstream effects of cell cycle-related gene targeting by the gammaherpesvirus transcription factors, perhaps due to the different cell lines and expression systems utilized (Figs. 2A and 3A).
1. Cell cycle control by EBV ZTA and RTA Both EBV ZTA and RTA have been reported to induce cell cycle arrest during lytic EBV infection at various stages and through diverse cellular pathways. Early work examining the effects of EBV on the cell cycle revealed a G0/G1 arrest upon lytic infection of NPC-derived cell lines or after ectopic expression of EBV ZTA in multiple cell lines (Cayrol and Flemington, 1996). The mechanism of arrest was characterized as an
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A
S G2
G1 M
B
CD 23a
CD 21 EBV coinfection
STAT3-inducible genes Apoptosis Immune supression
FIGURE 3 Gammaherpesvirus RTA proteins modulate host mRNA accumulation. Effects of EBV and KSHV RTA on (A) cell cycle, (B) immune system, and (C) cell proliferation and tumorigenesis are summarized. Downstream effects are hypothesized or confirmed.
upregulation of the cellular p53 gene, a potent tumor suppressor with roles in growth arrest, induction of apoptosis and initiation of DNA damage repair pathways. Activation of p53 in turn transactivated p21 (CIP1, WAF1, CDKN1A), a cyclin-dependent kinase (CDK) inhibitor (Cayrol and Flemington, 1996; Rodriguez et al., 1999). However, subsequent studies from the Kenney group found that G0/G1 arrest induced upon expression of ZTA from an adenovirus vector (Ad-ZTA) was cell type specific, as it occurred in primary human fibroblasts but not in primary human tonsil keratinocytes or gastric carcinoma (AGS) cells (Mauser et al., 2002b). Furthermore, even though infection of A549 (human lung adenocarcinoma) cells or primary fibroblasts with AdZTA induced p53 expression and a number of activation-associated p53 modifications, the transactivation function of p53 was repressed and thus it did not induce p21 (Mauser et al., 2002b,c). Additional reports favor a
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role for ZTA in repression of p53-mediated transactivation, linking this effect to the ability of ZTA to bind p53 (Sato et al., 2009b; Zhang et al., 1994) and stimulate its degradation (Sato et al., 2009b). The N-terminus of ZTA contains binding motifs for Cul2 and Cul5, subunits of the Elongin B/CCul2/5-SOCS-box protein (ECS) ubiquitin ligase complex, and ZTA functions as an adaptor component of the ECS complex, catalyzing the ubiquitin-mediated degradation of p53 (Sato et al., 2009a). Recent studies demonstrating a temporal component to p53 regulation by EBV ZTA during lytic reactivation may shed light on some of the seemingly conflicting results described above. Early in lytic infection, elevated p53 levels were shown to enhance viral genome replication (Chang et al., 2008; Sato et al., 2010), and its interactions with ZTA facilitated p53 binding and activation of target promoters, including p21 (Sato et al., 2010). Later in infection, when the cell would recognize the nascent viral genomes as DNA damage, ZTA may instead target p53 for degradation, as decreased p53 levels are required for efficient virion production (Sato et al., 2009a). Accordingly, p53 levels diminish over time (Guo et al., 2010) and p21 is transiently induced during lytic infection (Guo et al., 2010; Sato et al., 2010). EBV ZTA also influences cell cycle progression by reducing expression of the cell cycle regulators c-myc and E2F-1 (PBP3) in lytically infected NPC-derived cells (Rodriguez et al., 2001). Mutational analysis revealed that the DBD is required for suppression of c-myc and subsequent G0/G1 arrest, which could be overcome by overexpression of c-myc. Interestingly, c-myc and E2F-1 specifically oppose transactivation of viral genes containing ZREs by ZTA itself, thereby blocking lytic reactivation (Lin et al., 2004). Moreover, c-myc is required for the maintenance of KSHV latency and inhibits transcription of KSHV RTA (Li et al., 2010). Thus, cross-regulation between promoters of the cell cycle and the major EBV and KSHV lytic switch proteins could ensure that lytic reactivation occurs only under certain cellular conditions and that the optimal cellular environment would be maintained once lytic gene expression has initiated. An alternative pathway to p21-induced G1 arrest proceeds through C/EBPa, itself a bZIP transcription factor (reviewed in Johnson, 2005). C/EBPa regulates differentiation and G1 cell cycle arrest primarily through upregulation of p21, inhibition of E2F1-mediated transcription, and blocking cdk2 and cdk4 activity. EBV ZTA has been shown to elevate p21 mRNA and provoke arrest by inducing C/EBPa in lymphoma cell lines and in WT but not C/EBPa(/) mouse embryonic fibroblasts (MEFs; Wu et al., 2003a). Through its bZIP domain, ZTA interacts with and stabilizes C/EBPa protein, and this complex targets both the C/EBPa and p21 promoters (Wu et al., 2003a). Both DNA-binding and protein–protein interactions are likely important in C/EBPa-mediated transactivation by ZTA, as a DNA-binding mutant of ZTA that can interact
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with C/EBPa cannot induce G1 arrest (Schelcher et al., 2005). In this way, ZTA enhances the C/EBPa-dependent G1 arrest, both by stabilizing this transactivator of the CDK inhibitor p21 and by enhancing its transactivation function. Cell cycle progression may also be influenced by EBV RTA, which interacts with the transcription factor MBD1-containing chromatin-associated factor (MCAF1, ATF7IP) through its DBD and dimerization domains (Chang et al., 2005b). Though normally a transcriptional repressor, MCAF1 instead functions as a coactivator when bound to specificity protein 1 (Sp-1) and induces expression of genes containing Sp-1 binding sites (Fujita et al., 2003). RTA forms a ternary complex with MCAF1 and Sp-1, leading to upregulation of Sp-1-responsive promoters including those of the CDK inhibitors p21 and p16 (CDKN2A; Chang et al., 2005b). However, EBV RTA was also shown to activate the c-myc promoter in multiple cell lines (Gutsch et al., 1994b), which would downregulate p21, potentially leading to cell cycle resumption or apoptosis. As c-myc has been shown to be suppressed during lytic EBV infection (Rodriguez et al., 2001), induction by RTA may be opposed by ZTA, as mentioned above. Downstream of both p53 and p21, the retinoblastoma 1 (Rb) tumor suppressor binds and sequesters E2F1, preventing its transactivation of cell cycle promoting genes, resulting in G1 arrest. Phosphorylation of Rb by cyclin-dependent kinases triggers the release of E2F1, leading to cell cycle progression. Consistent with G1 arrest, hypophosphorylation of Rb in EBV ZTA- (Cayrol and Flemington, 1996) and RTA-expressing cells (Chen et al., 2009) has been reported. In contrast, the accumulation of phosphorylated Rb in lytically infected Akata (EBVþ BL) cells has also been described (Zacny et al., 1998), though other groups observe a reduction of overall Rb levels in lytically infected Raji (EBVþ BL) cells (Guo et al., 2010) and in Ad-RTA infected fibroblasts (Swenson et al., 1999). If E2F1 levels were maintained, either scenario could result in the progression to S phase, which has been demonstrated to occur in RTAexpressing cells (Guo et al., 2010; Swenson et al., 1999). EBV RTA binds Rb in infected cells and binding is contemporaneous with E2F1 release (Zacny et al., 1998). While the mechanism is unknown, E2F1 release is not likely achieved by competition for Rb binding, as E2F1 and RTA bind to distinct sites on Rb and the three can form stable complexes in vitro (Zacny et al., 1998). In HEK-293 (human embryonic kidney) and NPC-derived cells, EBV RTA has been demonstrated to trigger senescence, a state in which cells are still metabolically active yet arrested in G1 (Chen et al., 2009). Senescence was accompanied by the increased expression of the CDK inhibitors p21 and p27 and hypophosphorylation of Rb (Chen et al., 2009). Cyclin E expression was also induced, whereas cyclin D1 and cyclin-dependent kinases 4 and 6 were downregulated (Chen et al., 2009). Whether
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senescence confers an advantage to the virus or is strictly a cellular response to RTA-induced G1 arrest remains to be determined. In contrast to the reported G0/G1 cell cycle arrest, several reports document a ZTA-induced G2 arrest. In particular, Hela cells infected with Ad-ZTA exhibit a G2 arrest (Mauser et al., 2002a). Accordingly, genes expressed during S phase are induced in keratinocytes upon infection with Ad-ZTA (Mauser et al., 2002b) and pharmacological inhibition of S-phase CDK activity impedes expression of viral early genes in infected cells (Kudoh et al., 2004). The expression of Cyclin B1 (CCNB1), a promoter of the G2/M transition, was reduced, concurrent with the ZTA-induced G2 block, which could be partially reversed upon overexpression of cyclin B1 (Mauser et al., 2002a). Also downregulated was cyclin-dependent kinase 1 (CDK1, CDC2) protein (Mauser et al., 2002a). In contrast to previous results demonstrating reduced E2F-1 levels in lytically reactivated EBVþ NPC-derived cells (Rodriguez et al., 2001), E2F-1 is upregulated in EBV ZTA-expressing keratinocytes and AGS cells (Mauser et al., 2002b) and in lytically reactivated Akata (Zacny et al., 1998) and Raji cells (Guo et al., 2010). In fibroblasts, E2F1 levels are unchanged early in infection (Guo et al., 2010; Mauser et al., 2002b; Zacny et al., 1998), and reduced late in infection (Zacny et al., 1998). When primary fibroblasts were infected with Ad-ZTA, however, both a G0/G1 and G2/M block were observed (Mauser et al., 2002a,b), which suggests that cell cycle manipulation by EBV ZTA is indeed cell-type specific. Mutations in ZTA that relieve the block are unable to induce p53, p27, and p21, and do not repress c-myc (Rodriguez et al., 2001), which supports the model that arrest, at least in these cell lines, is achieved through these pathways.
2. Inhibition of cell cycle progression by KSHV K-bZIP Similar to EBV, lytic reactivation of KSHV in PEL and in primary dermal microvascular endothelial (DMVEC) cells also results in an increase in C/EBPa levels and a C/EBPa-dependent G1 arrest (Wu et al., 2002, 2003b). Expression of K-bZIP from an Ad vector was sufficient to induce p21 and inhibit S phase progression in human fibroblasts and DMVECs, but not in cells lacking C/EBPa (Wu et al., 2002). K-bZIP forms a complex through its bZIP domain with DNA-bound C/EBPa, leading to induction of C/EBPa and p21 (Wu et al., 2003b). Like EBV ZTA, GST-tagged K-bZIP binds and stabilizes in vitro translated C/EBPa, as well as p21. In ChIP assays with TPA-induced PEL cells, K-bZIP was found to associate in a C/EBPa-dependent manner with the C/EBPa promoter. Thus, K-bZIP likely induces G1 arrest in this system via both cooperative transcriptional upregulation of C/EBPa and subsequently p21, as well as direct stabilization of both proteins (Wu et al., 2003b). An additional mechanism of K-bZIP-induced growth arrest proceeds through its repressive interaction with CDK2 (Izumiya et al., 2003). CDK2
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induces cell cycle progression by phosphorylating Rb family members, thereby releasing and activating E2F to transcriptionally induce factors necessary for S phase. K-bZIP was shown to bind CDK2 in reticulocyte lysates and in BCBL-1 (PEL) cells, leading to an inhibition of CDK2 kinase activity. Both p21 and p27 are destabilized upon phosphorylation by CDK2 (Adams et al., 1996; Sheaff et al., 1997; Zhu et al., 2005), and these proteins were found to be upregulated upon TPA treatment of BCBL-1 cells (Izumiya et al., 2003). Given their direct interaction, K-bZIP may inhibit CDK2 by preventing access to its targets. Alternatively, K-bZIP might act as a pseudosubstrate, as is phosphorylated by CDK2 (Polson et al., 2001), which may modulate its transcriptional activity (see below). K-bZIP was recently shown to possess a SUMO interacting motif (SIM) and function as a poly-SUMO-specific E3 ligase (Chang et al., 2010). Sumoylation is a posttranslational modification that, analogous to ubiquitination, involves conjugation of a SUMO moiety to target proteins. K-bZIP itself is sumolyated in a manner critical for its ability to repress a number of viral promoters, and binds the E2 SUMO conjugating enzyme Ubc9 (UBE2I; Izumiya et al., 2005). Notably, p53 is one of the few transcription factors activated rather than suppressed by sumoylation (Li et al., 2006). K-bZIP enhances p53 and Rb sumoylation, and was shown by ChIP assay to be recruited in a SIM-dependent manner together with the SUMO moiety to p53 target promoters (Chang et al., 2010). While this did not affect the efficiency of p53 recruitment to these sites, it suggests that K-bZIP may stimulate the transcriptional activity of p53 by increasing its sumoylation. In a different study, K-bZIP was reported to bind p53 and repress its transcriptional activity (Park et al., 2000). These seemingly contradictory findings on how K-bZIP expression influences p53 function may be reconciled by a recent report showing that K-bZIP activity is modulated by phosphorylation (Izumiya et al., 2007). The authors propose a model in which phosphorylation of K-bZIP by the viral ORF36 protein kinase or CDK2 may serve as a switch to regulate the opposing transcriptional activities of this protein. While it is clear from the above work that EBV ZTA, as well as KSHV K-bZIP, is adept at directing the cell cycle, there is not yet a consensus as to where and how the blockade occurs. It is quite possible that different cell types are arrested at different stages in in vivo, and that doing so provides an advantage to the virus that is specific to that cell lineage. Moreover, others have found that while analysis of the cell cycle status of lytically reactivated cells is described as a block at one or more stages, the cells are in fact dividing, albeit more slowly than latently infected cells (Izumiya et al., 2003). It is possible that the lytic program simply slows rather than arrests the cell cycle in late G1 in order to delay genomic DNA replication while providing the virus with ample resources to replicate its own genome.
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C. Modulation of the immune response In order for a pathogen to successfully replicate in an immunocompetent individual, it must somehow evade the host’s powerful immune response. Pathogens accomplish this by diverting, evading, subverting and/or inhibiting various components of this system. After the acute phase of infection, most pathogens are cleared from the host, who may then be protected from reinfection by the adaptive immune response. Herpesviruses, in contrast, establish lifelong infections in their hosts, and, consequently, must continually avoid clearance by the adaptive immune system. Since the viral lytic cycle produces a large number of viral proteins and results in the destruction of the infected cell, it is likely the stage that is the most visible to the immune system. Accordingly, the lytic viral bZIP and RTA proteins coerce the infected cell into inducing and repressing immune-related genes to the advantage of the virus (Figs. 2B and 3B).
1. Major histocompatibility complex One mechanism by which the adaptive immune response recognizes a pathogen is by displaying pathogen-derived peptides on the cell surface in a process known as antigen presentation. Such peptides can be displayed directly by infected cells on major histocompatibility complex (MHC) class I, or by professional antigen presenting cells (APCs), such as monocytes/macrophages, dendritic cells, and B cells, on MHC class II. In humans, these molecules are more properly referred to as human leukocyte antigens (HLAs); however, for the sake of simplicity, we will refer to them here as MHCs. Since gammaherpesvirus infect a wide variety of cells, including APCs, the viruses have much to gain by controlling expression of both classes of MHC. A number of gammaherpesvirus genes target both MHC classes by various mechanisms during latent and lytic infection (Coscoy and Ganem, 2000; Croft et al., 2009; Di Renzo et al., 1993; Keating et al., 2002; Li et al., 2009; Rowe et al., 2007; Zuo et al., 2009). ZTA contributes indirectly to the reduction in MHC II during EBV infection, likely via transcriptional repression (Li et al., 2009). The cellular transcription factor MHC II transactivator (CIITA, NLRA), the master controller of MHC II expression, is constitutively expressed in dendritic cells and mature B cells but has to be activated in other cell lineages (reviewed in Drozina et al., 2005). In ZTAexpressing cells, the CIITA promoter is bound by the ZTA DBD through a promoter-proximal ZRE. ZTA binding correlates with a reduction in CIITA mRNA during lytic infection, presumably as a consequence of ZTA-mediated transcriptional repression (Li et al., 2009). siRNAmediated knockdown of ZTA in lytically infected cells partially restores CIITA levels (Li et al., 2009). ZTA thwarts MHC II induction in response to
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interferon gamma (IFNg) treatment as well, also by blocking upregulation of the CIITA transcript (Morrison et al., 2001). While MHC II is classically associated with the presentation of phagocytosed extracellular antigens, peptides derived from endogenous proteins are also loaded onto MHC II (Gannage and Munz, 2009). The downregulation of MHC II during lytic infection would therefore prevent the priming of the adaptive immune system by lytically infected lymphocytes.
2. Tumor necrosis factor alpha Tumor necrosis factor alpha (TNFa) is a multifunctional cytokine secreted primarily by macrophages, natural killer (NK) cells, and lymphocytes. In addition to stimulating the expression of MHCs, adhesion molecules, cytokines, and chemokines, TNFa can induce apoptosis in virus-infected cells (reviewed in Herbein and O’Brien, 2000). Extracellular TNFa binds to TNF receptors (TNFR), which are expressed constitutively in virtually all cells. In keratinocytes and epithelial cells, EBV ZTA inhibits TNFainduced expression of intercellular adhesion molecule 1 (ICAM-1) and the cleavage of caspase-3 (CASP3), protecting the cells from apoptosis (Morrison et al., 2004). The resistance to TNFa was determined to occur via repression of the TNFR-1 promoter, leading to reduced TNFR-1 mRNA levels in ZTA-expressing and in lytically infected cells. Since ZTA generally activates promoters, it was hypothesized that activation of a cellular repressor of TNFR-1 transcription would be the most likely mechanism for its suppression (Morrison et al., 2004). However, given the more recent result that EBV ZTA binds to ZREs in the promoter of CIITA, a gene that it inhibits (Li et al., 2009), it is possible that ZTA possesses intrinsic transcriptional repressor activity that has not yet been characterized. Interestingly, one downstream target of TNFa is NF-kB (Herbein and O’Brien, 2000; Li and Verma, 2002), which has been shown to antagonize lytic replication of gammaherpesviruses (Brown et al., 2003; Izumiya et al., 2009; Krug et al., 2007; Prince et al., 2003). Perhaps the objective of TNFR-1 repression by ZTA is twofold, serving both to inhibit apoptosis and to maintain lytic gene expression. In addition to reducing TNFR-1 levels, EBV lytic infection induces the expression of DcR3, a secreted decoy TNFR that inhibits FasL- and LIGHT-mediated apoptosis by immune cells (Pitti et al., 1998; Yu et al., 1999). EBV RTA stimulates transcription of DcR3 during lytic infection, likely by binding to an RRE in the DcR3 promoter (Ho et al., 2007). Overexpression of the limiting cellular transcription factor CBP stimulates RTA-mediated activation, suggesting a requirement for CBP in enhancement by RTA. Interestingly, some transactivation of DcR3 occurred in the presence of EBV RTA containing a mutated nuclear localization signal (NLS), perhaps indicating that RTA also induces DcR3 by a second mechanism from the cytoplasm (Ho et al., 2007). Whereas ZTA-mediated
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repression of TNFR-1 expression outlined above would reduce susceptibility of a lytically infected cell to TNFa-induced cell killing via an autocrine mechanism (Morrison et al., 2004), soluble DcR3 expression induced by EBV RTA could function in a paracrine manner, providing a potential benefit to tumor maintenance.
3. NF-kB One consequence of TNFa binding to the TNFR is the activation of the NF-kB pathway, which results in the expression of a multitude of cytokines, chemokines, and immune receptors (reviewed in Li and Verma, 2002; Pahl, 1999). In addition to downregulating expression of TNFR, EBV ZTA also acts farther downstream in the pathway, blocking NF-kBmediated activation of some of its targets (Dreyfus et al., 1999; Morrison and Kenney, 2004). In response to an activating stimulus, NF-kB is released from IkB and translocates to the nucleus where it stimulates expression of the genes it regulates (Li and Verma, 2002). In EBV ZTAexpressing cells, NF-kB is primarily nuclear and is competent to bind responsive promoters in vitro, while association of NF-kB with these promoters in intact cells is impaired and thus fails to activate NF-kBresponsive genes (Morrison and Kenney, 2004), though the mechanism by which this occurs is unclear. The p65 subunit of NF-kB binds EBV ZTA through its bZIP domain in vitro, but since NF-kB and ZTA reciprocally repress each other (Gutsch et al., 1994a), the effect of this binding is unknown. NF-kB is upregulated by latent membrane protein 1 (LMP-1) in EBV-infected cells (reviewed in Farrell, 1998), and high levels of NF-kB impair lytic reactivation of gammaherpesviruses in general (Brown et al., 2003; Izumiya et al., 2009; Krug et al., 2007; Prince et al., 2003). Inhibition of NF-kB activation by EBV ZTA may thus be a mechanism for maintaining lytic reactivation, in addition to dampening a cellular antiviral response.
4. Interferons and interferon-stimulated genes Interferons (IFNs) are powerful cytokines that fall into three classes. Type I interferons include IFN alpha and beta (IFNa and IFNb), and IFN gamma (IFNg) represents the sole member of type II IFNs. Type III IFNs (IFNl) will not be discussed here. IFNs mediate autocrine and paracrine antiviral effects and regulate the innate and adaptive immune responses by stimulating expression of IFN-stimulated genes (ISGs). IFNg regulates both the innate and adaptive immune response, resulting in activation of a number of immune effector cells and the induction of an antiviral state in nonimmune cells. Primarily produced by NK and T cells, secreted IFNg binds IFNg receptors (IFNgR) on target cells, initiating a signaling cascade culminating in the production of IFNg-inducible genes (reviewed in van Boxel-Dezaire and Stark, 2007). EBV ZTA has been found to block induction of IFN-stimulated genes (ISGs) in cells
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treated with IFNg, including CIITA and the regulator of apoptosis and tumor-suppressor interferon regulatory factor 1 (IRF-1; Morrison et al., 2001). Also repressed was the ISG interferon regulatory factor 9 (IRF-9, p48), a transcription factor, which, in complex with activated STAT1 and STAT2, mediates IFN-initiated signaling. While ZTA exhibited no effect on the overall levels of STAT1, STAT1 was hypophosphorylated, and thus inactive, under these conditions. However, STAT1 was successfully phosphorylated in the presence of ZTA in response to treatment with IFNa, which also signals through the STAT1/STAT2/IRF-9 complex, suggesting that the block was upstream of STAT1 activation and specific to IFNg. Further analysis revealed that IFNgRa (CD119, IFNGR1) was downregulated at the mRNA level and that both the DNA-binding and transactivation domains of EBV ZTA were required for this effect (Morrison et al., 2001). While the IFNgR is ubiquitous and highly expressed on some cell types, how ZTA alters IFNgR transcript levels remains to be determined. IFNb, a type I interferon, is a powerful cytokine with broad-spectrum antiviral and antitumor activity (reviewed in Takaoka and Yanai, 2006). Thus, the discovery that in various cell lines, KSHV K-bZIP induced the expression of IFNb up to 10-fold over background levels was somewhat surprising (Lefort et al., 2007). K-bZIP was shown to enhance expression of IFNb mRNA by binding the PRDIII region of the IFNb promoter, the site of interferon regulatory factor 3 (IRF-3) binding, although upregulation of IFNb mRNA was independent of IRF-3. However, despite this slight induction of IFNb, in the presence of K-bZIP, the IFNb promoter was unresponsive to other stimuli, such as infection with Sendai virus or induction of RIG-I (DDX58), MAVS (IPS-1, Cardif), and TBK1, indicating that K-bZIP might block IRF-3 binding to the IFNb promoter (Lefort et al., 2007). Thus, in this case K-bZIP strikes a compromise: an increase in the level of IFNb production in exchange for precluding a massive induction by other stimuli. This intriguing observation awaits confirmation in a lytic KSHV infection, where IFNb is antagonized by other viral factors, such as K9 (vIRF-1; Lin et al., 2001). Type I IFNs have also been demonstrated to inhibit lytic reactivation of KSHV in culture and MHV-68 in vivo (Barton et al., 2005; Chang et al., 2000; Monini et al., 1999). Thus, a low level of IFNb secretion might serve to maintain latency without precluding a limited amount of lytic reactivation. Interferon regulatory factor 7 (IRF-7) is expressed primarily in lymphocytes, dendritic cells, and cells of the secondary lymphoid organs, but can be induced in a variety of cell types by type I IFNs as well as by EBV LMP-1 (reviewed in Zhang and Pagano, 2002). IRF-7 activates ISGs and further stimulates expression of type I IFNs. Examination of OHL lesions revealed that IRF-7 was expressed in infected cells, and was localized to the nucleus, indicating that it had been activated (Hahn et al., 2005). In spite of IRF-7 expression and activation, however, in the presence of
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EBV ZTA, transactivation of IRF-7-responsive promoters was reduced. ZTA binds IRF-7 in cells, and its transactivation domain is required for inhibition of IRF-7 activity, whereas its DBD is dispensable. The observations that ZTA blocks transactivation by a constitutively active IRF-7, that it physically interacts with IRF-7 and that IRF-7 is activated and nuclear in ZTA-expressing cells indicate that the block is likely achieved by inhibiting IRF-7 transactivation activity (Hahn et al., 2005). KSHV inhibits IRF-7mediated transactivation via ORF45, which prevents its accumulation in the nucleus (Zhu et al., 2002). In contrast, the EBV ORF45 homolog BKRF4, had no effect on IRF-7 (Hahn et al., 2005), indicating that each virus has evolved a distinct strategy for inhibiting ISGs. It will be informative to determine whether ZTA binding of IRF-7 prevents its association with promoters or whether the IRF-7-ZTA complex binds IRF-7-regulated promoters and represses their transcription. Like the bZIP proteins, the gammaherpesvirus RTA homologs also target type I IFN signaling by distinct mechanisms. In addition to the block in IRF-7-mediated transactivation by EBV ZTA, EBV RTA transcriptionally represses both IRF-3 and IRF-7 (Bentz et al., 2010). Moreover, KSHV RTA induces the ubiquitin/proteasome-dependent degradation of IRF-7 (Yu et al., 2005) in addition to the block in transactivation by ORF45 (Zhu et al., 2002), resulting in the repression of some ISGs (Yu et al., 2005). The diversity of mechanisms for inhibiting IFN signaling by gammaherpesviruses reflects its potency in blocking viral replication in vivo. Interferons regulate gene expression in part through transcriptional activator sequences such as interferon stimulated response elements (ISREs). Curiously, a number of KSHV lytic genes feature ISREs in their promoters, and it is through these elements that KSHV RTA activates their expression (Zhang et al., 2005). Not surprisingly then, KSHV RTA also activates select cellular ISRE-containing promoters (ISG54 and IF16) and induces transcription of endogenous ISGs (ISG54, STAF50, MxA; Zhang et al., 2005). Apart from co-opting the IFN system to activate transcription of its own promoters, the significance of the induction of ISGs during lytic KSHV infection is unknown.
5. Tumor growth factor beta Tumor growth factor beta (TGFb) is an important controller of cell proliferation and differentiation, mediating G1 arrest or inducing apoptosis in virus-infected cells (Moustakas et al., 2002). KSHV K-bZIP counteracts these effects, as it enhances viability of cells cultured with TGFb (Tomita et al., 2004). Treatment of KSHV-infected cells with TGFb failed to activate TGFb-responsive genes, although unlike TNFa mentioned above, the block was determined to be downstream of TGFb receptor 1 (TGFbR1) signaling. TGFbR1 signals through the SMAD proteins, which were found to be activated and able to form complexes and bind DNA. Yet, in the
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presence of K-bZIP, transactivation by SMADs was repressed, perhaps indicating that K-bZIP interferes with the assembly of SMAD-containing complexes on TGFb-responsive promoters (Tomita et al., 2004). In contrast to KSHV, EBV ZTA was found to induce secretion of active TGFb by transactivating the TGFb promoter (Cayrol and Flemington, 1995). Upregulation of a TGFb-responsive gene, TGFbigh3, was also observed (Cayrol and Flemington, 1995), though it was not determined whether TGFbigh3 was induced directly by ZTA or by TGFb. Interestingly, TGFb has been shown to induce lytic reactivation in latently EBV-infected cells (Liang et al., 2002a), suggesting that the virus may enhance its secretion in order to stimulate overall virus production. Since TGFb features both immunostimulatory and anti-inflammatory activities, its relationship with gammaherpesvirus infection is expectedly complex and appears to be differentially regulated by KSHV and EBV.
6. Anti-inflammatory cytokines IL-10 and IL-13 Interleukin 10 (IL-10) is primarily an anti-inflammatory cytokine and suppresses both cell-mediated and humoral immune responses (reviewed in Mosser and Zhang, 2008). During lytic infection, both EBV ZTA and RTA upregulate IL-10 mRNA via stimulation of the IL-10 promoter, although the induction of IL-10 is greater in response to LMP-1 during EBV latency ( Jones et al., 2007b; Mahot et al., 2003). ZTA was found to associate with the IL-10 promoter, and both the transactivation and DBD domains of ZTA were required for promoter activation (Mahot et al., 2003). Neither ZTA nor EBV RTA appear to synergize with LMP-1 for IL-10 induction, although coexpression of these proteins with LMP-1 resulted in a reduction of LMP-1 levels, making it difficult to assess potential combinatorial effects ( Jones et al., 2007b). The fact that at least three viral proteins are capable of inducing IL-10 underscores the importance of this cytokine in latency, lytic reactivation, and potentially in pathogenesis and immune evasion. IL-13 promotes proliferation, maturation, and differentiation of B cells, stimulates MHC II expression by B cells and monocytes, and antagonizes the production of proinflammatory cytokines by macrophages (reviewed in Zurawski and de Vries, 1994). EBVþ B cells produce 50- to 1200-fold more IL-13 than uninfected cells, in part due to the activity of ZTA (Tsai et al., 2009). Enhanced IL-13 levels exert a paracrine effect in a mixed population of latently and lytically infected B lymphoblastoid cell lines (LCLs), as immunodepletion of IL-13 has a deleterious effect on cell proliferation in culture. Although a multitude of ZRE elements are predicted in the IL-13 promoter, only those most proximal to the transcriptional start site contribute to ZTA-mediated induction (Tsai et al., 2009).
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7. Proinflammatory cytokines IL-6 and IL-8 A proinflammatory cytokine, IL-6 functions in B cell differentiation, proliferation, and survival in both autocrine and paracrine manners. IL-6 plays a central role in the development and maintenance of neoplasms associated with both KSHV and EBV infection in cell culture and animal models of infection, and in the clinical setting (Foussat et al., 1999; Jones et al., 2007b; Scala et al., 1990; Tanner and Tosato, 1991). EBV ZTA has been shown to induce IL-6 at the RNA level, likely via enhanced transcription ( Jones et al., 2007b). IL-6 mRNA is also potently upregulated during lytic KSHV infection, as a consequence of both transcriptional activation and stabilization (discussed in section IVE). Ectopic expression of KSHV RTA in various cell lines stimulates IL-6 production and RTA can transactivate the IL-6 promoter in reporter assays (Deng et al., 2002; Roan et al., 2002). Mutational analysis of the IL-6 promoter reveals an array of elements that respond positively and negatively to coexpression of KSHV RTA. Of these, ATF, C/EBPb, and NF-kB response elements are not necessary for activation by RTA, although in their absence the magnitude of transactivation is reduced (Roan et al., 2002). Both IL-6 and IL-10 can induce ISGs by activating STAT3 in a paracrine manner. STAT3 is normally phosphorylated in response to extracellular cytokines, which stimulates dimerization and translocation to the nucleus, where it activates transcription of ISGs. KSHV RTA is itself capable of inducing STAT3-responsive promoters, and in addition, induces the dimerization and nuclear localization of STAT3 dimers in a phosphorylation-independent manner (Gwack et al., 2002). The effects of RTA on STAT3-dependent transcription are enhanced by activating factors IL-6 and v-src, and repressed in the presence of a dominant negative STAT3. RTA forms a complex with the nuclear STAT3 dimers through a region in its C-terminus (Gwack et al., 2002). Perhaps the case of STAT3 is similar to that of K-bZIP induction of IFNb and RTA upregulation of ISGs via ISREs, in which some, but not all STAT3-responsive promoters are activated, permitting KSHV to retain some measure of control over an inevitable antiviral response. EBV ZTA also induces production of IL-8 (CXCL8), an inflammatory chemokine and angiogenic factor (Hsu et al., 2008). Upregulation is achieved at the RNA level, and both the ZTA transactivation domain and DBD are required for stimulation. AP-1 and ZRE sites have been identified in the IL-8 promoter, each of which confers partial responsiveness to ZTA expression, and gel shift assays confirm that ZTA binds to both elements. Notably, although early growth response 1 (Egr-1, ZIF268) can stimulate IL-8 expression and Egr-1 is induced by ZTA (see below; Chang et al., 2006; Heather et al., 2009), in this context, IL-8 induction occurs independent of Egr-1 (Hsu et al., 2008). The existence of two
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potential mechanisms for inducing IL-8, one direct and one via the induction of Egr-1, points to the importance of IL-8 expression or secretion to the viral life cycle and/or to pathogenesis, particularly in light of its angiogenic activity.
8. CD21 and CD23a The immune receptors CD21 (C3DR) and CD23a (FCER2) were found to be highly upregulated by KSHV RTA in a microarray study of RTAexpressing BJAB (EBV-negative BL) cells (Chang et al., 2005a). Both genes are activated by KSHV RTA through their RBP-Jk binding sites, although for CD21 this occurs within an intronic silencer normally repressed by RBP-Jk, while the CD23a promoter instead possesses a binding site that confers upregulation by RBP-Jk (Chang et al., 2005a). The expression of membrane CD23, a receptor for IgE, which is primarily involved in allergy and the immune response to macroparasites, is a marker of EBV transformation (reviewed in Schwarzmeier et al., 2005). Although the role of membrane CD23 in gammaherpesvirus biology is unknown, the secreted form of CD23 (sCD23), which is also produced by RTA-expressing BJAB and KSHV-infected PEL cells (Chang et al., 2005a), can act as a mitogenic factor (Mossalayi et al., 1997), and this form may serve a function in EBV tumor development and maintenance. CD21, a receptor for complement component C3d, is also a receptor for EBV binding to lymphocytes. Interestingly, expression of exogenous KSHV RTA in BJAB cells and ensuing induction of surface CD21 results in a higher level of subsequent EBV infection (Chang et al., 2005a). Given that KSHVþ PEL isolates are commonly coinfected with EBV (Nador et al., 1996), induction of CD21 by transient expression of KSHV RTA could explain, in part, the frequency with which coinfection is observed.
D. Cell proliferation, differentiation, and tumorigenesis As a number of immune effectors serve as proliferation factors for lymphocytes, it is somewhat difficult to distinguish immunomodulation from tumorigenesis. However, in this section, we have included those genes that play roles in cell growth, differentiation, and tissue remodeling that are not primarily associated with the immune response. Expressed during the lytic cycle and transactivated directly or indirectly by the gammaherpesvirus transactivators, these genes serve a paracrine function or enhance lytic replication (Fig. 2C). In a microarray analysis of EBV-infected gastric carcinoma (AGS) cells, Jones et al. identified the dehydrogenase/reductase SDR family member 9 (DHRS9, RDHL) gene as responsive to EBV ZTA expression ( Jones et al., 2007a). DHRS9 converts retinol to retinal, an intermediate in the synthesis of retinoic acid. DHRS9 is upregulated 10-fold in WT- versus DZTA virus-
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infected cells and is also induced in response to ZTA expression in uninfected cells. Given the requirement for EBV ZTA’s DNA-binding activity and that it binds two promoter-proximal ZREs in vitro, it is likely that ZTA directly transactivates the DHRS9 gene ( Jones et al., 2007a). While the role of DHRS9 expression during lytic replication is unknown, this gene has been reported to drive differentiation of memory B cells to plasma cells (Ertesvag et al., 2007; Morikawa and Nonaka, 2005). Differentiation might provide a more suitable environment for lytic replication; indeed, differentiation into plasma cells has been shown to induce lytic reactivation of EBV-infected cells (Laichalk and Thorley-Lawson, 2005). Retinoic acid has been demonstrated to inhibit lytic reactivation of latently infected cells in culture (Roubal et al., 1980; Zeng et al., 1981) by blocking EBV ZTA-mediated transactivation of early genes (Sista et al., 1993), perhaps indicating that the upregulation of DHRS9 by EBV ZTA serves a regulatory function during lytic infection. Another cellular gene that is induced in EBV ZTA-expressing cells is discoidin domain receptor tyrosine kinase 2 (DDR2, TRK-related kinase, TKT, TYRO10; Lu et al., 2000). Whereas DDR2 is undetectable in nonZTA-expressing cells, it is induced at the transcript and protein level in epithelial cell lines in a manner that requires the bZIP and part of the transactivation domains of EBV ZTA (Lu et al., 2000). Examination of biopsy specimens revealed that DDR2 expression was highest in NPC and metastatic NPC tumors compared with other head and neck tumors and normal nasopharyngeal epithelial cells (Chua et al., 2008). Furthermore, the level of DDR2 transcripts in biopsies positively correlated with the levels of EBV ZTA mRNA. ZTA activated the DDR2 promoter in a reporter assay, indicating that the induction might be direct, though a predicted ZRE in the DDR2 promoter was not investigated (Chua et al., 2008). DDR2 is of particular interest because it induces the transcription of a number of genes, including matrix metalloproteinase-1 (MMP-1). Matrix metalloproteinases (MMPs) are secreted or membrane-bound proteases that digest the extracellular matrix (ECM), and are fundamental to migration and invasion of metastatic tumors (Kessenbrock et al., 2010). In addition to digestion of the ECM, secreted MMPs regulate growth signaling, apoptosis, and angiogenesis. MMP-1, in particular, is upregulated in NPC tumors, and EBV ZTA induces MMP-1 mRNA, leading to elevated protein and enzymatic activity (Lu et al., 2003). Interestingly, MMP-2 was also shown to be upregulated in NPC specimens (Lu et al., 2003) and cellular IL-8, which is induced by EBV ZTA (Hsu et al., 2008), is capable of inducing MMP-2. Since both MMP-1 and MMP-2 are secreted, low-level lytic reactivation within EBVþ neoplasms could enhance invasive tumor growth in a paracrine fashion. Egr-1 is a transcriptional activator of genes that govern cell proliferation, apoptosis, and differentiation. Normally, Egr-1 is upregulated by a
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number of stimuli, including cytokines, growth factors, and general cell stresses and is activated via the mitogen-activated protein kinase (MAPK) pathway (reviewed in Thiel and Cibelli, 2002). During lytic reactivation of EBV in epithelial and B cell lines, Egr-1 mRNA and protein are induced by ZTA (Chang et al., 2006). The Egr-1 promoter contains two ZREs and an SRE-Ets site, which binds the Elk-1 transcriptional activator. Coexpression of ZTA increases binding of Elk-1 to the SRE-Ets elements and both ZREs play a role in ZTA-dependent upregulation of Egr-1. Elk-1 activity is regulated by the extracellular signal-regulated kinase (ERK), which is itself activated in the presence of ZTA; inhibitors of ERK activation block ZTA-mediated induction of Egr-1 and binding of Elk-1. Furthermore, knockdown of Egr-1 results not only in a reduction of spontaneous lytic reactivation in infected HEK-293 cells, but in the levels of EBV ZTA as well, indicative of cross-stimulation between the two proteins (Chang et al., 2006). Surprisingly, although methylation is not generally associated with enhanced transcription factor binding, ZTA binding to the Egr-1 promoter is enhanced up to 10-fold under conditions of CpG methylation (Heather et al., 2009). Most of the binding occurs at the ZRE distal to the transcriptional start site, although these results may be cell line-specific. Interestingly, TGFb1 and p53, which are both upregulated by EBV ZTA (see above), can be induced by Egr-1. Furthermore, Egr-1 has been shown to stimulate expression of a wide variety of genes in primary endothelial cells, including growth factors such as the angiogenic vascular endothelial growth factor (VEGF), cell cycle promoters, and transcription factors (Fu et al., 2003). The Notch signal transduction pathway plays an important role in development, the immune response, and tumorigenesis. Activation of the Notch pathway induces genes involved in cell survival that function in an autocrine manner, and is an important regulator of angiogenesis in endothelial cells, upregulating a number of proangiogenic factors such as hypoxia inducible factor 1a (HIF-1a) (reviewed in Roy et al., 2007). One effector of Notch-mediated transcription is the transcription factor RBPJk, whose interaction with EBV latent proteins is well characterized (Hayward et al., 2006; Zimber-Strobl and Strobl, 2001). Notch activation converts RBP-Jk from a transcriptional repressor to an activator. KSHV RTA interfaces with the Notch pathway at several stages. RTA stimulates binding of RBP-Jk to viral and cellular promoters (Carroll et al., 2006; Liang et al., 2002b; Persson and Wilson, 2010; Wang and Yuan, 2007), including those of IL-6 and hairy and enhancer of split 1 (HES-1) (Carroll et al., 2006), obviating the need for Notch pathway activation. However, the effect is not uniform, as it does not enhance RBP-Jk binding to the RTA-responsive CD23 promoter (Carroll et al., 2006). KSHV RTA also regulates another effector of the Notch pathway, the transcriptional repressor hairy/enhancer-of-split related with YRPW motif 1 (Hey1;
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Yada et al., 2006). RTA transactivates the Hey1 promoter in cells expressing KSHV RTA alone and in lytically infected B cells. Interestingly, Hey1, in turn represses RTA transcription (Yada et al., 2006). In addition, RTA can induce the ubiquitin/proteasome-dependent degradation of Hey1 protein (Gould et al., 2009), indicating that Hey1 may play a tightly regulated role in the control of KSHV RTA expression and lytic replication. Thus, via regulation of RBP-Jk and Hey1, it appears that KSHV bypasses Notch to activate a subset of Notch-inducible genes, which allows the virus to fine-tune gene expression while remodeling the cellular environment.
III. EBV SM ALTERS SPLICING AND STABILITY OF CELLULAR MRNAS A. Introduction EBV SM, encoded by the BMLF1 gene, is an RNA-binding, nucleocytoplasmic shuttling protein that stimulates the cytoplasmic accumulation of viral mRNAs at a posttranscriptional level. Although some controversy exists as to the mechanisms by which this occurs, SM and its Rhadinovirus homologs known as MTA (ORF57), have been implicated in the regulation of splicing, nuclear export, translation, and stability of viral and select cellular mRNAs (Table I; Boyne et al., 2008, 2010; Conrad, 2009; Sergeant et al., 2008; Swaminathan, 2005), including a VEGF receptor mRNA (Gupta et al., 2000). While much work has focused on the role of SM and MTA in viral gene expression and on exogenous reporter genes, comparatively few studies have examined their effects on cellular genes and the mechanisms employed.
B. Induction of STAT1 and IFN-stimulated genes A microarray study of the effects of SM on cellular gene expression, specifically those involved in tumorigenesis, revealed that a small number of transcripts are differentially regulated in SM-expressing cells (Ruvolo et al., 2003). Of the 1700 genes examined, 8 (0.5%) transcripts were upregulated and 25 (1.5%) were downregulated at least twofold. Among the induced genes were several ISGs and STAT1. Since STAT1 binding to ISREs is enhanced in the presence of SM, induction of STAT1 likely drives the observed upregulation of ISGs (Ruvolo et al., 2003). Further analysis indicated that while overall STAT1 mRNA and protein levels are increased in SM-expressing cells, the ratio of the STAT1a and STAT1b splicing isoforms was reversed (Fig. 4; Ruvolo et al., 2003; Verma and Swaminathan, 2008). SM stimulates the usage of an alternative splice
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…
Stop codon
…
Poly(A) signal
…
…
Stop codon
…
IFNγ signaling
Poly(A) signal
AAAAAAAAAAAAn
IFNγ signaling
Stabilization of viral mRNAs
FIGURE 4 EBV SM alters splicing and stability of mRNAs. Effects of EBV SM on splicing of STAT1a/b mRNA and on the stability of viral mRNAs.
donor site while repressing the recognition of the constitutive splice site, leading to reduced expression of the STAT1a isoform. Although SM is reported to bind to STAT1 mRNA (Verma and Swaminathan, 2008), the mechanism by which it promotes the usage of one splice donor over the other is unknown. Finally, although SM drives the alternative splicing of STAT1, both the STAT1a and b mRNAs and proteins are induced, as is their phosphorylation (Ruvolo et al., 2003; Verma and Swaminathan, 2008), therefore SM may play multiple roles in the regulation of STAT1. STAT1a transactivates genes in response to both type I and type II IFNs, whereas STAT1b only transactivates IFN type I-responsive genes (Muller et al., 1993). Moreover, STAT1b can bind STAT1a and act in a dominantnegative fashion toward an IFNg response (Alvarez et al., 2003). Though this remains to be demonstrated, EBV SM may favor alternative splicing of this cellular mRNA in part to allow evasion of the host immune response to viral infection. While the effects of IFNg on lytic replication of KSHV and MHV-68 are varied (Chang et al., 2000; Steed et al., 2006), EBV obstructs the effects of IFNg in two ways: by suppressing expression of IFNgRa on lytically infected cells and by blocking activation of IFNginducible genes. Another mechanism by which EBV SM enhances expression of specific genes is by altering their stability. A cellular protein, Sp110b, is a cofactor in SM-mediated stabilization of viral mRNAs during lytic infection (Nicewonger et al., 2004). Sp110 is an ISG that functions as a transcriptional activator. Expression of the Sp110b isoform is induced in lytically infected cells and in cells expressing SM, perhaps via the activation of STAT1. In the presence of SM, Sp110b is required for the accumulation of a lytic viral mRNA, probably by stabilizing the transcripts (Nicewonger
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et al., 2004). Since Sp110 has not been reported to function in the stability of nuclear mRNAs, it is likely that its stabilizing effect is combinatorial with SM. Thus, besides eluding the effects of IFNg, an important result of the upregulation of STAT1 by EBV SM may be the enhanced expression via the stabilization of viral mRNAs.
IV. GLOBAL REPRESSION OF HOST GENE EXPRESSION BY THE ALKALINE EXONUCLEASES A. Introduction A broad downregulation in host gene expression (host shutoff) has been observed during lytic infection with EBV (Rowe et al., 2007), KSHV (Chandriani and Ganem, 2007; Glaunsinger and Ganem, 2004a,b, MHV68 (Covarrubias et al., 2009; Ebrahimi et al., 2003; Mages et al., 2008), and herpesvirus saimiri (Modrow and Wolf, 1983), a Rhadinovirus that naturally infects primates. For EBV, KSHV, and MHV-68, the primary source of the shutoff is the viral alkaline exonuclease (AE) homolog (Covarrubias et al., 2009; Glaunsinger and Ganem, 2004b; Rowe et al., 2007). All members of the Herpesviridae encode an AE, called BGLF5 in EBV, SOX in KSHV, and muSOX in MHV-68. The AE proteins have been shown to possess deoxyribonuclease (DNase) activity in vitro, a function that relates to their role in the resolution of branched structures that arise during replication of viral genomes (reviewed in Wilkinson and Weller, 2003). Similar to HSV-1 AE mutants, an EBV BGLF5 deletion mutant virus demonstrated reduced viral DNA synthesis, as well as defects in processing of linear genomes, packaging, and nuclear egress in HEK-293 cells— phenotypes that could be partially complemented with HSV AE (Feederle et al., 2009). Similarly, deletion of MHV-68 muSOX results in a lack of detectable viral replication in HEK-293T and other cell lines, and can also be partially rescued by expression of UL12 (Covarrubias et al., 2009; K. Clyde and B.A. Glaunsinger, unpublished observations). The AE proteins thus all appear to play critical roles in viral replication. However, the gammaherpesvirus SOX, muSOX, and BGLF5 proteins (Table I) have selectively evolved an additional host shutoff function, executed via mRNA destabilization in the cytoplasm, as well as hyperadenylation and nuclear retention of nascent messages (Fig. 5).
B. Degradation of cellular mRNAs The gammaherpesviral AE homologs are expressed with delayed early kinetics, and are detectable by 8–10 h post infection, a time coincident with the onset of host shutoff (Covarrubias et al., 2009; Glaunsinger and
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Cytoplasm AAA
AA A AA
Nucleus
AAA AAA
AA
AA
A
A
A AA AA AA AA A A
AA AA
A A
FIGURE 5 Gammaherpesvirus SOX homologs effect host shutoff. Summary of effects of KSHV SOX, MHV-68 muSOX, and EBV BGLF5 on cellular transcript abundance and on cellular PABPC localization. Proteins with a question mark indicate unknown cellular factors that are hypothesized to assist in targeting and/or degradation of cellular mRNAs.
Ganem, 2004b; Rowe et al., 2007). Expression of gammaherpesviral AE proteins alone in cells is sufficient to promote enhanced turnover of reporter and endogenous mRNAs, leading to a generalized dampening of host translation (Covarrubias et al., 2009; Glaunsinger and Ganem, 2004a,b; Lee and Glaunsinger, 2009; Rowe et al., 2007; S. Covarrubias and B.A. Glaunsinger, unpublished observations). HSV-1 AE lacks this RNA turnover activity, which in alphaherpesviruses is instead carried out by a viral ribonuclease (RNase) known as vhs (reviewed in Glaunsinger and Ganem, 2006). One notable difference between HSV-1 AE and the gammaherpesvirus AE homologs is their subcellular localization. While HSV-1 AE is nuclear, SOX, muSOX, and BGLF5 are present in both the nucleus and the cytoplasm (Covarrubias et al., 2009; Glaunsinger et al., 2005; Rowe et al., 2007) and cytoplasmic localization of SOX and muSOX is critical for host shutoff function (Covarrubias et al., 2009). A SOX nuclear localization signal (NLS) mutant that is predominantly cytoplasmic retains full host shutoff activity and removing the cytoplasmic population of muSOX selectively eliminates the host shutoff function (Covarrubias et al., 2009; Glaunsinger et al., 2005). Thus, the nuclear population likely functions in viral DNA replication and packaging, while the cytoplasmic population implements the block in cellular gene expression. However, cytoplasmic localization alone does not confer the host shutoff function in the gammaherpesvirus AE proteins, as a partially cytoplasmic HSV-1 AE NLS mutant still lacks host shutoff activity (Covarrubias et al., 2009). ˚ resolution, indicated a The crystal structure of BGLF5, solved at 3 A structural similarity to bacteriophage l-exonuclease and lead to the identification of a PD-(D/E)XK nuclease superfamily-like catalytic core, which ˚ crystal is essential for DNase function (Buisson et al., 2009). A 3.5-A
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structure was also obtained for a BGLF5 catalytic mutant (D203S), which ˚ resolution crystal superimposed well with the WT structure. A 1.85-A structure of KSHV SOX has also recently been solved and, like BGLF5, showed similarity to the bacteriophage l-exonuclease, with a type II PD(D/E)XK superfamily-like catalytic core (Dahlroth et al., 2009). Notably, whereas l-exonuclease is thought to function as a trimer (Kovall and Matthews, 1998), examination of crystal contacts and gel filtration analysis indicate that SOX is monomeric (Dahlroth et al., 2009). The observation that gammaherpesvirus AE homologs stimulate mRNA degradation raises the question of whether these proteins promote mRNA turnover in vivo via an intrinsic RNase activity, or whether shutoff relies in whole or in part on the activation of cellular RNA decay factors. In vitro, baculovirus-expressed BGLF5 possesses Mn2þ-dependent RNase activity on U-rich or capped mRNA substrates, but not on tRNA, with degradation proceeding in a 50 ! 30 direction (Buisson et al., 2009). The fact that BGLF5 could degrade a capped substrate from the 50 end suggests it may retain endonucleolytic activity as well, as a strict 50 exoribonuclease would require prior m7G cap hydrolysis by a decapping enzyme to generate an appropriate 50 monophosphorylated substrate. Significantly, a BGLF5 catalytic mutant (D203S) was defective for both DNase and RNase activity in vitro, indicating that the same active site catalyzes both DNA and RNA degradation. While the in vitro 50 ! 30 RNase activity of BGLF5 is consistent with the observed host shutoff phenotype in cells, several observations indicate that RNA degradation in vivo likely involves additional cellular cofactors. First, the gammaherpesvirus SOX homologs seem to preferentially target mRNA rather than noncoding RNAs in the cytoplasm (Buisson et al., 2009; Glaunsinger and Ganem, 2004b). While it is possible that noncoding RNAs are spared from shutoff by their extensive secondary and tertiary structure, host shutoff factors may directly target mRNAs while they are engaged in translation. Such a mechanism has been suggested for the HSV-1 vhs RNase, perhaps as a consequence of its interactions with the translation initiation complex (Elgadi and Smiley, 1999; Feng et al., 2001, 2005; Page and Read, 2010). Second, BGLF5 RNase activity is 1–2 orders of magnitude weaker than its DNase activity, and may require nonphysiologic levels of Mn2þ (Buisson et al., 2009). Significantly, similar in vitro RNase activity has been documented for recombinant HSV-1 AE with reduced efficiency on RNA substrates (Kehm et al., 1998; Knopf and Weisshart, 1990), while HSV-1 AE does not promote host shutoff in cells (Covarrubias et al., 2009; Glaunsinger and Ganem, 2004b). Moreover, we have observed that recombinant SOX and HSV-1 AE exhibit weak in vitro RNase activities, as do SOX mutants selectively defective for RNA turnover in cells, suggesting that specificity in vivo requires one or more cellular cofactors (B.A. Glaunsinger, unpublished
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observations). Random mutagenesis screens conducted with SOX and BGLF5 confirm that their in vitro DNase and in vivo host shutoff activities can be genetically separated, although many mutants lose both functions (Glaunsinger et al., 2005; Zuo et al., 2008). Interestingly, single function mutations map to the protein surface, distal to the catalytic core (Dahlroth et al., 2009). Thus, it is possible that while the active site is required both for host shutoff and DNase activity, additional residues modulate each distinct function and mediate specificity, perhaps through protein– protein or nucleic acid interactions.
C. Hyperadenylation and nuclear retention of nascent messages While the decay of mRNA in the cytoplasm may largely explain the global decrease in host protein synthesis during infection, such an effect could, in principle, be dampened by continual repopulation of the cytoplasm with newly transcribed messages. Indeed, many viruses that promote host shutoff also restrict upstream events such as transcription, RNA processing, or nuclear export. For example, in addition to vhs-mediated mRNA turnover, alphaherpesviruses encode a second host shutoff factor, ICP27, which inhibits splicing (reviewed in Sandri-Goldin, 2008), although its KSHV homolog MTA does not reduce expression of intronbearing transcripts (Kirshner et al., 2000). Interestingly, mRNAs with extended poly(A) tails (hyperadenylated) accumulate in SOX and muSOX-expressing cells (Covarrubias et al., 2009; Lee and Glaunsinger, 2009). SOX-induced hyperadenylation of reporter messages is reduced upon siRNA-mediated depletion of the nuclear poly (A) binding protein (PABPN) or the canonical mRNA poly(A) polymerase (PAP II), indicating that this process involves the normal cellular polyadenylation machinery (Lee and Glaunsinger, 2009). It is notable that like the other host shutoff-related phenotypes, hyperadenylation is achieved by the cytoplasmic fraction of SOX and muSOX, indicating that it occurs downstream of one or more cytoplasmic activities of SOX (Covarrubias et al., 2009). Given that poly(A) tails stabilize mRNAs, it may seem counterintuitive that host shutoff induces hyperadenylation. While hyperadenylation is not well characterized in higher eukaryotes, in yeast it has been shown to occur in the nucleus in association with mRNA processing and export defects (Glaunsinger and Lee, 2010; Hilleren and Parker, 2001; Jensen et al., 2001; Libri et al., 2002). Hyperadenylated mRNAs are generally retained in the nucleus as a consequence of nuclear quality control pathways and do not enter the translating pool of messages. Similarly, SOX and muSOX-expressing cells exhibit an accumulation of endogenous poly(A)þ RNA in the
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nucleus, indicative of an export failure. SOX mutants that lack shutoff activity fail to promote hyperadenylation of reporter mRNAs or endogenous nuclear poly(A)þ RNA accumulation (Covarrubias et al., 2009; Lee and Glaunsinger, 2009), indicating that this phenotype is linked to host shutoff.
D. Relocalization of poly(A) binding protein One clue as to how cytoplasmic SOX may influence nuclear events comes from the observation that during host shutoff, KSHV SOX and MHV-68 muSOX trigger relocalization of the cytoplasmic poly(A) binding protein (PABPC) to the nucleus (Covarrubias et al., 2009; Lee and Glaunsinger, 2009). PABPC forms an integral part of the cellular translation machinery, linking the 30 poly(A) tail of an mRNA to the cap-binding preinitiation complex, forming a closed loop (reviewed in Jackson et al., 2010). PABPC nuclear accumulation is also observed during lytic but not latent KSHV infection of endothelial and B cells (Arias et al., 2009; Lee and Glaunsinger, 2009). While the mechanism by which SOX causes PABPC relocalization has not yet been determined, no direct interaction between these two proteins has been observed. However, nuclear import of PABPC is linked to the host shutoff function of SOX, as host shutoff defective mutants fail to relocalize PABPC. Furthermore, similar to mRNA turnover activity, PABPC relocalization is accomplished by the cytoplasmic population of SOX and muSOX (Covarrubias et al., 2009). Removal of PABPC from the cytoplasm would be predicted to inhibit translation and destabilize mRNAs, two phenotypes observed during host shutoff. However, knockdown of PABPC reduces SOX’s mRNA depletion activity (Lee and Glaunsinger, 2009), suggesting that this protein plays an active role in host shutoff. Alternatively, PABPC may be retained in the cytoplasm as a consequence of its association with polyadenylated RNA, and SOXinduced degradation of mRNA could release PABPC from poly(A) tails allowing its accumulation in the nucleus. While PABPC and PABPN bear no significant homology, their shared capacity to bind poly(A) could create a competitive environment during polyadenylation, perhaps leading to mRNA processing defects. Indeed, nuclear accumulation of PABPC in the absence of SOX is sufficient to drive hyperadenylation (Kumar and Glaunsinger, 2010). It is notable that several other viruses that restrict host gene expression, including HSV-1, rotavirus, and Bunyamwera virus, have also been found to stimulate nuclear localization of PABPC during infection (Blakqori et al., 2009; Dobrikova et al., 2010; Harb et al., 2008; Salaun et al., 2010). Thus, PABPC relocalization may be a widely adopted mechanism by which viruses manipulate host gene expression.
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E. Evasion of host shutoff As detailed above, a number of viral factors induce expression of select cellular genes important for cell cycle control, immune evasion, and pathogenesis. However, to be effective, these gene expression changes must evade the downregulation of cellular gene expression during host shutoff. There are a number of mechanisms by which this can occur. The delayed onset of SOX expression likely provides a window of opportunity for early viral lytic proteins to upregulate cellular genes necessary for replication, or for encoding secreted factors that contribute to KSHV biology in a paracrine manner. Alternatively, a strong induction of a cellular gene might partially balance the loss to host shutoff. The viral G protein-coupled receptor (vGPCR, ORF74) is associated with enhanced expression of a variety of proangiogenic, proliferative, and cell survival paracrine factors (reviewed in Jham and Montaner, 2010). However, only a subset of genes induced by vGPCR in isolation are induced during lytic infection in the context of host shutoff (Chandriani and Ganem, 2007; Glaunsinger and Ganem, 2004a), although, of the remaining genes, not all are downregulated. Finally, specific cellular genes could be refractory to shutoff by SOX. Microarray analyses of lytically reactivated KSHVinfected cells revealed that while over 75% of cellular mRNA transcripts are susceptible to host shutoff, nearly one-quarter of mRNAs are either unaffected or upregulated throughout the lytic cycle (Chandriani and Ganem, 2007; Glaunsinger and Ganem, 2004a). Most prominent among the genes that escape host shutoff during KSHV infection is the cellular IL-6 mRNA. Though transcriptionally induced by KSHV RTA as described above, the IL-6 transcript is normally short-lived, featuring a number of destabilizing elements in its 30 UTR (Paschoud et al., 2006). In addition to IL-6, other host shutoff escapees were determined to be enriched for the normally destabilizing AU-rich element (ARE) in their 30 UTRs (Chandriani and Ganem, 2007). The KSHV kaposin B protein has been shown to stabilize ARE-bearing mRNAs by binding and activating the MK2 kinase, an inhibitor of ARE-stimulated decay pathways (McCormick and Ganem, 2005). Primarily a latent gene, kaposin B is also expressed during the lytic cycle (Sarid et al., 1998) and thus may contribute directly to the ability of IL-6 and other ARE-containing mRNA to remain abundant throughout host shutoff. However, in cotransfection experiments IL-6 was shown to be refractory to SOXinduced turnover even in the absence of kaposin B, suggesting it possesses cis-acting elements that confer escape from host shutoff (Glaunsinger and Ganem, 2004a). The wide variety of mechanisms for inducing IL-6 transcription and stabilizing its mRNA during both latent and lytic infection speak to its importance for EBV and KSHV biology.
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V. CONCLUSIONS Lytic cycle-induced regulation of host gene expression helps establish an appropriate environment for viral replication, immune evasion, and pathogenesis through both cell-autonomous and paracrine mechanisms. Analysis of these interactions has also revealed some common themes by which viruses intercalate their own gene expression mechanisms with those of the host. The inclusion of cellular response elements in viral promoters, for example, allows the virus to take advantage of pathways nonspecifically stimulated by viral infection, whereas viral factors can selectively interfere with those same networks to customize cellular gene expression. While much effort has gone into identifying host genes that are differentially regulated by individual viral proteins, it is important to evaluate such changes in the context of other viral factors, as some of the reported effects are clearly antagonistic. In addition, relating the abundance of these gene expression changes to the observed biology of the viruses in vivo continues to be an important challenge. There is also significant interest in dissecting the complexity of stimuli that induce lytic reactivation, as this represents a possible antiviral and antitumor strategy to eliminate the reservoir of dormant virus that is resistant to antiviral treatments (Swaminathan and Kenney, 2009). The role of lytic genes in tumor maintenance and viral transmission underscores the importance of studying the effects of lytic infection on cellular gene expression in order to elucidate gammaherpesvirus biology and tumorigenesis, while potentially also revealing novel cellular pathways that regulate diverse aspects of the cell and the organism.
ACKNOWLEDGMENTS We are grateful to members of the Glaunsinger lab for careful reading of this chapter and helpful comments. Funding for this work was provided by the Burroughs Wellcome Fund, Investigators in the Pathogenesis of Infectious Disease Award (to B. A. G.), and the Ruth L. Kirschstein National Research Service Award (F32AI080082 to K. C.).
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Steed, A. L., Barton, E. S., Tibbetts, S. A., Popkin, D. L., Lutzke, M. L., Rochford, R., and Virgin, H. W., IV (2006). Gamma interferon blocks gammaherpesvirus reactivation from latency. J. Virol. 80(1):192–200. Swaminathan, S. (2005). Post-transcriptional gene regulation by gamma herpesviruses. J. Cell. Biochem. 95(4):698–711. Swaminathan, S. (2008). Noncoding RNAs produced by oncogenic human herpesviruses. J. Cell. Physiol. 216(2):321–326. Swaminathan, S., and Kenney, S. (2009). The Epstein-Barr virus lytic life cycle. In ‘‘DNA Tumor Viruses’’, (B. Damania and J. M. Pipas, eds.), pp. 285–315. Springer Science/ Business Media, New York. Swenson, J. J., Mauser, A. E., Kaufmann, W. K., and Kenney, S. C. (1999). The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction. J. Virol. 73 (8):6540–6550. Takaoka, A., and Yanai, H. (2006). Interferon signalling network in innate defence. Cell. Microbiol. 8(6):907–922. Tang, S., and Zheng, Z. M. (2002). Kaposi’s sarcoma-associated herpesvirus K8 exon 3 contains three 5’-splice sites and harbors a K8.1 transcription start site. J. Biol. Chem. 277 (17):14547–14556. Tanner, J., and Tosato, G. (1991). Impairment of natural killer functions by interleukin 6 increases lymphoblastoid cell tumorigenicity in athymic mice. J. Clin. Investig. 88 (1):239–247. Thiel, G., and Cibelli, G. (2002). Regulation of life and death by the zinc finger transcription factor Egr-1. J. Cell. Physiol. 193(3):287–292. Tomita, M., Choe, J., Tsukazaki, T., and Mori, N. (2004). The Kaposi’s sarcoma-associated herpesvirus K-bZIP protein represses transforming growth factor beta signaling through interaction with CREB-binding protein. Oncogene 23(50):8272–8281. Tsai, S. C., Lin, S. J., Chen, P. W., Luo, W. Y., Yeh, T. H., Wang, H. W., Chen, C. J., and Tsai, C. H. (2009). EBV Zta protein induces the expression of interleukin-13, promoting the proliferation of EBV-infected B cells and lymphoblastoid cell lines. Blood 114 (1):109–118. van Boxel-Dezaire, A. H., and Stark, G. R. (2007). Cell type-specific signaling in response to interferon-gamma. Curr. Top. Microbiol. Immunol. 316:119–154. Verma, D., and Swaminathan, S. (2008). Epstein-Barr virus SM protein functions as an alternative splicing factor. J. Virol. 82(14):7180–7188. Wang, Y., and Yuan, Y. (2007). Essential role of RBP-J kappa in activation of the K8 delayedearly promoter of Kaposi’s sarcoma-associated herpesvirus by ORF50/RTA. Virology 359 (1):19–27. Wilkinson, D. E., and Weller, S. K. (2003). The role of DNA recombination in herpes simplex virus DNA replication. IUBMB Life 55(8):451–458. Wu, F. Y., Tang, Q. Q., Chen, H., ApRhys, C., Farrell, C., Chen, J., Fujimuro, M., Lane, M. D., and Hayward, G. S. (2002). Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-alpha. Proc. Natl. Acad. Sci. USA 99 (16):10683–10688. Wu, F. Y., Chen, H., Wang, S. E., ApRhys, C. M., Liao, G., Fujimuro, M., Farrell, C. J., Huang, J., Hayward, S. D., and Hayward, G. S. (2003a). CCAAT/enhancer binding protein alpha interacts with ZTA and mediates ZTA-induced p21(CIP-1) accumulation and G(1) cell cycle arrest during the Epstein-Barr virus lytic cycle. J. Virol. 77 (2):1481–1500. Wu, F. Y., Wang, S. E., Tang, Q. Q., Fujimuro, M., Chiou, C. J., Zheng, Q., Chen, H., Hayward, S. D., Lane, M. D., and Hayward, G. S. (2003b). Cell cycle arrest by Kaposi’s sarcoma-associated herpesvirus replication-associated protein is mediated at both the
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2 Adaptive Immunity to the Hepatitis C Virus Christopher M. Walker
Contents
Abstract
I. Introduction II. Patterns of HCV Replication III. Humoral Immunity to HCV A. Methods to study HCV entry and neutralization B. HCV attachment and entry C. Mapping of neutralization epitopes D. Antibody responses and infection outcome E. Attenuation and evasion of the humoral immune response IV. Cellular Immunity to HCV A. Methods to study HCV-specific T cell immunity B. Acute phase T cell responses C. Mechanisms for evading and silencing T cell responses V. Immunity Acquired by Natural Infection Can Protect Against HCV Persistence: Implications for Vaccination VI. Summary References
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The hepatitis C virus (HCV) is a global public health problem affecting approximately 2% of the human population. The majority of HCV infections (more than 70%) result in life-long persistence of the virus that substantially increases the risk of serious liver diseases, including
The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA Advances in Virus Research, Volume 78 ISSN 0065-3527, DOI: 10.1016/S0065-3527(10)78002-7
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2010 Elsevier Inc. All rights reserved.
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cirrhosis and hepatocellular carcinoma. The remainder (less than 30%) resolves spontaneously, often resulting in long-lived protection from persistence upon reexposure to the virus. To persist, the virus must replicate and this requires effective evasion of adaptive immune responses. In this review, the role of humoral and cellular immunity in preventing HCV persistence, and the mechanisms used by the virus to subvert protective host responses, are considered.
I. INTRODUCTION The existence of hepatitis C virus(es) was first predicted 35 years ago to explain transfusion-associated liver disease in individuals not infected with the hepatitis A or B viruses (Alter et al., 1975; Feinstone et al., 1975; Prince et al., 1974). The description in 1989 of a single hepatitis C virus (HCV) that caused most posttransfusion and community acquired non-A non-B hepatitis marked a significant turning point toward understanding the epidemiology, natural history, and pathogenesis of this disease (Choo et al., 1989; Houghton, 2009). Seroepidemiology studies indicate that HCV has infected approximately 2% of the world’s population. The virus establishes persistent, life-long viremia in about 75% of infected humans and significantly increases the risk of progressive liver diseases, including inflammation, cirrhosis, and hepatocellular carcinoma. The discovery of HCV has also facilitated the development of new small molecule inhibitors of virus replication (designated STAT-C agents) that will soon be an adjunct to, and perhaps eventually replace, current standard therapy with pegylated type I interferon and ribavirin that is toxic, expensive, and frequently ineffective (Shimakami et al., 2009). HCV is a member of the Flaviviridae and the prototype virus in the hepacivirus genus (Moradpour et al., 2007). It has a small RNA genome of about 10,000 nucleotides encoding a single polyprotein of 3000 amino acids that is processed by host cell and viral proteases into 10 in-frame proteins (Moradpour et al., 2007). At least one small frame-shifted protein of unknown function is also produced. Structural proteins include a core or nucleocapsid and two envelope glycoproteins. Seven nonstructural proteins are important for HCV replication. There are at least six distinct genotypes that can be further classified into subtypes defined by phylogenetic relationships (Simmonds et al., 2005). HCV circulates as a population of different but closely related genomes in infected individuals (Simmonds et al., 2005). How the virus manages to avoid immune responses and establish life-long persistence is still a mystery. It is apparent that most viral proteins important for HCV replication also participate in evasion of innate and/or adaptive immune responses. As an example, the NS3 helicase/protease is critical for HCV replication and a prime
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target for small-molecule STAT-C inhibitors. NS3 protease activity also disrupts induction of innate immune defenses through RIG-I (retinoic acid inducible gene 1) and toll-like receptor 3 (TLR-3) sensors by cleavage of cellular intermediates important in signal transduction (Foy et al., 2003; Gale and Foy, 2005; Li et al., 2005). Despite the tremendous efficiency of HCV in establishing persistence, spontaneous clearance of infection in some individuals provides optimism that chronic hepatitis C can be prevented by vaccination and perhaps treated by immunotherapeutic approaches.
II. PATTERNS OF HCV REPLICATION HCV replication and adaptive immune responses have been studied in humans and chimpanzees, the only species other than man with known susceptibility to infection. Transmission of non-A non-B hepatitis from humans to chimpanzees provided an animal model for initial characterization of the agent as a small enveloped RNA virus and paved the way for molecular cloning of the HCV genome (Alter et al., 1978; Hollinger et al., 1978; Tabor et al., 1978). Although there have been few detailed studies of the issue, spontaneous resolution of HCV infection may be more common in chimpanzees than in humans (Bassett et al., 1998; Lanford et al., 2001). Moreover, these animals do not develop serious progressive liver disease, at least in the time frame described for most human infections. Despite these differences, chimpanzees have been invaluable for comparison of immunity in HCV infections that spontaneously resolve or persist. The chimpanzee model has several important advantages for the study of HCV-specific humoral and cellular immunity. Animals can be infected with genetically defined strains of HCV, including molecular clones that are sequence-matched with antigens used to probe immunity. Moreover, serial liver and blood samples can be collected from the earliest times after virus challenge for studies of immunity. Patterns of acute phase HCV replication are well defined in both species (Abe et al., 1992; Fang et al., 2003; Larghi et al., 2002; Spada et al., 2004; Thimme et al., 2001, 2002). HCV RNA is detectable in serum within a few days of exposure to the virus and usually peaks 8–12 weeks later when serum transaminases are elevated. Three common patterns of viremia have been observed (Fig. 1). The first pattern leads to spontaneous resolution of infection. Peak viremia and serum transaminase levels are typically observed 8-12 weeks after infection and then drop sharply. This is followed by permanent clearance of HCV RNA from serum, sometimes after several weeks or months of low-level, fluctuating viremia. Spontaneous resolution appears to result in long-lived immunity that, at least for some, provides protection against HCV persistence upon reexposure to the virus (see Section IV). The second pattern is
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Polyclonal
Phenotype PD-1 ++ − CD127 Sustained
+/− ++
Oligoclonal
++ − Transient
+++ +/−
Weak/absent
Viremia
Function IFN-g
Poly/oligoclonal
FIGURE 1 Three patterns of viremia have been described during the acute phase of HCV infection. In individuals who clear the infection (left panel), viremia peaks several weeks after infection when functional polyclonal CD4þ and CD8þ T cell responses targeting epitopes in most viral proteins are first detected in blood. CD8þ T cells lose expression of the coinhibitory molecule PD-1 and gain expression of the CD127 IL-7 receptor that is required for self-renewal of memory populations. Transient control of viremia can be observed for several months to a year after infection (middle panel). As indicated in the panel, these infections frequently persist, but resolution has also been observed after a prolonged period of low-level, fluctuating viremia. The end of transient virus control is associated with loss of CD4þ T helper cell function. Infections that persist without transient control of viremia have been described (right panel). T cell responses, if detected, are not sustained and usually target a limited number of epitopes. Most CD8þ T cells express high levels of PD-1. CD127 is low or absent. This exhausted phenotypic profile may be attenuated if the targeted viral epitope acquires an escape mutation (see Section IV, C1 and C2 for details).
difficult to distinguish from the first because the sharp initial decline in acute phase viremia is also followed by a period of partial control so effective that HCV RNA is often intermittently undetectable in serum (Fig. 1). However, after a variable period of time (sometimes up to 1 year or more), viremia that is low and fluctuating transitions to a high, stable pattern characteristic of chronic hepatitis C (Abe et al., 1992; McGovern et al., 2009; Mosley et al., 2008; Thimme et al., 2001, 2002). In the third pattern of viremia, limited or no control of virus replication is observed before persistence is established (Fig. 1). Viremia is remarkably stable in the chronic phase of infection, although steady-state levels vary over a 2–3 log10 range among infected individuals (Arase et al., 2000; Fanning et al., 2000; Gordon et al., 1998; Nguyen et al., 1996; Thomas et al., 2000). In this review, recent evidence that adaptive immunity influences the pattern of acute phase virus replication and the outcome
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of infection is considered. Mechanisms of immune evasion important to persistence of this small RNA virus are also discussed.
III. HUMORAL IMMUNITY TO HCV It has been known for two decades that seroconversion is substantially delayed during acute hepatitis C, with serum antibodies appearing several weeks after the initiation of virus replication regardless of infection outcome (Chen et al., 1999). Progress toward understanding the role of antibodies in HCV infection has been slow. This is due almost entirely to the technically challenging task of studying HCV attachment and entry into host cells, and whether this process is susceptible to neutralization by antibodies generated during infection. In the absence of a cell culture system supporting HCV replication, surrogate assays for virus binding, entry, and neutralization were developed using cell lines presumed to express viral receptors. As reviewed below, HCV ligands used in these cell culture models gradually evolved in sophistication from soluble recombinant envelope glycoproteins to synthetic virus-like particles (VLP) and finally retrovirus particles pseudotyped with HCV envelope glycoproteins (HCVpp). With the relatively recent advent of HCV strains that can complete the entire replication cycle in cell culture (designated HCVcc), it has been possible to validate and extend insights into the requirements for HCV entry, antibody-mediated neutralization, and evasion mechanisms.
A. Methods to study HCV entry and neutralization Chinese hamster ovary (CHO) cell production of a soluble recombinant E2 protein that was fully glycosylated and capable of binding conformationdependent antibodies provided the first key reagent for studying HCV attachment to cells (Rosa et al., 1996). Binding of recombinant E2 to the Molt-4 T cell line as quantified by flow cytometry represented a key breakthrough in early efforts to identify cellular receptors for HCV and develop surrogate assays for antibody neutralization (Rosa et al., 1996). Expression cloning of cDNA libraries from Molt-4 cells revealed that the tetraspanin CD81 bound soluble recombinant E2 with high affinity (Pileri et al., 1998) and this interaction could be blocked by antibodies to E2 or CD81 (Rosa et al., 1996). Noninfectious VLP produced from insect cells that expressed the HCV core, E1 and E2 proteins have also been used to assess antibody-mediated blockade of cellular entry (Baumert et al., 2000). The approaches were largely superseded by the development of pseudoparticles containing rhabdo- (Lagging et al., 2002; Meyer et al., 2000) or retro(Bartosch et al., 2003c; Hsu et al., 2003) virus core particles bearing HCV envelope glycoproteins. HCVpp containing retrovirus core particles are
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now widely used in entry and neutralization assays. They offer distinct advantages for studying the virus–host cell interaction, including the ability to pseudotype retrovirus core particles with E1 and E2 glycoproteins from different HCV strains and direct visualization of target cell transduction by incorporation of a reporter gene (Bartosch et al., 2003c; Hsu et al., 2003). As detailed below, HCVpp facilitated studies of the molecular interaction between E2 and cellular receptors, and neutralization of HCV entry into hepatoma cells by antibodies from infected or vaccinated humans and animals (Bartosch et al., 2003a; Logvinoff et al., 2004). HCVpp assays were validated by correlating in vitro neutralization with antibody-mediated protection of chimpanzees from infection (Bartosch et al., 2003a). While the HCVpp model remains important, the demonstration that select genotype 1a (Yi et al., 2006), 1b (Silberstein et al., 2010), and 2a (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005) HCVcc strains productively infect cultured cells provided a major new approach to dissect virus–host cell interactions. An HCV replicon containing all structural and nonstructural genes of a genotype 2a virus from a Japanese patient with fulminant hepatitis replicated in hepatoma cells without adaptive mutations and produced particles that could initiate infection in chimpanzees. HCVcc containing complete or recombinant JFH-1 genomes are now the most widely used adjunct to HCVpp for studies of virus neutralization and receptor-mediated entry into cells (Lindenbach et al., 2005; Zhong et al., 2005).
B. HCV attachment and entry There is a consensus that E1 and E2 are critical for cellular attachment and entry because infection fails if HCVpp lack one of these envelope glycoproteins (Bartosch et al., 2003c; Drummer et al., 2003; Hsu et al., 2003). E1 and E2 exist as heterodimers in the lipid bilayer of the virus (Dubuisson and Rice, 1996; Lavie et al., 2007) and mediate cellular entry via clathrindependent endocytosis, delivering the viral genome from the early endosome to the cytoplasm by a pH-dependent fusion process (Blanchard et al., 2006; Meertens et al., 2006). One recent study integrated a model of E2 tertiary structure based on the position of nine disulfide bonds with published functional data to obtain a tentative map of the CD81 binding site and identify a candidate loop involved in fusion (Krey et al., 2010). However, crystal structures of the envelope glycoproteins have not yet been solved and so there is limited information on the location of neutralizing B cell epitopes relative to the functional domains of E2. Internalization of HCVpp and HCVcc is, with few exceptions, restricted to cultured cells of the hepatocyte lineage, suggesting that the HCV host range is defined at least in part by the distribution of cellular receptor(s) for the virus (Bartosch et al., 2003b; Flint et al., 2006; Hsu et al., 2003; Lavillette
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et al., 2005b). The low-density lipoprotein (LDL) receptor (Molina et al., 2007), the C-type lectins dendritic cell (DC), and liver/lymph-node (L) specific intracellular adhesion molecule-3-grabbing integrin (DC-SIGN and L-SIGN, respectively; Cormier et al., 2004; Lozach et al., 2003, 2004) and/or glycosaminoglycans (Barth et al., 2006) may facilitate virus attachment to target cells. For instance, ApoE associated with the virion can bind the LDL receptor on hepatocytes and blockade of this interaction interferes with HCV entry (Burlone and Budkowska, 2009; Owen et al.,2009). Four cellular proteins are required for virus internalization in cell culture models. These include CD81 (Bartosch et al., 2003b; Hsu et al., 2003; Lindenbach et al., 2005; Pileri et al., 1998), the scavenger receptor class member B1 (SR-B1; Bartosch et al., 2003b; Grove et al., 2007; Kapadia et al., 2007; Scarselli et al., 2002) and the tight junction proteins, claudin-1 (Evans et al., 2007) and occludin (Benedicto et al., 2009; Liu et al., 2009; Ploss et al., 2009). Claudin-6 and claudin-9 may substitute for claudin-1 in this process (Meertens et al., 2008; Zheng et al., 2007). Successful HCVpp and HCVcc infection of nonpermissive cells that coexpressed SR-B1, CD81, claudin-1, and occludin demonstrated that all four proteins were necessary for viral entry (Ploss et al., 2009). This approach involving transfection of human genes into rodent cells also facilitated mapping of cellular receptors that govern the species specificity of HCV entry. Receptor orthologues of rodent origin bind E2 with reduced efficiency and may interact less efficiently with human receptor components (Catanese et al., 2010; Haid et al., 2010; Ploss et al., 2009). How HCV is thought to interact with these cellular proteins for attachment, entry, and release of the viral genome into the cytoplasm of target cells is reviewed below. CD81 and SR-B1 were first identified as viral receptors based on their physical association with soluble recombinant E2 proteins. The CD81 tetraspanin is widely expressed on human tissues and is important for signal transduction in a variety of cells, including B and T lymphocytes (Levy and Shoham, 2005). SR-B1 is restricted to liver cells and steroidogenic tissue and serves as a primary receptor for multiple ligands, including high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL; Krieger, 2001). Experimental evidence implicating these proteins in virus entry is strong. Antibodies directed against CD81 or SR-B1, and siRNA-mediated silencing of the genes encoding these cellular proteins, substantially impair entry of HCVpp and/or HCVcc (Bartosch et al., 2003b; Kapadia et al., 2007; Lavillette et al., 2005b; Lindenbach et al., 2005; Zhang et al., 2004). It is likely that they cooperate in the entry process because combination of antibodies to these cellular proteins also synergistically blocks HCVcc infection of hepatoma cells (Kapadia et al., 2007; Zeisel et al., 2007). Antibodies directed against CD81 can also block HCV infection of human hepatocytes grafted into immunodeficient mice (Meuleman et al., 2008). E2 binds the large extracellular loop (LEL) of CD81 via multiple
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discontinuous amino acids that have been mapped using monoclonal antibodies and by mutagenesis of E2 domains (Stamataki et al., 2008), and by identification of residues required for adaptation of E2 binding to murine CD81 (Bitzegeio et al., 2010). SR-B1 is thought to bind hypervariable region 1 (HVR-1) that is located at the N-terminus of the E2 glycoprotein. Cellular entry of HCVpp that lack this E2 sequence cannot be blocked by SR-B1-specific antibodies (Bartosch et al., 2005). HCV and HDL appear to interact with distinct regions of SR-B1 because mutagenesis of the receptor impaired HCV infectivity without loss of HDL binding (Catanese et al., 2010). SR-B1 is involved in very early steps in virus entry, perhaps including initial virus attachment (Catanese et al., 2010), but a role in postbinding steps is also suggested by SR-B1 antibody blockade studies (Zeisel et al., 2007). Finally, infection of hepatoma cells can be enhanced or inhibited by HDL and oxidized LDL, respectively, indicating that the interplay between SR-B1, its natural ligands, and the virus is complex and not yet fully understood (Bartosch et al., 2005; Dreux et al., 2006; Voisset et al., 2006; von Hahn et al., 2006). Soluble recombinant E2 was not used to identify claudin-1 and occludin as HCV receptors. Instead, these tight junction proteins were implicated in HCV entry by an iterative cloning strategy that depended on expression of cDNA libraries in cells that were not fully permissive for HCV infection. Physical interaction of these cellular proteins with E2, if it occurs, is poorly understood. An association between occludin and E2 in the endoplasmic reticulum has been reported (Benedicto et al., 2009), although the nature of the interaction and how it governs the entry process remains to be defined. Reduced expression of claudin-1 and occludin inhibited HCV glycoprotein-dependent cell-to-cell fusion, suggesting that both tight junction proteins act at a late stage of virus entry (Benedicto et al., 2009; Evans et al., 2007). How the virus contacts claudin1 and occludin is unknown. The interaction of E2 and CD81 activates GTPase and this may mediate actin-dependent relocalization of the E2CD81 complex to cell contact sites that contain tight junction proteins (Brazzoli et al., 2008). A physical interaction between claudin-1 and CD81 that is critical for HCV infection has been documented by mutagenesis (Harris et al., 2010) and antibody blockade studies (Krieger et al., 2010). Whether HCV enters cells via tight junctions is unknown, but two recent studies suggest that this is not essential at least in culture models involving hepatoma cell lines. Pools of nonjunctional claudin-1 that can complex with CD81 have been observed at the basolateral membrane of polarized hepatoma cells (Mee et al., 2009). Visualization of fluorescent-labeled HCV particles as they are internalized has provided evidence of a complex involving the virus, claudin-1, and CD81 that forms outside tight junctions (Coller et al., 2009; Harris et al., 2010; Mee et al., 2009). This model is attractive as it is consistent with virus entry into the liver through the
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sinusoidal blood supply and subsequent association with receptors on the basal surface of polarized hepatocytes (Stamataki et al., 2008).
C. Mapping of neutralization epitopes E2 contains continuous (linear) and discontinuous (nonlinear) neutralizing epitopes involved in binding to CD81 and/or the SR-B1 viral receptors. Two of the best characterized regions containing linear epitope(s) are (i) the HVR-1 spanning amino acids 384–401 and (ii) an adjacent sequence spanning amino acids 413–420 of E2 (Owsianka et al., 2001, 2005; Tarr et al., 2006, 2007). Antibodies against HVR-1 are found in most if not all naturally infected humans but are isolate-specific and evolve continuously. A temporal correlation between the appearance of anti-HVR antibodies and the emergence of HCV variants provided the first presumptive evidence of an acute phase neutralizing response (Farci et al., 2000; Kato et al., 1993, 1994; Taniguchi et al., 1993; Weiner et al., 1992). Conversely, limited or no HVR-1 diversification was observed in viruses from humans with hypogammaglobulinemia or chimpanzees that failed to generate antibodies to E2 (Bassett et al., 1998; Booth et al., 1998; Penin et al., 2001; Puntoriero et al., 1998; Ray et al., 2000). Passive neutralization of HCV infectivity by antiHVR antibodies before challenge of chimpanzees provided direct evidence that HVR-1 contains neutralizing epitope(s) (Farci et al., 1996). The adjacent epitope spanning amino acids 413–420 that is involved in CD81 binding was first defined by monoclonal antibodies generated from rodents immunized with recombinant E2 (Owsianka et al., 2001, 2005; Tarr et al., 2006, 2007). In comparison to HVR-1, it is poorly recognized in naturally infected humans. Only 2.5% of sera from subjects with resolved or chronic hepatitis C recognized amino acids 413–420 of E2 (Tarr et al., 2007). The epitope defined by the rodent antibodies also differs from HVR-1 because it is highly conserved across most HCV genotypes (Broering et al., 2009; Owsianka et al., 2005). HCVcc with mutations in this conserved epitope can arise spontaneously (Dhillon et al., 2010) or by selection in the presence of the cognate neutralizing antibodies (Dhillon et al., 2010; Gal-Tanamy et al., 2008) during in vitro replication in hepatoma cells. Expanded use of these models of virus adaptation should provide new insights into the interaction of the virus with host cell receptors as well as mechanisms of antibody neutralization and evasion. A number of conformation-dependent epitopes in E2 were also defined using antibodies from HCV-infected humans ( Johansson et al., 2007; Keck et al., 2005, 2007; Law et al., 2008; Op De Beeck et al., 2004). Alanine-scanning mutagenesis and competitive binding assays with panels of human monoclonal antibodies, or antigen-binding fragments generated from phage display libraries, provided evidence for three conformational domains or regions (operationally designated A, B, and C)
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within E2 that are antigenic (Keck et al., 2004; Law et al., 2008). Antibodies targeting domain A epitopes are nonneutralizing and may be derived from isolated envelope proteins rather than virions (Keck et al., 2005; Law et al., 2008). Antibodies specific for domain B and C epitopes neutralized infectivity, probably by competitive inhibition of the CD81–E2 interaction. In support of this concept, many of the amino acids required for binding of the domain B antibodies (G530, D535, and W529; Johansson et al., 2007; Keck et al., 2008a,b, 2009; Law et al., 2008; Owsianka et al., 2008; Perotti et al., 2008) were identical to residues critical for E2 binding to CD81 ( Johansson et al., 2007; Owsianka et al., 2006; Rothwangl et al., 2008). One recent study documented that a broadly cross-reactive monoclonal antibody directed against domain B provided protection from HCV infection in mice reconstituted with human hepatocytes (Law et al., 2008).
D. Antibody responses and infection outcome Whether antibodies modify the course of HCV infection or contribute to spontaneous resolution is controversial. Direct evidence supporting involvement of the humoral immune response in control of HCV replication or resolution of infection is sparse. Spontaneous resolution without seroconversion has been observed in chimpanzees (Cooper et al., 1999) and humans (Christie et al., 1997; Post et al., 2004), including some with primary antibody deficiency (Christie et al., 1997). Although the number of subjects with hypogammaglubulinemia who permanently cleared viremia was small, this study provided support for the concept that anti-HCV antibodies are not necessarily required for a successful infection outcome (Christie et al., 1997). On the other hand, passive transfer of serum containing HCV immunoglobulins to chimpanzees substantially delayed, but did not prevent, replication of the virus upon challenge (Krawczynski et al., 1996), suggesting that antibodies can alter the trajectory of infection. Studies conducted before validated neutralization assays were available suggested an association between the rapid onset of an antibody response to E2 and infection outcome. As an example, subjects who spontaneously resolved HCV infection were more likely to have serum antibodies against the E2 HVR within the first 6 months of infection when compared with those who developed persistent viremia, even when the timing of antibody responses to the core or nonstructural proteins was approximately the same (Dittmann et al., 1991; Zibert et al., 1997). This result was corroborated and extended by more recent studies employing the HCVpp neutralization assay. In a single-source outbreak of hepatitis C involving multiple subjects, HCV clearance was associated with rapid induction of neutralizing antibodies in the early phase of infection (Pestka et al., 2007). However, it remains uncertain if the neutralizing antibodies
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had a primary role in containment of infection or were simply a surrogate marker of a more effective T cell response. A broad pattern of cross-reactivity against genetically diverse HCV strains by serum antibodies from persistently infected humans suggests continuous evolution of the virus. (Bartosch et al., 2003a; Kaplan et al., 2007; Lavillette et al., 2005b; Logvinoff et al., 2004; Meunier et al., 2005; Netski et al., 2005; Steinmann et al., 2004; von Hahn et al., 2007; Wakita et al., 2005). Few longitudinal studies have addressed this issue because construction of multiple HCVcc or HCVpp that incorporate serial envelope sequences that emerge over time is technically challenging. Nevertheless, HCVpp were used to study the evolution of neutralizing antibodies and envelope glycoprotein E2 in one subject, designated H, who provided serial serum samples over 26 years of acute and persistent HCV replication (von Hahn et al., 2007). Analysis of neutralization at the time of seroconversion revealed a response that was narrowly directed against the autologous virus (Logvinoff et al., 2004; von Hahn et al., 2007). Broadly cross-reactive antibodies were present 8 months later as persistent infection was established, but they did not neutralize HCVpp containing contemporaneous envelope glycoproteins. Emergence of the cross-reactive response was associated with diversification of E2 and loss of reactivity against the early HVR sequences encoded by the virus during the acute phase of infection (von Hahn et al., 2007). One recent detailed study of persistent viruses from subject H identified mutations in E2 that also facilitated escape from neutralizing antibodies targeting conformation-dependent domain B epitopes (Keck et al., 2009). Taken together, these data indicate that broadening of the neutralizing response was associated with loss of recognition of timematched envelope glycoproteins, and are consistent with continuous escape of HCV under selection pressure from antibodies (von Hahn et al., 2007). Another study documented that neutralizing antibodies drive HCV envelope sequence evolution in humans that develop chronic infections (Dowd et al., 2009). Importantly, resolution of acute hepatitis C was correlated with an antibody response that effectively neutralized autologous time-matched viruses (Dowd et al., 2009).
E. Attenuation and evasion of the humoral immune response Studies in subject H indicate that HCV evades antibody responses in part by mutational escape of conformation-dependent epitopes like those in domain B and linear epitopes like HVR-1. HVR-1 may also play a unique role in mutational escape by serving as a decoy for neutralizing antibody responses against other functionally important but less mutable epitopes (Mondelli et al., 2001; Ray et al., 1999; von Hahn et al., 2007). It may be well adapted for this role because chemicophysical properties of HVR-1 important to receptor-mediated cellular entry appear to be highly
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conserved (Penin et al., 2001; Puntoriero et al., 1998) despite exceptional immunogenicity and capacity for rapid mutation (Mondelli et al., 2001; Ray et al., 1999; von Hahn et al., 2007). In addition to serving as a decoy for neutralizing antibody responses, HVR-1 may physically protect or mask adjacent epitopes from B cell recognition. For instance, most humans fail to recognize the epitope spanning amino acids 413–420 defined by the rodent monoclonal antibodies (Tarr et al., 2007). It has been proposed that this CD81 binding site is concealed from immune recognition by residues 384–401 that comprise the HVR-1 domain (Bankwitz et al., 2010). At least two other mechanisms that attenuate antibody recognition of E2 neutralizing epitopes have been described. The 11 N-linked glycosylation sites of E2 are occupied and mutation of those sites located near residues important for CD81 binding increased the sensitivity of HCV to neutralization by monoclonal antibodies and patient serum (Falkowska et al., 2007; Helle et al., 2007). Finally, it appears that nonneutralizing antibodies can interfere with humoral responses against epitopes critical to virus entry. For instance, antibodies to two E2 epitopes (mapped to amino acids 412–426 and 434–446) were recently isolated from the plasma of humans with chronic hepatitis C. Peptide blockade or immunoaffinity depletion of serum antibodies against the 434–446 epitope revealed potent residual neutralizing activity against the adjacent 412–426 epitope of multiple HCV genotypes (Zhang et al., 2009). HCV may also evade neutralization by direct cell-to-cell transfer of virus (Timpe et al., 2008; Witteveldt et al., 2009) or masking of E2 epitopes by lipids. Density and sedimentation properties of virions in human serum provided evidence for a physical association with lipoproteins (Kanto et al., 1995; Nielsen et al., 2006; Prince et al., 1996), including b-lipoprotein (Agnello et al., 1999; Prince et al., 1996; Thomssen et al., 1992; Wunschmann et al., 2000). Furthermore, involvement of the VLDL pathway in the assembly and release of HCVcc particles from cultured hepatoma cells has now been documented (Chang et al., 2007; Huang et al., 2007; Lavillette et al., 2005a). The hypothesis that virions associated with lipoproteins are less sensitive to antibody-dependent neutralization is supported by the observation that HDL facilitates HCVpp infection, and reduces the sensitivity of the virus to antibody-mediated neutralization (Bartosch et al., 2005; Dreux et al., 2006; Lavillette et al., 2005a).
IV. CELLULAR IMMUNITY TO HCV Several observations indicate a critical role for cell-mediated immunity in spontaneous resolution of HCV infection. To summarize, early studies of infection in humans and chimpanzees established a temporal kinetic
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relationship between initial control of acute phase viremia and expansion of functional CD4þ helper and CD8þ cytotoxic T cells (Bowen and Walker, 2005a; Rehermann, 2009). To prevent persistence, these responses must be sustained past the point that viral genomes are eradicated from host cells. Indeed, as reviewed below, premature failure of the acute phase CD4þ T helper cell response may be the best predictor of persistence. Immunogenetic associations between the outcome of infection and expression of specific HLA class I and II alleles also support the concept that T cells influence the course of infection (Singh et al., 2007). As an example, one recent study confirmed that the DRB1*0101 class II allele and certain alleles belonging to the HLA-B*57 class I group were associated with an absence of HCV RNA in a large multiracial group of HCV seropositive women (Kuniholm et al., 2010). Animal studies have provided the most direct evidence for involvement of T cells in infection outcome. Antibodymediated depletion of CD4þ or CD8þ T cells from immune chimpanzees resulted in prolonged or persistent infection upon rechallenge with HCV (Grakoui et al., 2003; Shoukry et al., 2003). Moreover, vaccination of chimpanzees with genes encoding nonstructural proteins substantially blunted acute phase viremia after experimental challenge with HCV (Folgori et al., 2006). Because this genetic vaccine did not encode envelope glycoproteins that elicited neutralizing antibodies, it is reasonable to conclude that T cells primed by the nonstructural proteins enhanced acute phase control of HCV replication (Folgori et al., 2006).
A. Methods to study HCV-specific T cell immunity State of the art methods for determining the frequency, function, and phenotype of antiviral T cells have been adapted for the study of HCV infection in humans and chimpanzees (Yoon and Rehermann, 2009). Briefly, mononuclear cells are typically stimulated ex vivo with synthetic recombinant proteins or overlapping peptide sets that are usually closely matched to the polyprotein encoded by the HCV strain or genotype circulating in the infected individual(s). Virus-specific T cell responses are then measured using readouts like proliferation (for instance, incorporation of 3H-tdr or dilution of a membrane marker dye like CFSE) or cytokine production (by ELISpot or intracellular cytokine staining; Yoon and Rehermann, 2009). A key advantage of the cytokine readout is accurate quantification of functional HCV-specific T cells. If T cells lack function, as is frequently the case in chronic infections, direct visualization of virus-specific populations is preferable. Soluble tetrameric class I molecules that incorporate viral epitopes have been essential for visualization of HCV-specific T cells in persistent HCV infection (Shiina and Rehermann, 2009). Conjugation of a fluorophore to a class I ‘‘tetramer’’
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facilitates detection of HCV-specific T cells isolated from blood or liver by flow cytometry regardless of whether they retain effector functions. Lack of a cell culture model supporting virus replication hindered studies of the interaction between HCV-specific T cells and infected hepatocytes. This situation has improved with the development of genomic and subgenomic HCV replicons and HCVcc strains that replicate in hepatoma cells ( Jo et al., 2009; Soderholm et al., 2006; Uebelhoer et al., 2008). For instance, these models can be used to assess activation or inhibition of T lymphocytes by infected cells, susceptibility of virus replication to T cell cytotoxic activity or cytokine production, and the impact of immune escape mutations on viral fitness for replication ( Jo et al., 2009; Soderholm et al., 2006; Uebelhoer et al., 2008). Finally, there are some important limitations and caveats in the study of HCV-specific T cell immunity, particularly in persistently infected individuals. First and foremost, blood is the most accessible tissue but HCVspecific T cells are generally absent or present at low frequency in circulation (Bowen and Walker, 2005a). Whether circulating T cells are representative of intrahepatic populations is controversial. Most HCV-specific T cells are highly localized to the liver where effector functions and phenotype might differ. With the exception of explanted liver obtained during transplant, the tissue is commonly accessed only by percutaneous biopsy. From these small tissue samples, the number of recovered mononuclear cells is usually too low for direct assessment of T cell frequency, phenotype, or function. Moreover, serial liver biopsy of humans is uncommon, particularly during the acute phase of infection, and so evolution of the cellular immune response at the site of infection is difficult to study.
B. Acute phase T cell responses T cells are present in blood and liver of most humans and chimpanzees with acute hepatitis C but the response is remarkably delayed when compared with many other viral infections. Although the kinetic is highly variable, expansion of T cells in blood is often not evident until 8–12 weeks after infection, coincident with the peak in serum transaminases and initial control of viremia (Fig. 1; Bowen and Walker, 2005a; Rehermann, 2009). This exceptionally long delay in generation of primary T cell immunity is unexplained, but is observed in most infections regardless of outcome. Available data strongly suggest that successful control of infection requires cooperation between CD8þ cytotoxic and CD4þ helper T cells.
1. CD8þ T cells Acute phase CD8þ T cell responses in chimpanzees and humans that clear the infection are remarkably broad, targeting multiple unique class I restricted epitopes in all viral proteins (Cooper et al., 1999; Lechner et al.,
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2000b; Thimme et al., 2001, 2002), with the possible exception of the alternative reading frame protein designated F (Drouin et al., 2010). In blood CD8þ T cells frequencies against individual epitopes can exceed 3–4% when measured in functional assays or by tetramer analysis (Lechner et al., 2000b). Frequencies are almost certainly higher in the liver, the site of virus replication. During the acute phase of infection CD8þ T cells appear to expand in blood several days or weeks before they acquire effector functions (Lechner et al., 2000b; Shoukry et al., 2003; Thimme et al., 2001). The significance of this phenomenon is uncertain because effector activity (most notably the ability to produce IFN-g) eventually recovers and is evident regardless of whether infections ultimately resolve or persist. CD8þ T cells that expand during the acute phase of infection transiently express the coinhibitory molecule programmed cell death 1 (PD-1; Kasprowicz et al., 2008; Radziewicz et al., 2008; Urbani et al., 2006b) and activation markers including HLA class II, CD38, and CD69 (Appay et al., 2002; Lechner et al., 2000a; Thimme et al., 2001; Urbani et al., 2006b). Resolution of HCV infection results in contraction of the CD8þ T cell response and enhanced expression of molecules associated with long-term memory including BcL-2 that inhibits apoptosis and CD127, a component of the IL-7 receptor that is required for selfrenewal of memory populations (Badr et al., 2008; Golden-Mason et al., 2006; Urbani et al., 2006a). PD-1 expression is also lost or substantially reduced as virus load declines and the infection is terminated (Bowen et al., 2008; Kasprowicz et al., 2008; Radziewicz et al., 2007). There is considerable heterogeneity in the strength of acute phase CD8þ T cell responses in those who follow a chronic course. Responses can be transiently detected in the blood of many subjects during acute hepatitis C and sometimes target multiple epitopes (Cox et al., 2005a; Kaplan et al., 2007; Lauer et al., 2005; Lechner et al., 2000a). Differences in the vigor of acute phase CD8þ T cell activity are illustrated by studies in chimpanzees experimentally infected with the virus (Gottwein et al., 2010; Thimme et al., 2002). Two animals in a recent study developed a persistent infection after HCV challenge, but one had CD8þ T cell activity against all HCV proteins and transient control of replication. CD8þ T cells from the other animal targeted one HCV protein and exerted limited if any antiviral activity (Gottwein et al., 2010). Where CD8þ T cell populations have been tracked from the acute to chronic phase of infection, responses that initially target several epitopes narrow dramatically as persistence is established (Cox et al., 2005a; Lauer et al., 2005; Lechner et al., 2000a). Even though responses are often more narrowly focused in the chronic phase of infection, there is no apparent preference for epitopes in the structural, nonstructural, and alternative reading frame proteins (Bain et al., 2004; Ward et al., 2002). It is likely that effector functions are lost sequentially during the transition from the acute to chronic phase of
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infection, as is the case in LCMV infection of mice where cytotoxicity and production of IL-2 fail early in infection, followed by IFN-g production just before exhaustion is fully established (Shin and Wherry, 2007). Intrahepatic CD8þ T cells that are a remnant of an earlier acute phase response can be recovered from the liver of humans and chimpanzees several years after persistence is established (Grabowska et al., 2001; He et al., 1999; Koziel et al., 1992; Meyer-Olson et al., 2004; Nelson et al., 1997; Wong et al., 1998). Many of these populations target class I epitopes that acquired escape mutations years earlier. However, some recognize intact epitopes but provide no apparent control of HCV replication (Erickson et al., 2001; He et al., 1999; Koziel et al., 1992; Nakamoto et al., 2008; Nelson et al., 1997; Neumann-Haefelin et al., 2008; Spangenberg et al., 2005; Wong et al., 1998), probably because they lack effector functions due to exhaustion (Golden-Mason et al., 2007a; Gruener et al., 2001; Kasprowicz et al., 2008; Nakamoto et al., 2008; Radziewicz et al., 2007; Spangenberg et al., 2005). Exhausted HCV-specific CD8þ T cells are prone to apoptosis unless rescued in cell culture by cytokines like IL-2 (Radziewicz et al., 2007). Unlike CD8þ T cells from those who resolve infection, CD127 is usually reduced or undetectable and PD-1 is constitutively expressed (Golden-Mason et al., 2007a; Kasprowicz et al., 2008; Nakamoto et al., 2008; Radziewicz et al., 2007; Urbani et al., 2006b). How T cells targeting intact and mutated epitopes survive in the persistently infected liver without any apparent mechanism for self-renewal is uncertain but probably depends on constant stimulation with HCV antigens. One intriguing possibility is that lymph nodes draining the liver are a site of CD8þ T cell renewal in chronic hepatitis C. Perihepatic lymph nodes from individuals with advanced hepatitis C were recently shown to harbor HCV-specific CD8þ T cells that appeared to retain effector functions when compared with those in the liver and blood (Moonka et al., 2008). While most HCV-specific CD8þ T cells are exhausted, there is evidence that some populations have alternate functions that could facilitate persistence or modulate the course of infection. For instance, HCV-specific CD8þ T cells that produce IL-10 (Abel et al., 2006; Accapezzato et al., 2004; Kaplan et al., 2008; Rowan et al., 2008) and/or TGF-b (Alatrakchi et al., 2007; Rowan et al., 2008) have been observed in the blood and liver of persistently infected subjects. These cytokines are considered anti-inflammatory and have the potential to block effective antiviral immunity in acute or chronic phases of infection. Why CD8þ T cells would produce this set of suppressive cytokines is unclear, but one recent study has shown that ligation of the viral receptor CD81 drives naı¨ve CD4þ and CD8þ T cells to produce IL-13 (Serra et al., 2008). Antigen-specific stimulation of PBMC from individuals with chronic hepatitis C can also elicit IL-17, a proinflammatory mediator (Billerbeck et al., 2010; Rowan et al., 2008). Production of this cytokine appears to be associated with an expansion of CD8þ T cells that
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express CD161, a type C lectin also known as NKRP1A (Billerbeck et al., 2010; Northfield et al., 2008). Interestingly, CD8þ T cells with intense cell surface expression of CD161 had a unique phenotypic profile that included expression of the transcription factor retinoic acid-related orphan receptor g-t, cytokine receptors (IL-23R and IL-18R) and chemokine receptors including CXCR6 that is important for liver homing (Billerbeck et al., 2010). CD8þ T cells that expressed CD161 were markedly enriched in liver and have the potential to modulate HCV replication or liver disease by production of IL-17 and IL-22 (Billerbeck et al., 2010).
2. CD4þ T cells HCV-specific CD4þ T cells are particularly important to the outcome of infection, at least in part because they provide support for effector CD8þ T cells in acute hepatitis C. Loss of helper activity has been associated with poor CD8þ T cell function (Francavilla et al., 2004; Kaplan et al., 2007, 2008; Urbani et al., 2006a). Moreover, antibody-mediated depletion of CD4þ T cells from immune chimpanzees resulted in failure of CD8þ T cellmediated control of HCV replication and emergence of viruses with escape mutations in class I restricted epitopes (Grakoui et al., 2003). A key role for CD4þ T cells is also indicated by the striking temporal relationship between control of acute viremia and detection of a functional, multispecific CD4þ T cell response in blood as first documented using proliferation assays (Diepolder et al., 1995; Gerlach et al., 1999; Missale et al., 1996). Acute phase CD4þ T cells are generally present at lower frequency and target fewer epitopes in infections that persist, even though transient responses that are indistinguishable from acute resolving infections have been described (Thimme et al., 2001). More recently, HLA class II tetramers were used to visualize HCV-specific CD4þ T cells in the blood of all HCV-infected subjects regardless of infection outcome (Lucas et al., 2007). CD4þ T cell populations remain detectable in the circulation of subjects who resolved the infection but frequencies dropped below the level of detection in those who followed a persistent course (Day et al., 2003; Lucas et al., 2007). This is consistent with comprehensive mapping of CD4þ T cell responses in individuals with chronic versus resolved infections (Day et al., 2002; Schulze zur Wiesch et al., 2005). CD4þ T cells targeting multiple epitopes were detected in the blood of subjects with resolved but not chronic infections (Day et al., 2002; Schulze zur Wiesch et al., 2005). Studies in humans subjects have demonstrated that transient control of HCV replication is lost if CD4þ T cells lose the ability to proliferate or produce antiviral (proinflammatory) cytokines like IFN-g or IL-2 during the acute phase of infection (Gerlach et al., 1999; Ulsenheimer et al., 2003, 2006). HCV-specific CD4þ T cells can be recovered from the blood and liver of humans with chronic hepatitis C after stimulation with antigens and cytokines (Penna et al., 2002; Schirren et al., 2000) but whether they are functional in situ is unknown.
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An analysis of CD4þ T cells in peripheral blood demonstrated a selective loss in the ability to produce IL-2 even though IFN-g production was retained (Semmo et al., 2005). Explanations for spontaneous CD4þ T cell failure in some infections remain poorly developed even though it is perhaps the most reliable predictor of HCV persistence.
C. Mechanisms for evading and silencing T cell responses As noted above, CD4þ and CD8þ T cells are primed in most if not all HCV infections that persist. Deletion of primed HCV-specific T cells from the repertoire has not been formally demonstrated, although the virus can exploit holes in the TcR repertoire in some individuals who develop a persistent infection (Wolfl et al., 2008). Multiple mechanisms have been proposed to explain the failure of intrahepatic T cells to control HCV infection. Inadequate priming by dysfunctional professional antigen presenting cells and/or an absence of innate signals may impair generation of an adaptive immune response (Szabo and Dolganiuc, 2008), but these features of HCV infection have not yet been precisely correlated with suboptimal T cell differentiation, survival, or effector function. Recombinant viral proteins can modulate T cell function in cell culture models by binding surface receptors. For instance, the HCV core protein can bind the gC1qR complement receptor on T cells, impairing proliferation and interferon-g production by inhibiting Stat phosphorylation of SOCS signaling molecules (Cummings et al., 2009). Similarly, ligation of CD81 on naive CD4þ and CD8þ T cells by the E2 glycoprotein was recently reported to drive production of IL-13 (Serra et al., 2008). It is important to emphasize that the relevance of these mechanisms identified in cell culture models to HCV persistence has not yet been established in humans or chimpanzees. How binding of an HCV protein to a receptor that is widely expressed on resting or activated lymphocytes leads to a very specific lesion in HCV-specific T cell immunity, without imposing a more global suppressive effect on all T cells, remains to be explained. Three mechanisms contributing to the evasion and silencing of T cell immunity against HCV will be considered in detail. They include (i) mutational escape of epitopes, (ii) expression of coinhibitory molecules that deliver negative signals to T cells, and (iii) regulatory T cell activity. Emerging data suggest that these mechanisms are interconnected and together may contribute to a profound, virus-specific defect in adaptive cellular immunity. Most published studies describe mechanisms of CD8þ T cell failure, but the fate of CD4þ T cells is also considered where data are available.
1. Mutational escape The positive-stranded RNA genome of HCV is replicated by the errorprone RNA-dependent RNA polymerase encoded by the viral NS5b gene. The lack of a proofreading mechanism imparts on the virus a remarkable
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capacity for adaptive mutation. Mutations in class II epitopes have been reported to skew patterns of cytokine production (Wang et al., 2003), and antagonize (Frasca et al., 1999) or abrogate recognition (von Hahn et al., 2007; Wang and Eckels, 1999) by HCV-specific CD4þ T cells. Nevertheless, a pervasive pattern of class I epitope escape has not been observed in naturally infected humans (Fleming et al., 2010) or chimpanzees (Fuller et al., 2010), except perhaps in animals vaccinated before virus challenge (Puig et al., 2006). It is possible that CD4þ T cells do not consistently exert selection pressure against the virus, perhaps because they fail very early in acute infection. These results reinforce the view that helper activity is silenced by other as yet unidentified mechanisms. CD8þ T cells, on the other hand, are a potent selective force that act on HCV genomes to enrich for immune escape variants (Bowen and Walker, 2005b). This was first demonstrated in chimpanzees experimentally infected with a genetically defined HCV inoculum (Weiner et al., 1995). Identification of class I MHC epitopes and sequencing of viruses that emerged during the acute and chronic phases of infection provided the first statistical evidence that CD8þ T cells exert Darwinian selection pressure against some (but not all) class I epitopes (Erickson et al., 2001). This mechanism is also operational in humans where a persistent outcome of infection has been correlated with emergence of HCV escape variants (Cox et al., 2005b; Ray et al., 2005; Tester et al., 2005; Timm et al., 2004). Escape mutations that arise after several years of chronic infection have been described (Erickson et al., 2001; von Hahn et al., 2007). For instance in subject H an escape mutation in a class I epitope was observed more than two decades after persistent infection was established (von Hahn et al., 2007). Nevertheless, late escape may not be a common occurrence. Studies in chimpanzees (Fernandez et al., 2004) and humans (Kuntzen et al., 2007) indicate that the rate of nonsynonymous mutation is highest in the first few weeks or months of persistent infection, consistent with the view that CD8þ T cells can exert selection pressure for a limited period of time before they lose function (Cox et al., 2005a; Urbani et al., 2005). Large populations of HCV sequences derived from HLA-defined humans have provided phylogenetic evidence of class I mutational escape (Neumann-Haefelin et al., 2008; Poon et al., 2007; Rauch et al., 2009; Timm et al., 2007). While these studies have yielded strong evidence for divergent evolution caused by CD8þ T cell selection pressure, many changes are often in the direction of an ancestral HCV sequence (Ray et al., 2005; Salloum et al., 2008; Timm et al., 2004). This pattern has been interpreted as evidence for reversion of escape variants that impair replicative fitness of the virus. Cell culture models of HCV replication support this view (Ray et al., 2005; Salloum et al., 2008; Timm et al., 2004). Growth of replicons or HCVcc engineered to contain some class I escape mutations is substantially impaired in cultured cells (Dazert et al., 2009; Soderholm
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et al., 2006; Uebelhoer et al., 2008). One of these studies examined a series of sequential escape mutations that appeared in one epitope of a genotype 1a virus as persistence was established in a chimpanzee (Erickson et al., 2001). Mutations that appeared in the epitope at the earliest time points after infection of the chimpanzee were least fit for replication in the cell culture model (Uebelhoer et al., 2008). It remains to be determined if these escape mutations also impair replication of the virus after it is transmitted to a new host, and the extent to which compensating adaptive mutations increase stability of amino acid substitutions that facilitate escape from antiviral CD8þ T cells. The issue of whether mutational escape in class I epitopes is a cause or consequence of persistent infection remains unsettled. As noted above, not all class I epitopes targeted by CD8þ T cells escape and so it may not be a requirement for persistence. The percentage of epitopes that undergo mutation is highly variable, ranging from less than 20% of epitopes in some studies (Komatsu et al., 2006; Kuntzen et al., 2007) to greater than 60% in others (Cox et al., 2005b). The importance of escape mutations in some key epitopes is favored by studies in chimpanzees, where there was a statistically significant increase in the rate of nonsynonymous mutation in class I epitopes of viruses that persist when compared with those that are cleared (Erickson et al., 2001). In support of this statistical analysis, an association between escape mutation and loss of immune control early in infection has been documented in one study involving a limited number of subjects (Guglietta et al., 2009). Finally, immune selection pressure driven by CD8þ T cells might be altered by vaccination (Zubkova et al., 2009) or new antiviral therapies that target nonstructural HCV proteins important for virus replication. As an example, known drug resistance sites in the HCV protease and polymerase may overlap with class I epitopes, raising the possibility of HLA-associated immune resistance to the drugs (Gaudieri et al., 2009; Salloum et al., 2010).
2. Inhibitory receptor signaling As noted above, some HCV class I epitopes remain intact during chronic infection even though cognate CD8þ T cells persist in the liver, sometimes at high frequency (Erickson et al., 2001; Spangenberg et al., 2005). This indicates that mechanism(s) other than mutational escape silence CD8þ T cells in persistent HCV infection. Functional exhaustion of HCV-specific CD8þ T cells targeting intact epitopes is almost certainly critical for persistence and may be mediated or reinforced by signaling through PD-1 that has an intracellular domain containing an immunoreceptor tyrosine-based inhibitory (ITIM) as well as an immunoreceptor tyrosinebased switch (ITSM) motif. PD-1 ligation by its receptors, PD-L1 or PD-L2, impairs induction of the cell survival factor Bcl-xL and expression of
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transcription factors associated with effector cell function, including GATA-3, Tbet, and Eomes (Keir et al., 2008). Establishing an association between PD-1 expression on T cells during acute hepatitis and the outcome of infection is complicated because this receptor is naturally upregulated on activated CD8þ T cells. PD-1 expression is elevated on HCV-specific CD8þ T cells during acute hepatitis C, but has not been consistently associated with resolution or persistence of infection (Kasprowicz et al., 2008; Radziewicz et al., 2008; Urbani et al., 2008). Very high levels of PD-1 expression are associated with caspase-9 dependent apoptosis of CD8þ T cells from individuals that ultimately develop a chronic infection (Radziewicz et al., 2008). Regulation of T cell function and survival during the acute phase of infection is probably determined by other positive and negative signals in addition to those delivered by PD-1. As an example, the costimulatory B7 ligand CD86 that is normally present on antigen presenting cells was coexpressed with PD1 on HCV-specific CD8þ T cells and served as a unique marker of effective IL-2 signaling as measured by the phosphorylation state of STAT-5 (Radziewicz et al., 2010). Loss of CD86 and sustained expression of PD-1 on the T cell surface was observed as HCV persistence was established. Whether expression of molecules like CD86 transiently tempers or counter-balances inhibitory PD-1 signals during acute hepatitis C remains to be determined. As noted above strong circumstantial evidence implicates PD-1 in maintenance of the persistent state once it has been established. HCVspecific CD8þ T cells in the blood and liver of persistently infected humans display high levels of PD-1 (Golden-Mason et al., 2007b; Penna et al., 2007; Urbani et al., 2006b, 2008) and the intrahepatic populations are prone to apoptosis (Radziewicz et al., 2007). Importantly, antibody-mediated blockade of PD-1 signaling in cell culture models restores proliferation if not function to these exhausted CD8þ T cells (Golden-Mason et al., 2007b; Nakamoto et al., 2008; Radziewicz et al., 2007; Urbani et al., 2008). High, sustained expression of PD-1 on HCV-specific T cells, combined with restoration of function in the cell culture model by receptor blockade, suggests that anti-PD-1 antibodies could provide a therapeutic effect in patients with chronic infection. Early phase clinical trials of PD-1 blockade have been initiated to test in humans the safety of a strategy that could provide an important adjunct to direct suppression of HCV replication by antiviral agents (see ClinicalTrials.gov identifier NCT00703469 for details). Studies in the LCMV model of persistent infection predict that multiple inhibitory receptors coregulate CD8þ T cell exhaustion (Blackburn et al., 2009). PD-1 is not the only inhibitory molecule expressed by HCV-specific CD8+ T cells. Inhibitory receptors such as PD1, 2B4 (a SLAM-receptor that can deliver activating or inhibitory signals), CD160 (a coinhibitory glycoylphosphatidylinositol-anchored receptor), and KLRG1 (killer cell-lectin-like
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receptor G1) were coexpressed by many HCV-specific CD8þ T cells in one study (Bengsch et al., 2010). Collectively, they impaired proliferative capacity and effector functions in persistent infection. The inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) was also preferentially upregulated on PD-1-positive T cells from the liver but not blood of chronically HCV-infected patients (Nakamoto et al., 2009). Coexpression of PD-1 and CTLA-4 inhibitory receptors on intrahepatic T cells was associated with a profound loss of virus-specific effector functions that could be reversed in cell culture by simultaneous blockade of the PD-1 and CTLA-4 signaling pathways (Nakamoto et al., 2009). In this study antibody-mediated inhibition of either receptor alone did not restore T cell function. Other receptors may also contribute to HCV-specific T cell exhaustion. Recently, coexpression of PD-1 and Tim-3 (the T cell immunoglobulin and mucin domain-containing molecule 3) was described on CD4þ and CD8þ T cells in chronic hepatitis C, including some intrahepatic populations that were HCV-specific (Golden-Mason et al., 2009). Interference with TIM-3 signaling also restored antigen-stimulated proliferation of T cells and increased IFN-g production while decreasing IL-10 output (Golden-Mason et al., 2009). Finally, it is very likely that mutational escape of epitopes and exhaustion mediated by coinhibitory receptor signaling are interdependent. As an example, CD8þ T cells targeting escaped epitopes tend to express higher levels of CD127 and lower levels of PD-1 than those targeting intact epitopes in the acute (Rutebemberwa et al., 2008) and chronic (Bengsch et al., 2010; Kasprowicz et al., 2010) phases of infection. These studies suggest that continuous stimulation with viral antigens reinforces the exhausted phenotype. Expression of CD127, even at low levels, could explain persistence of CD8þ T cells targeting epitopes that have escaped. Whether these T cells truly retain some effector functions or a capacity for self-renewal that distinguishes them from those targeting intact epitopes remains unknown.
3. Regulatory CD8þ T cell activity Two types of regulatory T cells (Treg) have been defined. They include natural Treg that develop in the specialized environment of the thymus and adaptive Treg that mature extrathymically (Sakaguchi et al., 2010). Phenotypic markers that uniquely define Treg are controversial, but most studies operationally use CD4, the transcription factor forkhead box P3 (FoxP3), and the IL-2Ra chain (CD25) to identify and manipulate these populations (Sakaguchi et al., 2010). Treg are thought to act by production of suppressive cytokines like IL-10 or TGF-b, or by receptor-mediated sequestration of IL-2 that is required for effector T cell activity (Bluestone and Abbas, 2003; Sakaguchi et al., 2010). These functional activities are important for maintenance of immune tolerance but also have the potential to modulate the course of HCV infection (Bluestone and Abbas, 2003;
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Sakaguchi et al., 2010). It has been proposed that they dampen HCVspecific effector T cell activity during the acute or chronic phases of infection, simultaneously contributing to a persistent state and tempering immunopathological damage to the liver. At present, there is no direct experimental evidence that Treg activity during acute hepatitis C results in persistent infection. HCV infection of chimpanzees did provoke proliferation of Treg because T cell receptor excision circles in FoxP3-positive cells were diluted when compared to uninfected animals (Manigold et al., 2006). Functional FoxP3-positive CD4þ T cells have been expanded from blood of human subjects during the acute phase of HCV infection, but frequencies did not differ in subjects with acute resolving versus persistent outcomes (Smyk-Pearson et al., 2008). Similar results were obtained when HCV-specific Treg that expressed FoxP3 were visualized with MHC class II tetramers (Heeg et al., 2009). Only transient expansion of adaptive Treg was observed in blood and did not correlate with development of persistent viremia (Heeg et al., 2009). While it is possible that analysis of intrahepatic Treg function would reveal an influence on the outcome of acute hepatitis C, it is perhaps more likely that changes in the frequency and function of these regulatory T cells represent a response to the inflammatory environment in the liver of most infected individuals. Treg may be active in the chronic phase of infection to reinforce exhaustion of effector T cells. Production of anti-inflammatory mediators like TGF-b and IL-10 by natural and adaptive CD4þ Treg was documented in chronically infected subjects (Ebinuma et al., 2008; Kaplan et al., 2008; Langhans et al., 2010; MacDonald et al., 2002; Ulsenheimer et al., 2003). Virus-responsive Treg from the blood of individuals with chronic hepatitis C have a stable FoxP3 phenotype and function, and may be genetically programmed for survival (Li et al., 2009). Others have documented that depletion of CD25-positive mononuclear cells from PBMC of subjects with chronic hepatitis C increased the frequency of T cells responding to HCV antigen stimulation in proliferation or IFN-g ELISpot assays (Boettler et al., 2005; Cabrera et al., 2004; Rushbrook et al., 2005; Sugimoto et al., 2003). While this ex vivo experimental approach provided some preliminary circumstantial evidence for enhanced Treg activity in chronic hepatitis C, their importance in maintaining persistent HCV replication in the liver of infected humans has remained unresolved. Immunohistochemical staining has revealed that a high proportion of CD4þ T cells in the liver express the transcriptional regulator FoxP3 (Ward et al., 2007). One recent study confirmed that the liver harbors high frequencies of Foxp3 positive-Treg, and that they suppress effector T cell activity via a contact-dependent mechanism (Franceschini et al., 2009). Impressively, the frequency of intrahepatic Foxp3 positive-Treg correlated directly with serum HCV virus load and inversely with
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hepatocellular injury. These Treg also expressed exceptionally high levels of PD-1 that modulated suppressive activity upon ligation of PD-L1 that is expressed on liver parenchymal cells. Antibody-mediated inhibition of PD-1 signaling relieved a block on IL-2 driven STAT-5 phosphorylation, resulting in enhanced proliferation and suppressive activity by Treg (Franceschini et al., 2009). Finally, while some of the intrahepatic Treg proliferated in response to HCV antigens, the proportion that are natural versus adaptive (i.e., antigen-specific) remains to be determined.
V. IMMUNITY ACQUIRED BY NATURAL INFECTION CAN PROTECT AGAINST HCV PERSISTENCE: IMPLICATIONS FOR VACCINATION HCV-specific T cells are detectable in blood for at least two decades after resolution of infection even in humans who no longer have detectable antibody responses to the virus (Takaki et al., 2000). There is evidence that memory T cells primed naturally by successful resolution of infection protect against persistence upon reexposure to the virus. Studies involving humans serially exposed to the virus through intravenous drug use have provided valuable insight into this issue. Virus replication following reinfection of humans who spontaneously cleared a prior HCV infection is well documented so sterilizing immunity is probably uncommon if it occurs at all (Aberle et al., 2006; Aitken et al., 2008a; Grebely et al., 2006; Mehta et al., 2002; Micallef et al., 2007; Mizukoshi et al., 2008; Osburn et al., 2009; Page et al., 2009; van de Laar et al., 2009). However, the magnitude and duration of viremia is substantially decreased in secondary versus primary infection and is associated with HCV-specific T cell immunity (Aberle et al., 2006; Bharadwaj et al., 2009; Mehta et al., 2002; Mizukoshi et al., 2008; Osburn et al., 2009). Critical support for the role of memory T cells in HCV reinfection was generated using the animal model. Rechallenge of immune chimpanzees often resulted in low-level transient virus replication (Bassett et al., 2001; Lanford et al., 2004; Major et al., 2002) that was associated with recall of CD4þ and CD8þ T cell responses (Grakoui et al., 2003; Nascimbeni et al., 2003; Shoukry et al., 2003). Antibodymediated depletion of CD4þ helper (Grakoui et al., 2003) or CD8þ cytotoxic (Shoukry et al., 2003) T cells from immune chimpanzees caused prolonged or even persistent infection. It is important to note that protection afforded by successful resolution of one HCV infection is not absolute in humans or chimpanzees. This is perhaps best illustrated by an animal study where transient virus replication was observed after multiple sequential infections before persistence was finally established (Bukh et al., 2008). Moreover, some humans with chronic hepatitis C harbor T cells that target HCV strains or genotypes unrelated to the persistent
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virus (Schulze Zur Wiesch et al., 2007; Sugimoto et al., 2005). It is likely that these T cells are a marker of an earlier resolved infection that ultimately failed to protect against persistence. Documented persistent infection in humans who had spontaneously cleared earlier infection(s) supports this interpretation (Mehta et al., 2002; van de Laar et al., 2009). It is possible that immunity is less effective against heterologous HCV genotypes (Prince et al., 2005), although sequential infection and clearance of unrelated HCV genotypes has been documented in animals (Bukh et al., 2008; Lanford et al., 2004) and humans (Aitken et al., 2008b). A key unanswered question is whether immunity primed by natural infection can be recapitulated by vaccination. As noted above, vaccination of chimpanzees with nonstructural HCV proteins does not prevent infection with HCV (Folgori et al., 2006). However, the course of infection was altered because acute phase viremia was substantially reduced when compared to unvaccinated control animals (Folgori et al., 2006). These preliminary studies suggest that priming of T cells alone could be sufficient to shift the balance of most HCV infections away from persistence and toward resolution. However, the ability of the virus to undermine the response even when replication is substantially controlled cannot be underestimated and a comprehensive approach that involves priming of humoral and cellular immunity may be desirable (Frey et al., 2010; Houghton and Abrignani, 2005).
VI. SUMMARY HCV is somewhat unique amongst human viruses in its ability to establish either persistent life-long infection or durable immunity that can protect against persistence after reexposure to the virus. This has provided a unique opportunity to define mechanisms of protective immunity and evasion by a small human RNA virus. Mutational escape from humoral and cellular immune responses is a common finding in humans and chimpanzees with a persistent outcome of infection. However, this mechanism alone cannot explain the remarkable ability of HCV to persist. Failure of the CD4þ T helper cell activity early in infection is likely a central event in subversion of B and CD8þ T lymphocyte responses and establishment of persistence. Mechanisms underpinning an HCV-specific defect in CD4þ T cell immunity remain very poorly understood. Closing this gap in knowledge would likely accelerate development of safe and effective vaccines. HCV can also rapidly acquire resistance to STAT-C inhibitors targeting the protease and polymerase enzymes and so it is likely that they will be used in combination with type I interferon and ribavirin for the foreseeable future. How interferon and ribavirin inhibit the virus is not known, but could involve direct interference with replication, modulation
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of immunity, or both. Replacement of interferon and ribavirin is desirable because of toxicity and lack of a predictable therapeutic effect in humans. Further studies of adaptive immunity may suggest immunotherapeutic approaches to chronic hepatitis C that could be used in combination with small molecule inhibitors of HCV replication may provide substitutes. For instance, a detailed picture of how the virus utilizes CD81, SR-B1, and the tight junction proteins to initiate infection should facilitate development of attachment and entry inhibitors that may be useful in combination with STAT-C inhibitors. Finally, modulation of cellular immunity by interference with inhibitory signaling pathways like PD-1 and CTLA-4 could conceivably provide a well-defined approach to restoration of host responses capable of eradicating the infection.
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Timpe, J. M., Stamataki, Z., Jennings, A., Hu, K., Farquhar, M. J., Harris, H. J., Schwarz, A., Desombere, I., Roels, G. L., Balfe, P., and McKeating, J. A. (2008). Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 47:17. Uebelhoer, L., Han, J. H., Callendret, B., Mateu, G., Shoukry, N. H., Hanson, H. L., Rice, C. M., Walker, C. M., and Grakoui, A. (2008). Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness. PLoS Pathog. 4:e1000143. Ulsenheimer, A., Gerlach, J. T., Gruener, N. H., Jung, M. C., Schirren, C. A., Schraut, W., Zachoval, R., Pape, G. R., and Diepolder, H. M. (2003). Detection of functionally altered hepatitis C virus-specific CD4 T cells in acute and chronic hepatitis C. Hepatology 37:1189. Ulsenheimer, A., Lucas, M., Seth, N. P., Tilman Gerlach, J., Gruener, N. H., Loughry, A., Pape, G. R., Wucherpfennig, K. W., Diepolder, H. M., and Klenerman, P. (2006). Transient immunological control during acute hepatitis C virus infection: Ex vivo analysis of helper T-cell responses. J. Viral Hepat. 13:708. Urbani, S., Amadei, B., Cariani, E., Fisicaro, P., Orlandini, A., Missale, G., and Ferrari, C. (2005). The impairment of CD8 responses limits the selection of escape mutations in acute hepatitis C virus infection. J. Immunol. 175:7519. Urbani, S., Amadei, B., Fisicaro, P., Tola, D., Orlandini, A., Sacchelli, L., Mori, C., Missale, G., and Ferrari, C. (2006a). Outcome of acute hepatitis C is related to virus-specific CD4 function and maturation of antiviral memory CD8 responses. Hepatology 44:126. Urbani, S., Amadei, B., Tola, D., Massari, M., Schivazappa, S., Missale, G., and Ferrari, C. (2006b). PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J. Virol. 80:11398. Urbani, S., Amadei, B., Tola, D., Pedrazzi, G., Sacchelli, L., Cavallo, M. C., Orlandini, A., Missale, G., and Ferrari, C. (2008). Restoration of HCV-specific T cell functions by PD-1/ PD-L1 blockade in HCV infection: Effect of viremia levels and antiviral treatment. J. Hepatol. 48:548. van de Laar, T. J., Molenkamp, R., van den Berg, C., Schinkel, J., Beld, M. G., Prins, M., Coutinho, R. A., and Bruisten, S. M. (2009). Frequent HCV reinfection and superinfection in a cohort of injecting drug users in Amsterdam. J. Hepatol. 51:667. Voisset, C., Op de Beeck, A., Horellou, P., Dreux, M., Gustot, T., Duverlie, G., Cosset, F. L., Vu-Dac, N., and Dubuisson, J. (2006). High-density lipoproteins reduce the neutralizing effect of hepatitis C virus (HCV)-infected patient antibodies by promoting HCV entry. J. Gen. Virol. 87:2577. von Hahn, T., Lindenbach, B. D., Boullier, A., Quehenberger, O., Paulson, M., Rice, C. M., and McKeating, J. A. (2006). Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells. Hepatology 43:932. von Hahn, T., Yoon, J. C., Alter, H., Rice, C. M., Rehermann, B., Balfe, P., and McKeating, J. A. (2007). Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo. Gastroenterology 132:667. Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, H. G., Mizokami, M., Bartenschlager, R., and Liang, T. J. (2005). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11:791. Wang, H., and Eckels, D. D. (1999). Mutations in immunodominant T cell epitopes derived from the nonstructural 3 protein of hepatitis C virus have the potential for generating escape variants that may have important consequences for T cell recognition. J. Immunol. 162:4177. Wang, J. H., Layden, T. J., and Eckels, D. D. (2003). Modulation of the peripheral T-Cell response by CD4 mutants of hepatitis C virus: Transition from a Th1 to a Th2 response. Hum. Immunol. 64:662. Ward, S., Lauer, G., Isba, R., Walker, B., and Klenerman, P. (2002). Cellular immune responses against hepatitis C virus: The evidence base 2002. Clin. Exp. Immunol. 128:195.
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Ward, S. M., Fox, B. C., Brown, P. J., Worthington, J., Fox, S. B., Chapman, R. W., Fleming, K. A., Banham, A. H., and Klenerman, P. (2007). Quantification and localisation of FOXP3þ T lymphocytes and relation to hepatic inflammation during chronic HCV infection. J. Hepatol. 47:316. Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J. E., Mason, T. J., Saracco, G., Bonino, F., Crawford, K., Marion, C. D., Crawford, K. A., et al. (1992). Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: Potential role in chronic HCV infections. Proc. Natl. Acad. Sci. USA 89:3468. Weiner, A., Erickson, A. L., Kansopon, J., Crawford, K., Muchmore, E., Hughes, A. L., Houghton, M., and Walker, C. M. (1995). Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant. Proc. Natl. Acad. Sci. USA 92:2755. Witteveldt, J., Evans, M. J., Bitzegeio, J., Koutsoudakis, G., Owsianka, A. M., Angus, A. G., Keck, Z. Y., Foung, S. K., Pietschmann, T., Rice, C. M., and Patel, A. H. (2009). CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells. J. Gen. Virol. 90:48. Wolfl, M., Rutebemberwa, A., Mosbruger, T., Mao, Q., Li, H. M., Netski, D., Ray, S. C., Pardoll, D., Sidney, J., Sette, A., Allen, T., Kuntzen, T., et al. (2008). Hepatitis C virus immune escape via exploitation of a hole in the T cell repertoire. J. Immunol. 181:6435. Wong, D. K., Dudley, D. D., Afdhal, N. H., Dienstag, J., Rice, C. M., Wang, L., Houghton, M., Walker, B. D., and Koziel, M. J. (1998). Liver-derived CTL in hepatitis C virus infection: Breadth and specificity of responses in a cohort of persons with chronic infection. J. Immunol. 160:1479. Wunschmann, S., Medh, J. D., Klinzmann, D., Schmidt, W. N., and Stapleton, J. T. (2000). Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J. Virol. 74:10055. Yi, M., Villanueva, R. A., Thomas, D. L., Wakita, T., and Lemon, S. M. (2006). Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc. Natl. Acad. Sci. USA 103:2310. Yoon, J. C., and Rehermann, B. (2009). Determination of HCV-specific T-cell activity. Methods Mol. Biol. 510:403. Zeisel, M. B., Koutsoudakis, G., Schnober, E. K., Haberstroh, A., Blum, H. E., Cosset, F. L., Wakita, T., Jaeck, D., Doffoel, M., Royer, C., Soulier, E., Schvoerer, E., et al. (2007). Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology 46:1722. Zhang, J., Randall, G., Higginbottom, A., Monk, P., Rice, C. M., and McKeating, J. A. (2004). CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J. Virol. 78:1448. Zhang, P., Zhong, L., Struble, E. B., Watanabe, H., Kachko, A., Mihalik, K., VirataTheimer, M. L., Alter, H. J., Feinstone, S., and Major, M. (2009). Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity. Proc. Natl. Acad. Sci. USA 106:7537. Zheng, A., Yuan, F., Li, Y., Zhu, F., Hou, P., Li, J., Song, X., Ding, M., and Deng, H. (2007). Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus. J. Virol. 81:12465. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Wieland, S. F., Uprichard, S. L., Wakita, T., and Chisari, F. V. (2005). Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. USA 102:9294. Zibert, A., Meisel, H., Kraas, W., Schulz, A., Jung, G., and Roggendorf, M. (1997). Early antibody response against hypervariable region 1 is associated with acute self-limiting infections of hepatitis C virus. Hepatology 25:1245. Zubkova, I., Choi, Y. H., Chang, E., Pirollo, K., Uren, T., Watanabe, H., Wells, F., Kachko, A., Krawczynski, K., and Major, M. E. (2009). T-cell vaccines that elicit effective immune responses against HCV in chimpanzees may create greater immune pressure for viral mutation. Vaccine 27:2594.
CHAPTER
3 Molecular Biology of Kaposi’s Sarcoma-associated Herpesvirus and Related Oncogenesis Qiliang Cai,* Suhbash C. Verma,† Jie Lu,* and Erle S. Robertson*
Contents
I. General Background A. Discovery of KSHV/HHV-8 B. Diseases associated with KSHV C. Epidemiology of KSHV infection II. Life Cycle of KSHV A. Latent infection B. Lytic replication III. KSHV Primary Infection A. KSHV entry and internalization B. KSHV interaction with cellular signaling pathways C. Animal and virus models IV. KSHV-Mediated Oncogenesis A. Induction of cellular growth and survival B. Regulation of angiogenesis C. Immune evasion D. Response to microenvironmental stress V. Potential Therapies Against KSHV-Associated Malignances Acknowledgments References
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* Department of Microbiology and the Tumor Virology Program, Abramson, Comprehensive Cancer Center, {
University of Pennsylvania Medical School, Philadelphia, Pennsylvania, USA Department of Microbiology and Immunology, School of Medicine, University of Nevada, Reno, Nevada, USA
Advances in Virus Research, Volume 78 ISSN 0065-3527, DOI: 10.1016/S0065-3527(10)78003-9
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2010 Elsevier Inc. All rights reserved.
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Kaposi’s Sarcoma-associated Herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is the most recently identified human tumor virus,and is associated with the pathogenesis of Kaposi’s sarcoma and two lymphoproliferative disorders known to occur frequently in AIDS patients—primary effusion lymphoma and multicentric Castleman disease. In the 15 years since its discovery, intense studies have demonstrated an etiologic role for KSHV in the development of these malignancies. Here, we review the recent advances linked to understanding KSHV latent and lytic life cycle and the molecular mechanisms of KSHV-mediated oncogenesis in terms of transformation, cell signaling, cell growth and survival, angiogenesis, immune invasion and response to microenvironmental stress, and highlight the potential therapeutic targets for blocking KSHV tumorigenesis.
I. GENERAL BACKGROUND A. Discovery of KSHV/HHV-8 Kaposi’s sarcoma (KS) was first described by a Hungarian dermatologist, Moritz K. Kaposi, in 1872 (Kaposi, 1872). Kaposi published the case histories of elder male patients in Vienna with ‘‘idiopathic multiple pigmented sarcoma of the skin’’ which is now referred to as classic KS (Antman and Chang, 2000). Prior to the acquired immunodeficiency syndrome (AIDS) epidemic, KS was thought of as a rare, slow progressing neoplasm which affects mainly elderly men of Mediterranean and Eastern European region. However, the AIDS epidemic triggered KS to become the most aggressive AIDS-associated cancer which can present with lymphoadenopathy rather than skin lesions (Oettle, 1962). Unlike classic KS, this endemic KS often can be rapidly fatal and is associated with significant morbidity and mortality, and is now recognized as one of the leading cancers in African children with HIV (Bayley, 1984; Downing et al., 1984). Importantly, KS is also known to develop after organ transplantation (posttransplant or iatrogenic KS; Penn, 1983). The fact that the immunosuppressed patients born in countries where classic KS occurs continues to develop KS after a transplant suggests that there is a genetic predisposition or an infectious agent transmitted and responsible for KS development. Studies of AIDS case surveillance showing that KS occurs predominantly in gay and bisexual men with AIDS instead of AIDS patients with hemophilad or intravenous drug users further support the existence of an infectious cofactor (Beral et al., 1990). According to the epidemiological data, several groups set out to identify a ‘‘KS agent’’ in the early 1990s. In 1994, Chang and Moore’s group successfully identified the infectious cause of KS as a new herpesvirus called Kaposi’s sarcoma-associated herpesvirus (KSHV) using
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polymerase chain reaction-based subtractive analysis between the KS lesions and unaffected skin from the same patient (Chang et al., 1994). Analysis of conserved herpesviral genes showed that KSHV belongs to a clade of primate herpesviruses within the gamma2 sublineage, and it is ranked eighth known human herpesvirus (HHV-8) which is closely related to rhesus rhadinovirus (RRV) in nonhuman primates and was with their hosts 25 million years ago (McGeoch and Davison, 1999).
B. Diseases associated with KSHV In the several years since the original identification of KSHV from KS lesions by Chang and Moore, KSHV sequence has been described in an array of disease entities. They are included marrow hypoplasia, haemophagocytic syndrome (HPS), multiple myeloma, sarcoidosis, angio-immunoblasticlymphoma, and most recently primary pulmonary hypertension (PPH; Cool et al., 2003; Low et al., 1998; Pastore et al., 2000; Schulz, 2006). Since these results are largely based upon PCR analysis for detection of KSHV DNA which can be froth with false positivity, and none of these findings have been confirmed by several other investigating groups, it is still yet to be accepted that KSHV plays a significant role in any of these diseases. However, KSHV has been demonstrated to be present in endothelial/spindle cells and the cells that appear to constitute the primary derivation of the tumor (Boshoff et al., 1995). In addition to KS, two B-lymphocyte disorders: multicentric Castleman’s disease (MCD, Soulier et al., 1995) and primary effusion lymphoma (PEL; Cesarman et al., 1995) are consistently linked with KSHV infection.
1. Kaposi’s sarcoma KS is an unusual multifocal neoplasm characterized by dark purple lesions, which differs from most other common tumors in that the lesions contain multiple cell types (Boshoff et al., 1997). KS lesions contain extensive neoangiogenesis, infiltrating inflammatory cells, erythrocyte extravasation, endothelial cells, and ‘‘spindle’’ cells typical for KS. There are four distinct clinical variants of KS which is based on the extent of immunosuppression and severity of infection. These include classic KS, endemic KS, iatrogenic KS, and AIDS-associated KS (Boshoff and Chang, 2001). The form of KS that was originally described by Kaposi is now referred to as classic KS. Classic KS most commonly presents in HIVnegative elderly male patients of Mediterranean and Eastern European decent and is relatively indolent (Franceschi and Serraino, 1995). Endemic KS is the prevalent form of the disease in Equatorial, Eastern, and Southern Africa, and is a substantially more aggressive form than classic KS (Wabinga et al., 1993). Unlike classic KS, endemic KS often presents in HIV-negative children as a lymphadenopathy rather than skin lesions (Ziegler and Katongole-Mbidde, 1996). Latrogenic or posttransplant KS is
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developed in patients undergoing immunosuppressive therapy to prevent graft rejection after organ transplantation (Regamey et al., 1998). The most aggressive form of KS is the AIDS-associated KS, which is the most common neoplastic manifestation of AIDS in the United States and Europe (Martin et al., 1993). This form of KS is most commonly presented in gay and bisexual men, suggesting that the disease transmission is likely through high-risk sexual routes (Gao et al., 1996b; Kedes et al., 1996; Simpson et al., 1996). Distinct from the classic form of KS localized to the lower extremities; AIDS-KS commonly occurs throughout the body, which includes skin of face, torso, the extremities, and mucous membranes of the oral cavity (Cheung, 2004). However, despite these different clinical manifestations of KS, the histology of KS lesions from skin, lymph nodes, respiratory tract, and intestines are very similar.
2. Primary effusion lymphoma PEL, also referred to as body cavity-based lymphoma (BCBL), is a rare, rapidly fatal lymphoma associated with KSHV infection and commonly found in HIV-infected patients (Cesarman, 2002). PEL is a unique form of NHL found more commonly in immunocompromised AIDS patients and unlike KS, PEL is generally presented as a pleural or pericardial effusion without a detectable tumor mass (Carbone and Gaidano, 1997), or can present as a solid mass in the lymph nodes, lungs, or the gastrointestinal tract (Arvanitakis et al., 1996). Due to the presence of hypermutated immunoglobulin genes and markers of late stage B cell differentiation like CD30 and CD138, PEL cells are thought to be usually monoclonal and originated from postgerminal center B cells (Arvanitakis et al., 1996; Carbone et al., 1997; Gaidano et al., 1996). In PEL cells, it has frequently been seen that KSHV presents as single positive or KSHV/Epstein Barr Virus (EBV) double positive, and that the KSHV genome is maintained at a relatively high copy number (50–150 per cell; Arvanitakis et al., 1996; Cesarman et al., 1995; Renne et al., 1996a).
3. Multicentric Castleman’s disease Multicentric Castleman’s disease (MCD) is a usual lymphoproliferative disorder characterized by expanded germinal centers with B cell proliferation and vascular proliferation. KSHV-positive MCD is now recognized as a distinct subset of MCD, called plasmablastic MCD, which contains large plasmablastic cells (Dupin et al., 2000). Dysregulated IL-6 levels are considered a likely contributor to the clinicpathophysiology of MCD (Parravicini et al., 1997). Like KS and PEL, KSHV genomes are detectable in almost all HIV-seropositive MCD cases and approximately 50% HIVseronegative MCD cases (Dupin et al., 1999; Soulier et al., 1995). However, different from PEL cells, coinfection of EBV with KSHV has not been detected in MCD plasmablasts.
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C. Epidemiology of KSHV infection Seroepidemiologic studies have clearly shown that KSHV is prevalent throughout the world, although there appears to be striking variation in local seroprevalence. The association between KS prevalence and KSHV seroprevalence is high. In an attempt to understand the divergence and preference of KSHV in certain human populations, several groups have been working on the pattern of KSHV variability (Alagiozoglou et al., 2000; Biggar et al., 2000; Lacoste et al., 2000; Poole et al., 1999). Unlike other herpesvirus, KSHV contains a highly variable gene K1 which is located at the region next to the terminal repeat (TR) in viral genome. This makes it be a good marker to trace KSHV variants that are associated with particular populations. Based on the phylogenetic analysis of K1 gene, KSHV has been classified into five main branches (termed A–E) which appears to relate with different human populations (see Table 1). Generally, the sequence variation between these different clades is very low and less than 3% at nucleotide level in most regions of the viral genome. In view of the fact that KSHV is highly coevolved with the human population, the geographical distribution would be interesting to further investigate. In 1996, the TABLE 1
World-wide distribution and divergence of KSHV subtype infection
KSHV subtype Populations related
References
A
Lacoste et al. (2000), Poole et al. (1999)
B
C D
E
Z
Northern European, Americans, Asian African
Biggar et al. (2000), Cook et al. (1999), Kakoola et al. (2001), Lacoste et al. (2000), Meng et al. (2001), Poole et al. (1999) Lacoste et al. (2000), Poole et al. (1999)
Northern European, Americans, Asian Old Asian, Polynesian Biggar et al. (2000), Cook et al. (1999), (old pacific island) Kakoola et al. (2001), Lacoste et al. (2000), Meng et al. (2001), Poole et al. (1999) Brazilian Amerindian Biggar et al. (2000), Cook et al. (1999), Kakoola et al. (2001), Lacoste et al. (2000), Meng et al. (2001), Poole et al. (1999) Small cohort of Lacoste et al. (2000), Poole et al. (1999) Zambian children
According to K1 differ by 0.4–44%.
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antibodies produced by recombinant capsid protein or the latent nuclear antigen (LANA) made it possible to study the distribution of KSHV seroprevalence among the different risk groups (Gao et al., 1996a,b; Kedes et al., 1996; Lennette et al., 1996; Simpson et al., 1996). The epidemiology data showed that KSHV remains widespread in sub-Saharan Africa, where KSHV is found in more than 50% of adults (Simpson et al., 1996), and relatively frequent in countries from the Mediterranean region which ranges from 3% of northern Italy to 30% in Sicily (Calabro et al., 1998; Whitby et al., 1998). In contrast, seroprevalence rates are lower than 5% in northern Europe, Asia, and most parts of North America. In endemic areas like Africa, it has been found that transmission from the mother to the child or among siblings or transmission through sexual contact is the most important route (Eltom et al., 2002; Martin et al., 1998; Simpson et al., 1996). However, in nonendemic areas, KSHV is commonly found in persons who have multiple sexual partners (especially homosexual men) or who immigrate from endemic areas (Dukers et al., 2000; Melbye et al., 1998). Although KSHV transmission by blood transfusion or transplanted organs is documented, based on cost-analysis most countries do not yet routinely screen blood or organ donors for KSHV infection (the epidemiological studies related to KSHV transmission are reviewed elsewhere; Corey et al., 2002; Henke-Gendo and Schulz, 2004).
II. LIFE CYCLE OF KSHV KSHV, like other herpesviruses, exhibits a biphasic life cycle with predominant lifelong latent infection and a typical short-lived lytic reactivation cycle. Latent infection is the quiescent state of the viral life cycle, which is characterized by the expression of a limited number of viral genes with no production of virus particles. Majority of the herpesviruses other than the members of gamamaherpesvirus (KSHV and EBV) family do not cause any obvious pathology during latent infection. Members of the gammaherpesvirus have the ability to drive cell proliferation and transformation and are broadly referred to as oncogenic viruses. KSHV is the latest member of the human herpesviruses, which belongs to the gammaherpesvirus subfamily, rhadinovirus genera and have a significant genetic similarity with EBV, a member of the lymphocryptovirus genera (see reviewed in Damania, 2004). Full genome sequence of KSHV virus from KS biopsy samples and PEL cells reveal that its genome is approximately 165 kbp in size with a central region of low GC (L-DNA) flanked by GC-rich (H-DNA) TR units (Neipel et al., 1997b; Russo et al., 1996). The L-DNA is the viral protein-coding region, which encodes for at least 90 ORFs, some with homology to cellular genes (Neipel et al., 1997a,b; Russo et al., 1996;
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Fig. 1). These viral homologs seem to be the pirated copies of the human genome, which were acquired during the process of evolution by the viruses to have growth and immune evasion advantages (Moore and Chang, 1998a,b; Neipel et al., 1997a,b). The past 15 years of research have certainly advanced our understanding of the biology and pathogenesis of KSHV. The KSHV genome exists as linear dsDNA copy in the virion particle. It is delivered into the infected cells by a mechanism which is likely to be a multistep process which is yet to be completely resolved (Chandran, 2010). The virion particle attaches to the host cell surface by temporal interaction of multiple cellular receptors with surface glycoproteins. Attachment is followed by penetration of the viral capsid into the cytosol, achieved by either direct fusion of the virion envelope with the plasma membrane or by internalization through endocytosis through fusion of viral envelope with endosomal membrane (reviewed in Chandran, 2010). Virion capsid delivered to the cytoplasm is transported to the nuclear periphery, which is followed by disassembly of virion capsid and release of viral DNA into the nucleus (Naranatt et al., 2004, 2005; Raghu et al., 2007). The entry of virion into the targets’ cells also brings cellular and viral proteins as well as RNA (Bechtel et al., 2005a,b; Zhu et al., 2005). These proteins include replication and transcription activator (RTA), ORF8, ORF21, ORF24, ORF25, ORF26, ORF33, ORF75, and heat shock proteins 70 and 90 (Bechtel et al., 2005b; Zhu et al., 2005). These proteins are considered to be important in establishing early infection. Since RTA is an immediate early protein, the incoming RTA may boost lytic replication and increase the viral copy number. A quantitative real-time PCR analysis of the early transcripts detected RTA as early as 2 h postinfection, which declines sharply at 24 h postinfection (Krishnan et al., 2004). In contrast, expression of ORF73 encoded LANA is detected at very low levels within 6 h postinfection but the levels increase significantly 24 h postinfection (Cai et al., 2007; Lan et al., 2005). Newly synthesized and virally delivered RTA promotes expression of LANA by binding to the LANA promoter (Lan et al., 2005). Expression of LANA subsequently blocks the expression of lytic proteins and pushes the cells to enter into latent phase. Fine tuning of LANA and RTA expression levels decides the fate of the virus which undergoes the latent to lytic cycle (Lan et al., 2005). The distinct viral gene expression profiles during latent and lytic phase of the viral life cycle will be discussed in following sections.
A. Latent infection Latency is characterized by the expression of subset of viral transcripts and persistence of the viral genome as a circular episome attached to the host chromatin. Another characteristic feature of latency is that the cells
K2 K1
K4.1 K6 K5 K7 K4 K4.2 K3
K10.6 K10.1 K11.1 K9 K10 K10.5 K11
K8
K12
K13
6
7
9
74
40
16
ORFs
8
19 10 11
4
2
70
21
22
26 28 29 32 33
25 23 24
17 18
20
30 31
63 43 44
37 39
29a
35 36 38
41
48 46 47
49
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DNA Pol KCP ssDBP
1
TS
vCCL1
vBcl2
MIR1 vCCL3 gB
10
vIL-6
20
vCCL2
Major capsid protein
TK
vIAP
Tegument protein gH
30
40 IE lytic
Minor capsid protein
Kinase Packaging proteins
50 Early lytic
Helicase, primase
ZEBRA
gL
Alkaline exonuclease
60 Late lytic
70
58
62
65
67
75 69
71 72
RTA
vIRF-1 vIRFs DNA replication proteins
80
Uncharacterized
73
TR LANA-2 vIRF3
dUTPase gM
57
53
UDG
MIR2
DHFR
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66
59 60 61
56
TR KIS
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64
54
45
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34
27
K15
K14
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90
TRI-1
SCIP
(DUB) Glycoprotein Large Kaposin Ribonucleotide reductases
Small
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LAMP miRNA cluster
120
vGPCR vCycD vFLIP
vOx2
LANA
130
140 kb
Latent
FIGURE 1 A schematic of the KSHV genome consisting of 145 kb long unique, gene-coding region flanked with terminal repeat units. The coding regions contain over 90 open reading frames (ORFs; Russo et al., 1996). The gene-encoded proteins are labeled in the bottom from left to right. ORFs unique to the KSHV are given the prefix K. The miRNA cluster encoding for 12 microRNAs (yellow) is located between K12 (Kaposin) and ORF71 (vFLIP). The blocks representing the ORFs are also labeled in color according to gene class [latent, immediate early (IE), early and late lytic, and unclassified] (based on previous reviews of Coscoy, 2007; Moore and Chang, 2001).
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harboring the viral genome downregulate cell surface makers which can be typically detected by the host immune surveillance. Importantly, viralencoded latent genes have been shown to play an important role in modulating viral and cellular gene expression to successfully establish latent infection.
1. KSHV latent genes/transcripts Due to the lack of a true latency animal model for studying KSHV infection, cell lines derived from KSHV-infected patients have proved useful in characterizing the expression profiles of latency-associated genes (Cannon et al., 2000; Flore et al., 1998; Foreman et al., 1997; Renne et al., 1996b). PEL cells, which maintain high levels (98–99%) of latently infected cells (a fraction of KSHV-infected cells can undergo spontaneous lytic reactivation) restrict transcription to specific viral genes, including the latency-associated nuclear antigen encoded by ORF73, viral cyclin D encoded by ORF72, vFLIP encoded by ORF71, Kaposin encoded by K12, and viral miRNA (Burysek and Pitha, 2001; Dittmer et al., 1998; Kedes et al., 1997; Rainbow et al., 1997; Sadler et al., 1999). Another latent protein that consistently detected in KSHV-induced tumors is viral interferon regulatory factor (vIRF; Russo et al., 1996). Genes encoded by ORF71, ORF72, and ORF73 are expressed from the same locus in polycistronic, differentially spliced mRNAs whose transcription is regulated by a common promoter upstream of LANA (Dittmer et al., 1998; Grundhoff and Ganem, 2001). Interestingly, LANA promoter is bidirectional and also controls the transcription of ORF73, 72, and 71 during latency and a bicistronic transcript encoding the expression of K14 and ORF74/ vGPCR during lytic cycle (Chiou et al., 2002). Intriguingly, the bicistronic transcripts for ORF74-K14 were detected in some KS lesions and latent biopsy samples. However, it is somewhat enigmatic that ORF74-K14 can be expressed in latently infected cells that coexpress LANA–vCyclin– vFLIP from the same locus ( Jeong et al., 2001; Nador et al., 2001). In addition, the Kaposin is expressed from the promoter present downstream of ORF73 (Li et al., 2002). Among these latent proteins, LANA is the most consistently detected antigen in KSHV-infected cells of KS, PELs, and MCD origins and is a hallmark of KSHV genome persistence (Ballestas et al., 1999; Cotter and Robertson, 1999; Dupin et al., 1999). LANA is a large (220–230 kDa) heavily posttranslationally modified nuclear protein detected as specific punctate dots in immunofluorescence assays (Lennette et al., 1996). Colocalization of these LANA punctate dots with the hybridization signals for the KSHV genome by immnuo-FISH (fluorescence in situ hybridization) assay in the chromosomes of the infected cells suggested a role for LANA in viral genome attachment to the host chromatin (Cotter and Robertson, 1999). Studies from a number of laboratories later identified LANA binding sites (LBS) in the KSHV
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genome, which mapped to the TR units (Ballestas and Kaye, 2001; Cotter et al., 2001). In addition, binding of LANA to the TR DNA was mapped to the DNA-binding domain of LANA at the carboxyl terminus (Cotter et al., 2001; Hu and Renne, 2005).
2. KSHV latent replication and persistence Linear KSHV genome circularizes to make episomal DNA through a notyet-known mechanism upon entry into the nucleus following infection. The viral genome persists as a circular episome in the form of highly ordered chromatin structure during latent infection (Stedman et al., 2004, 2008). LANA has been shown to be important for attaching the viral episomal structure to the host chromatin. The amino-terminal domain of LANA has a chromatin binding sequence (5–22 aa) that attaches to chromatin surface by binding to a multiprotein complex including histones on the cellular chromatin (Barbera et al., 2006; Cotter and Robertson, 1999; Krithivas et al., 2002; Lim et al., 2003; Matsumura et al., 2010; Ottinger et al., 2006). The carboxyl terminal domain of LANA directly binds to the LANA binding sequence (LBS) located in the TRs of the viral genome (Cotter et al., 2001; Hu and Renne, 2005; Komatsu et al., 2004; Srinivasan et al., 2004). Binding of LANA at the TR is critical to efficient initiation of latent replication at the TRs as demonstrated by short-term replication assays using a construct with the TR cloned onto a plasmid (Hu et al., 2002; Komatsu et al., 2004; Lim et al., 2002; Verma et al., 2006). In an attempt to identify the minimal DNA element required for replication at the TR, Hu et al. performed short-term replication assays with the deletion mutants of the TR and mapped a 71-bp unit of the TR as the minimal replicator element (Hu and Renne, 2005). This unit comprises LBS1/2 and an adjacent 29- to 32-bp-long GC-rich element referred to as replication element (RE) upstream of the LBSs (Hu and Renne, 2005). We recently identified a latent replication origin in the long unique region of the viral genome, which initiates replication independent of viral proteins in trans, suggesting that this is an autonomously replicating element in the LUR (Verma et al., 2007a). In contrast to the LANA-dependent replication origin of TR, this autonomously replicating element is high in AT content and can recruit the host cellular replication machinery to initiate replication (Verma et al., 2007a). Although LANA does not seem to have any enzymatic activity for replication function, it is essential for TR-mediated replication primarily because it recruits the required cellular replication machinery to the RE element of the TR (Stedman et al., 2004; Verma et al., 2006). The primary replication protein, origin recognition complexes (ORCs), which serves as the launching pad for the recruitment of other proteins including MCMs gets recruited by LANA at the RE site of the TR (Verma et al., 2006). Chromatin immunoprecipitation assay with hyperacetylated histone H3 and H4 antibodies primarily detects the TR region
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suggesting a loose chromatin structure at the TR which is indicative of replicative origin (Stedman et al., 2004). LANA-mediated TR replication as well as replications mediated by the autonomous elements was done on a plasmid-based assay. Therefore, we cannot conclusively state that these sites are firing during replication of the viral genome. A comprehensive study to demonstrate the usage of these replication sites would be important in understanding the mechanism of latent viral DNA replication. KSHV-infected PEL cells maintain similar number of copies of the viral episome over multiple rounds of cell division suggesting a faithful mechanism of viral genome segregation after each cell division (Skalsky et al., 2007). As discussed earlier, LANA associates with the KSHV genome in infected cells and colocalizes with the viral genome at interphase nuclei and on mitotic chromosome as a punctuate pattern (Ballestas et al., 1999; Cotter and Robertson, 1999). The ability of LANA to tether viral episomes to the host chromatin is important for the establishment of latent infection. Studies with a KSHV genome deleted for LANA ORF cloned in a bacterial artificial chromosome showed loss of viral episomes from the infected cells without establishment of latency (Ye et al., 2004). Another study where LANA expression was depleted using the shRNA strategy in PEL cells showed reduction of viral episomes to 20% compared to the control shRNA treated cells (Godfrey et al., 2005). These studies clearly indicate that the presence of LANA in the host cells is required for persistence of the viral DNA. LANA binding to the host chromatin at nucleosomal surface through histones and other cellular proteins throughout the cell cycle confirms that the LANA:cellular proteins interactions are critical in maintaining the viral genome (Barbera et al., 2006; Cotter and Robertson, 1999; Krithivas et al., 2002; Lim et al., 2003; Matsumura et al., 2010; Si et al., 2008; Verma et al., 2007b; ViejoBorbolla et al., 2005; You et al., 2006). A yeast-2-hybrid assay using the LANA-N terminus as bait identified the Nuclear Mitotic Apparatus (NuMA) protein as a LANA interacting protein (Si et al., 2008). NuMA is an essential protein for cellular genome segregation, which interacts with a number of essential mitotic components including microtubule proteins dynein and dynactin during mitosis to efficiently segregate the daughter chromatids (Du et al., 2002; Merdes et al., 1996). Depletion of NuMA by siRNA and dominant-negative approach to block NuMA function has resulted in decreased copies of latently maintained KSHV epiosome (Si et al., 2008). Proteomic analysis of LANA binding proteins from KSHV-infected cells identified a centromeric protein F, a component of multiprotein complex that assembles on centromeric DNA and links the chromosome to microtubule during mitosis and associates with the kinetochore (Cheeseman et al., 2005; Liao et al., 1995). LANA associates with CENP-F and a kinetochore protein, Bub1 in KSHV-infected cells.
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Interestingly, depletion of Bub1 by shRNA dramatically reduced the number of latently persisting KSHV genomes (Xiao et al., 2010). These studies suggest that KSHV episomal DNA segregates in a fashion similar to the case of the host genome at the same time usurping the cellular segregation mechanism.
B. Lytic replication Lytic cycle is characterized by the expression of most of the viral genes in an orderly fashion (immediate early, early, and late) and the production of infectious virion particles. Gene expression profiles of KSHV have been studied in biopsies from KS tissue, PELs, and MCD, and also in in vitro setting by de novo infection of the cultured cells. These studies show that the majority of tumor cells in the KS biopsies contain KSHV viral DNA and express viral latent transcripts, and that a subpopulation (1–3%) of tumor cells undergo lytic reactivation as demonstrated by the expression of early and late viral transcripts (Staskus et al., 1997; Sun et al., 1999). These lytic transcripts include viral macrophage inflammatory protein-I, viral interleukin 6, viral Bcl-2 homolog, and polyadenylated nuclear RNA (PAN RNA). The subpopulation of cells undergoing lytic reactivation also express the late viral transcripts major capsid protein (MCP) and the small viral capsid (sVCA) which indicates production of virion particles and potentially go on to infect the surrounding cells. Additionally, the cells undergoing lytic reactivation produce cellular cytokines, which may create a favorable microenvironment to enhance the growth of latently infected cells and contribute to tumor progression. In order to study the full-blown lytic reactivation, PEL cells (BC-1, BC-3, and BCBL-1) can be treated with chemical agents such as phorbol esters or sodium butyrate (NaB) to induce the cascade of lytic cycle genes and the detection of viral transcripts shows immediate early, early, and late gene patterns like other herpesvirus (Gradoville et al., 2000; Renne et al., 1996b; Sarid et al., 1998; Zhong et al., 1996).
1. KSHV lytic genes The lytic genes are classified into three major groups, immediate early (IE), early (E), and late (L) genes. The immediate early genes are the first group of genes expressed during lytic replication whose transcription generally does not require de novo protein synthesis. The immediate early genes encode for proteins with regulatory functions in activating cascade of downstream genes and also modulating the host cell environment for viral replication. PEL cells treated with TPA and NaB to activate lytic a cascade as well as primary cells, TIME, HFF, and 293 cells infected with the KSHV virus have identified a number of immediate early genes which include ORF50, ORF45, ORF K4.2 (Gradoville et al., 2000; Krishnan
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et al., 2004; Lukac et al., 1999; Sarid et al., 1998; Sharma-Walia et al., 2005). ORF50, also called RTA is the best-characterized immediate early gene. RTA is 691aa long, which has an N-terminal DNA binding and dimerization domain, C-terminal activation domain, and two nuclear localization signals (Lukac et al., 1998; Sun et al., 1998). The molecular weight (110 kDa) of RTA is higher than the predicted (73.3 kDa) size because of various posttranslational modifications (Lukac et al., 1999; Song et al., 2002). Studies from various laboratories have shown that RTA serves as a molecular switch from latent to lytic cycle and ectopic expression of RTA into PEL cells induces the cascade of lytic gene expression including vIL-6, PAN RNA, ORF59, ORF65, and K8.1 and production of DNase-resistant encapsidated viral DNA, providing proof that RTA is capable of driving complete viral lytic cycle (Gradoville et al., 2000; Lukac et al., 1998; Sun et al., 1998). The expression of RTA is controlled by various cellular and viral proteins, including, RTA itself representing an important strategy used by KSHV to express sufficient amounts of RTA to activate the lytic cycle (Gradoville et al., 2000; Lan et al., 2004). RTA activates its promoter by binding to the Oct-1 transcription factor and RBP-Jk, a known cellular partner of RTA bound to the RTA promoter (Deng et al., 2000; Liang et al., 2002). Even though RTA has a DNA-binding domain, direct binding of RTA to its promoter is not required for it autoactivation as Oct-1–RTA complex was not detected in gel shift assay and a defective DNA-binding RTA mutant was capable of autoactivating through binding with RBPJk (Chang et al., 2005; Liang et al., 2002; Sakakibara et al., 2001). Latent protein, LANA also controls RTA expression and suppress lytic reactivation by repressing basal RTA promoter activity as well as RTA-mediated autoactivation (Lan et al., 2004). LANA-mediated suppression of RTA promoter is dependent on RBP-Jk, which is also involved in RTA autoactivation suggests that LANA may be suppressing RTA autoactivation by competing with RTA for binding to RBP-Jk (Lan et al., 2005). Since RBPJk is one of the key molecules in both positive and negative regulation of RTA expression, the levels of LANA and RTA stringently controls the latent to lytic switch. Additionally, RTA upregulates LANA expression to suppress lytic reactivation and thus acts as a negative feedback regulator of RTA (Lan et al., 2005; Matsumura et al., 2005). The early (E) genes generally encode proteins that are involved in nucleic acid metabolism, modulation of cellular functions, and their expression is controlled by the IE genes. RTA activates a number of early lytic genes by either direct or indirect mechanisms and they include PAN RNA, Kaposin, ORF57, K-bZIP (K8), vIL-6, K5, K9, K14, K15, ORF6, ORF59, ORF21, and ORF74 (Chang et al., 2000; Chen et al., 2000; Haque et al., 2000; Jeong et al., 2001; Lukac et al., 2001; Wong and Damania, 2006; Zhang et al., 1998).
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Among the early gene transcripts, PAN RNA is most abundant transcript comprising approximately 80% of the total polyadenylated RNA in the infected cells. PAN RNA is a novel 1.1–1.2-kb noncoding polyadenylated transcript whose expression is controlled by RTA (Song et al., 2001). The function of PAN RNA in lytic reactivation and pathogenesis is yet to be resolved. Kaposin, which is expressed during latent infection but strongly induced by lytic reactivation, has the ability to drive cell transformation (Kliche et al., 2001; Muralidhar et al., 1998). RTA controls the expression of Kaposin (E) through RRE (RTA response element) site within the Kaposin promoter (Song et al., 2003). ORF57 encodes a posttranscriptional regulator, a conserved protein in herpesviruses, which is upregulated by RTA expression (Duan et al., 2001; Kirshner et al., 1999). ORF57 promotes the accumulation (stabilization) and export of viral intronless RNA transcripts by a mechanism, which is yet to be clearly defined. vMIP-1, a virus encoded chemokine homologue, is also expressed during early lytic cycle and its expression is controlled by RBPJk and RTA protein–protein interaction and formation of a macromolecular complex at the RBP-Jk binding site at the vMIP promoter (Chang et al., 2005). KSHV encodes vIL-6 (encoded by ORFK2) which is an early protein of lytic cycle. vIL-6 has 25% amino acid similarity with the human homologue (IL-6) and promotes growth and proliferation of IL-6-dependent human B cells similar to the human IL-6 (Moore et al., 1996; Nicholas et al., 1997). The immediate early gene, RTA strongly induces the expression of vIL-6 by binding to the RRE site making vIL-6 one of the most abundant transcripts in PEL cells during lytic reactivation (Deng et al., 2002; Sun et al., 1999). KSHV-encoded G protein-coupled receptor (vGPCR) is also an early gene which plays an important role in angiogenesis. vGPCR is expressed from a bicistronic RNA with K14 at the 50 end and vGPCR at the 30 end (Nador et al., 2001). The promoter controlling K14/vGPCR is highly responsive to RTA, which binds to RBP-Jk to upregulate the transcription of this K14/vGPCR transcript (Liang and Ganem, 2004). K-bZIP (K8), a basic leucine zipper protein, plays an important role in lytic viral DNA replication, and is an early protein whose expression is controlled by RTA (Lin et al., 2003). RTA also controls the expression of other early and late gene by either directly binding to the RTA response elements or through binding with other cellular factors (West and Wood, 2003).
2. KSHV reactivation KSHV establishes lifelong latency with the persistence of viral genome as chromatin with the expression of only latency-associated genes. These genes maintain latency by blocking the expression of the immediate early gene. However, certain physiological conditions including hypoxia and
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pharmaceutical agents may trigger the expression of RTA (Cai et al., 2006a; Chen et al., 2001; Haque et al., 2003). During latency, viral genomes are assembled into a nucleosomal structure with DNA wrapped around histones (Shinohara et al., 2002; Stedman et al., 2004). Tail modifications of histones play an important role in determining chromatin structure and condensation, both of which are important in regulating transcriptional activity. Acetylation of core histone tail by histone acetyltransferases (HATs) leads to the loosening of the chromatin and thus makes it transcriptionally active (Niedermeier et al., 2006). In contrast, deacetylation of histone tails by histone deacetylases (HDACs) condenses the chromatin making it transcriptionally inactive (Lu et al., 2003; Stedman et al., 2004). HDACs associate with the RTA promoter during latency resulting in hypoacetylated histones and an inactive promoter (Lu et al., 2003). Treatment of latently infected PEL cells with HDAC inhibitors, NaB, and TPA leads to hyperacetylation of histones with the recruitment of HATs and expression of RTA and other lytic genes (Lu et al., 2003). Methylation of DNA also plays an important role in controlling lytic reactivation as the inhibitor of DNA methyltransferases, 5-azacytidine (5-AzaC) stimulates KSHV lytic cycle (Chen et al., 2001). KSHV genome does not seem to have extensive methylation but promoters of specific genes like RTA and LANA have been shown to be controlled by methylation (Chen et al., 2001). BCBL-1 cells showed extensive methylation at the RTA promoter in latent cells and treatment with 5-AzaC results in demethylation of ORF50 promoter and expression of ORF50 and early (vIRF) and late gene (K8.1; Chen et al., 2001). As indicated above, chromatin remodeling due to histone modification can modulate transcriptional activity. The HAT inducer, TPA can induce lytic cycle by activating and enhancing the DNA-binding activity of transcription factors (Wang et al., 2004c). TPA can induce the expression of C/EBPalpha transcription factor and enhance its transactivation activity. Since RTA promoter has C/EBPalpha binding site, expression is enhanced by TPA (Wang et al., 2004c). TPA can also enhance the binding of the AP-1 transcription factor at the RTA promoter to induce RTA expression (Wang et al., 2004c). A number of KSHV proteins including vIRF, LANA, RTA, and K-bZIP have been shown to interact with the transcriptional coactivators p300 and CBP (CREB-binding protein; Huang et al., 2001; Lim et al., 2001; Seo et al., 2000). CBP/p300 has intrinsic HAT activity and RTA binding positively regulates HAT activity (Gwack et al., 2001a). However, binding of LANA, vIRF, and K-bZIP leads to a reduction in protein’s HAT activity (Hwang et al., 2001; Lim et al., 2001; Wang et al., 2003). NaB-mediated activation of ORF50 transcription is due to the binding of the Sp1 transcription factor at the RTA promoter (Ye et al., 2005). LANA regulates ORF50 activity by directly binding to ORF50 promoter and binds to Sp1 during latency (Lu et al., 2006; Verma et al.,
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2004). Treatment of PEL cells with NaB results in acetylation of LANA resulting in the disruption of LANA from ORF50 promoter bound to Sp1 (Lu et al., 2006). Therefore, it can be speculated that removal of LANA from the ORF50 promoter and Sp1 may allow ORF50 and Sp1 to form a complex which enhances transcription. Posttranslational modification including sumoylation, phosphorylation, and ADP-ribosylation of viral proteins, primarily RTA, plays an important role in viral reactivation. Poly(ADP-ribose) polymerase 1 (PARP-1) and a kinase, hKFC interacts with RTA to ribosylate and phosphorylate which reduces the transcriptional activity of RTA by abolishing binding to RRE (Gwack et al., 2003). Sumoylation of K-bZIP also plays a role in modulating k-bZIP-mediated RTA activation of KSHV-specific promoters (Izumiya et al., 2005). These studies propose that posttranslation modifications of viral proteins are required to regulate KSHV lytic replication.
III. KSHV PRIMARY INFECTION Studies for characterization of KSHV primary infection totally relies on the development of systems with high infection efficiency. KSHV produced from PEL and sometimes KS lesions has been showed to infect various cell types but with limited primary infection efficiency or failure of long-term infection. Based on all the systems examined so far, KSHV was found to eventually establish latency after primary infection (Fig. 2). However, depending on cell types or infection conditions, it has been shown that KSHV either enters latency immediately or starts the full productive replication phase followed by establishment of latency infection (Dezube et al., 2002; Foglieni et al., 2005; Gao et al., 2003). Interestingly, the first description of KSHV primary infection system was tested in 293 cells (Foreman et al., 1997). However, due to the direct involvement of endothelial cells in KS tumors, many groups have mainly focused on investigation of KSHV infection in human primary endothelial cells (Ciufo et al., 2001; Flore et al., 1998; Lagunoff et al., 2002; Moses et al., 1999). Initially, it was reported that KSHV infected only a small number of human primary endothelial cells in vitro, like bone marrow microvascular endothelial cells and human umbilical vein endothelial cells (HUVEC; Flore et al., 1998). Subsequent study showed that KSHV was also able to infect primary human dermal microvascular endothelial cells (DMVEC; Moses et al., 1999). The KSHV-infected cells present a typical KS spindle morphology and are able to survive for many months while the uninfected cells go to senescence within a few weeks of been in culture (Flore et al., 1998). The efficiency of primary cell infection in these systems remains very low, although some studies are suggesting strategies for
gB KSHV
gH/L
gpK8.1A gM/N MAPK Kaposin A xCT
JAK-STAT
NOTCH
gp 130
vGPCR
HIF
Wnt
CD98
Heparan integins sulfate
JAK2 ERK1/2
Virus
vFLIP
STAT3
GSK-3
vFLIP MEK ERK IKK
HIF1a
Kaposin B Dynein
MK2
IKB
JNK
p38 JAG1 cIL-6
c-Jun
hNIC
LANA Microtubule
Cytoplasm
vIRF3
GSK-3 LANA
RBP-JK
HIC
Nucleus AP1
Rho GTPase
ERK1/2
DLL4
STAT3
LANA CSL
RTA HIF1a
Pre-miRNA miR-K12-11 miR-132 miR-146a miR-K1
b-cat
xCT p300 CXCR4 p21
FIGURE 2 KSHV primary infection and the cell signaling pathways targeted. Infection is initiated by cell fusion or endocytosis. For cell fusion, KSHV fuses with the plasma membrane by gB, gH, gL, gM, gN, and gpK8.1A, then release of virus particles to the cell plasma. For endocytosis, KSHV binds to the cell surface via interactions with heparan sulfate (HS) followed by temporal interactions with integrins and xCT (CD98) molecules, then formation of endocytic vesicles, virus entry, movement in the cytoplasm, release of virus particles, delivery into the nucleus by dynein along the microtubule. Viral gene expression interferes with the cellular signaling pathways such as MAPK, JAK–STAT, Notch, HIF, Wnt, and miRNA, consequently reprograms host cell gene expression.
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improvement. To further investigate whether the other genetic factors contribute to KSHV primary infection, several similar studies were performed in the E6/E7 or telomerase-immortalized DMVEC culture (Moses et al., 1999). Surprisingly, KSHV infection in the telomerase-immortalized DMVEC shows that it is high. However, the growth of the virus in these cells is not sustained in long-term culture or reactivation to induce lytic replication (Moses et al., 1999). Overall, the limitations in the systems mentioned above have restricted their use to be able to use them for further characterization of KSHV infection. Fortunately, in contrast to the systems above, the recombinant KSHV Bac36 is able to efficiently infect HUVEC cultures and produces large amounts of infectious virion as well as can establish latency at a later stage of infection (Gao et al., 2003). The latently infected cells can be induced to lytic replication by TPA and NaB. This provides a possible path for examining KSHV latent and lytic replication via primary infection (Gao et al., 2003).
A. KSHV entry and internalization Similar to other herpesvirus, the process of KSHV primary infection includes two steps: attachment (or binding) and entry (Spear and Longnecker, 2003). The attachment allows viral proteins to contact with host cell coreceptors, and then stimulate the entry step by either a fusion event between envelope and cell membrane, or receptor-mediated endocytosis (Spear and Longnecker, 2003). KSHV encodes several transmembrane glycoproteins that are involved in attachment and entry into target cells. Some of them are conserved among the herpesvirus like gB (ORF8), gH (ORF22), gL (ORF47), gM (ORF39), and gN (ORF53; Koyano et al., 2003; Krishnan et al., 2005). Some are unique and share no significant homology with glycoproteins of other herpesvirus like K1, K8.1A, K8.1B, and vOX-2(K14) (see Table 2; Chandran et al., 1998; Chung et al., 2002; Li et al., 1999; Luna et al., 2004). KSHV was also shown to attach to the cell surface molecules heparin sulfate (Akula et al., 2001a,b), integrin a3b1 (Akula et al., 2002), and DC-SIGN (dendritic cell-specific ICAM-3-3-grabbing nonintegrin; Rappocciolo et al., 2006). After attachment, KSHV enters the target cell via fusion at the plasma membrane or via endocytosis. KSHV fuses with the plasma membrane to enter target cells as shown for 293 cells and MVDECs (Dezube et al., 2002; Inoue et al., 2003; Pertel, 2002). KSHV envelope proteins, gB, gH, gL, gM, gN, and gpK8.1A, are important and play key roles in the cell–cell fusion process (Chandran, 2010; Pertel, 2002). Endocytosis can occur through four major pathways which include clathrin-mediated endocytosis, caveolae, macropinocytosis, and novel nonclathrin, noncaveolae pathways (Kirchhausen, 2000; McPherson et al., 2001; Sieczkarski and Whittaker, 2002). KSHV entry is via interactions with heparan sulfate
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Major cellular homologs encoded by KSHV
Gene product
ORF
Function
vIL-6
K2
vCCL1
K6
vCCL2
K4
vCCL3
K4.1
vIAP
K7
vBcl-2
ORF16
vIRF1
K9
vIRF2
K11.5
LANA2
K10.5
vIRF3
K10.6
vFLIP
K13/
Constitutively activate gp130 independently of IL-6R; B cell proliferation; auto/paracrine growth factors; angiogenic CCR5 and CCR8 agonists; chemoattraction of Th2 cells and monocytes; angiogenic CCR3 and CCR8 agonists; chemoattraction of Th2 cells and monocytes; angiogenic CCR4 agonist; chemoattraction of Th2 cells; induction of VEGF-A and angiogenesis Inhibitor of apoptosis; inhibition of vGPCR expression and function Inhibition of Bax-mediated and virally induced apoptosis Transformation; inhibition of p300, p53, and TGF-b; inhibition of type I interferon Inhibition of type I interferon and NF-kB; inhibition of Fas-mediated apoptosis via inhibition of CD95L expression Inhibition of type I interferon production; inhibition of PKR- and caspase 3-mediated apoptosis Inhibition of p53 and NF-kB; inhibition of Fas-mediated apoptosis via inhibition CD95L expression ORF71 Transactivator of NF-kB; antiapoptotic activity via FADD and TRADD binding; transformation Constitutively activate Cdk6; resistant to Cdk inhibitors, destabilizes p27 Downregulation of myeloid cell activation; regulation of inflammatory cytokine production (IL-1b, TNF-a, IL-8, IFN-g, and IL-6) Constitutively active; induces VEGF secretion; transformation
vCyclin ORF72 vOX-2
K14
vGPCR
ORF74
Expression pattern
Productive
Productive
Productive
Productive
Productive Productive Productive
Productive
Productive
Productive
Latent
Latent Productive
Latent/ productive
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(HS) followed by temporal interactions with integrins and xCT (CD98) molecules (Kaleeba and Berger, 2006), followed by formation of endocytic vesicles. A recent report showed that KSHV infection induces RhoA GTPase as well as rearrangements of microtubules and the actin cytoskeleton by clathrin-mediated endocytosis pathway in endothelial cells (Sharma-Walia et al., 2004). Thus, the actin dynamics play a pivotal role in internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in HUVEC cells (Greene and Gao, 2009). KSHV also utilizes the actin polymerization-dependent macropinocytic pathway that involves a Rob34 GTPase-dependent late endosome and low-pH environment to entry into HMVEC-d and HUVEC cells (Raghu et al., 2009). KSHV virions are seen in large endocytic vesicles within 5 min of HMVEC-d and HFF cell infection and fusion of the virions envelope with endocytic vesicles (Akula et al., 2003; Raghu et al., 2009).
B. KSHV interaction with cellular signaling pathways 1. MAPK signaling The activation of the MEK/ERK, JNK, and p38 mitogen-activated protein kinase (MAPK) pathways is pivotal at several stages during KSHV infection. Their activation immediately following infection enables successful establishment of KSHV infection (Pan et al., 2006; Sharma-Walia et al., 2005). Subsequently, MAPK pathways are activated during reactivation of latent infection (Ford et al., 2006; Xie et al., 2008; Yu et al., 2007). LANA is a major activator of the serum response element and MAPK pathways via interactions with a mediator complex (Roupelieva et al., 2010). Extracellular heat shock protein 90 localizes to the cells surface (csHsp90) and is a cofactor for MAPK activation and latent viral gene expression during de novo infection by KSHV (Qin et al., 2010a). These studies suggest that KSHV may utilize MAPK pathways to regulate viral infection and switch from viral latency to lytic replication.
2. JAK–STAT signaling Cytokine-mediated JAK–STAT signaling controls a number of important biological processes like immune response, cell growth, and differentiation. It has been shown that KSHV infection constitutively activates receptor-associated Janus tyrosine kinases (JAKs) and thereby results in the subsequent phosphorylation of signal transducers and activators of transcription (STATs). For instance, KSHV infection upregulates gp130 receptor expression and leads to constitutive phosphorylation of JAK2/ STAT3 activation (Morris et al., 2008; Punjabi et al., 2007). Further studies have indicated that both LANA and vGPCR play a role in regulation of JAK2/STAT3 signaling to produce angiogenic factors (Burger et al., 2005; Muromoto et al., 2006). Recent studies showed that not only is the IL-6
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induced by the Tat protein of HIV-1which is dependent on activated STAT3 signaling, but also that IL-4/STAT6 signaling contributes to KSHV lytic replication (Chen et al., 2009; Zeng et al., 2007). This is further confirmed by our findings that KSHV LANA plays a role in inhibition of IL-4-mediated STAT6 phosphorylation for maintenance of latency and response to apoptosis stress (Cai et al., 2010a).
3. Notch signaling KSHV infection is essential for the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and entry of KSHV into the lytic phase. KSHV-encoded RTA binds to RBP-Jk and is a major end point of the Notch signal transduction pathway (Liang et al., 2002; Persson and Wilson, 2010). Moreover, the KSHV-encoded LANA protein can stabilize activated forms of the Notch receptor by targeting the Sel10 protein (Lan et al., 2007). KSHV also manipulates the Notch signaling pathway by directly increasing the expression of two Notch ligands (JAG1 and DLL4) through two KSHV genes expressed during latent and lytic infection, respectively (Emuss et al., 2009). These results showed that KSHV infection can manipulate the Notch signaling pathway to influence cell proliferation and differentiation.
4. HIF signaling Hypoxia-inducible factor (HIF) is a ubiquitously expressed transcriptional regulator that involves an induction of numerous genes associated with angiogenesis and tumor growth. HIF is a heterodimer which composes of inducible a subunit and a constitutively expressed b subunit. It has been demonstrated that there are at least three isoforms of HIFa (HIF1a, HIF2a, and HIF3a) in human cells. In the presence of oxygen, HIFa is hydroxylated and ubiquitylated for proteasomal degradation (Maxwell et al., 1999; Ravi et al., 2000). However, under hypoxic conditions, HIFa hydroxylation is blocked and become stable to activate large number of downstream genes associated with angiogenesis, erythropoiesis, and glycolysis (Lee et al., 2004; Seagroves et al., 2001). Due to the powerful activation of HIF signaling, it has been demonstrated that HIFa is aberrantly overexpressed in many cancers and there is a striking correlation with tumor grade and vascularization (Zagzag et al., 2000; Zhong et al., 1999). In the KSHV-associated cancers, we and other groups have found that HIF1a and HIF2a are overexpressed in KSHV latently infected cells and tissues (Cai et al., 2007; Carroll et al., 2006). Furthermore, both latent antigens LANA and vIRF3 play a role on the HIF1a stabilization via protein–protein interaction (Cai et al., 2006b, 2007; Shin et al., 2008). Recent analysis of clinical patient samples further supported a role for LANA in stabilization of HIF1a (Long et al., 2009). Interestingly, the lytic antigen vGPCR has also been shown to upregulate VEGF
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expression through activation of HIF1a expression (Sodhi et al., 2000). These results suggest that HIF1a is stringently targeted by KSHV during both latent and lytic replication.
5. Wnt signaling KSHV uses components of Wnt pathways to regulate their own viral gene expression and additionally alter cell gene expression through mimicry or manipulation of downstream pathway responses after entry the cells. LANA stabilizes b-catenin by interacting with GSK-3b and inducing its nuclear translocation, thereby preventing phosphorylation of b-catenin in the cytoplasm and stimulation of TCF/LEF-dependent transcription (Fujimuro and Hayward, 2003, 2004). And the I-mfa domain proteins, HIC (human I-mfa domain-containing protein), and I-mfa (inhibitor of MyoD family) interact with LANA and negatively regulate LANAmediated activation of Wnt signaling-dependent transcription (Kusano and Eizuru, 2010). These data suggest that LANA-mediated dysregulation of b-catenin can play an important role in KSHV-mediated transformation after primary infection.
6. miRNA Like all herpesviruses, KSHV has a large, double-stranded DNA genome ( 165 kb; Russo et al., 1996). KSHV encodes more than 85 protein-coding genes, and at least 12 pre-miRNAs that give rise to at least 17 different miRNAs (16 different 5p or 3p miRNAs, and a single-nucleotide-edited miRNA) that are highly conserved (Lin et al., 2010; Marshall et al., 2007). These viral miRNAs interact with cellular factors important for establishment and/or maintenance of KSHV latent infection. For instance, miRK12-11 upregulates xCT expression in both KSHV-infected macrophages and endothelial cells via suppression of BACH-1 (Qin et al., 2010b). miR132 regulates the innate antiviral immunity by inhibiting expression of the p300 transcriptional coactivator (Lagos et al., 2010). KSHV-encoded viral FLICE inhibitory protein vFLIP suppresses CXCR4 expression by upregulating miR-146a (Punj et al., 2010). KSHV-encoded miRNAs target the leucine zipper transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog) and downregulates its expression during primary KSHV infection (Hansen et al., 2010). MicroRNA miR-K1 inhibits p21 expression and attenuates p21-mediated cell-cycle arrest (Gottwein and Cullen, 2010). miR-K1 also regulates NF-kB inhibitor IkBa and viral replication by targeting the 30 UTR of its transcript (Lei et al., 2010), and KSHV miRNA cluster can derepress the ORF50 (RTA) transcription (Lu et al., 2010). These studies suggest that viral miRNAs play a critical role in establishment and/or maintenance of KSHV latent infection.
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C. Animal and virus models To date, many animal and virus models have been developed to investigate the in vivo behavior of KSHV-related malignancies. For animal models, BCBL-1 and infected PEL cells were injected alone or with human peripheral blood mononuclear cells (PBMCs) into SCID mice (Boshoff et al., 1998; Picchio et al., 1997). SCID-Hu Thy/Liv mice were utilized to study viral transcription as well as the susceptibility of the mice to infection with BCBL-1-derived KSHV (Dittmer et al., 1999; Parsons et al., 2006). Similarly, injection of KSHV in human skin engrafted on SCID mice induces KS-like lesions (Foreman et al., 2001). FVB/N transgenic mouse lines that express constitutively active Rac1 (V12 mutant or RacCA) under the control of the a-smooth muscle actin (a-SMA) promoter can develop tumors resembling KS (Ma et al., 2009). Recently, one study reported the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with the recombinant KSHV rapidly seroconvert and maintain a vigorous antiKSHV antibody response. KSHV DNA and LANA were readily detected in the PBMCs and tissues of the infected marmosets (Chang et al., 2009). Recently, Lossos group developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells, showing that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions (Sarosiek et al., 2010). For virus models, rhesus rhadinovirus (RRV) infection results in the development of abnormal cellular proliferations and can coinfect rhesus macaques with simian immunodeficiency virus. This has been suggested as an excellent primate model to investigate KSHV-like pathogenesis (Orzechowska et al., 2008; Wong et al., 1999). Another simian homolog of KSHV—retroperitoneal fibromatosis-associated herpesviruses (RFHV), is its ability to develop a malignancy closely resembling KS and retroperitoneal fibromatosis in animal that become immunodeficient after infection (Bruce et al., 2006). Murine gammaherpesvirus 68 (MHV-68) is a small mouse model, but its infection is not associated with KS-like and related diseases (Flach et al., 2009; Virgin et al., 1997). Herpesvirus saimiri (HVS) mainly infects New World primates and results in T-lymphoproliferative disorder ( Jung et al., 1999). Additionally, two types of cell lines carrying KSHV are able to generate KSHV-associated tumors. One is based on HUVECs that express telomerase (TIVE-LTC; An et al., 2006; Sadagopan et al., 2009), and the other is based on normal mouse bone marrow endothelial lineage cells (Meck36) transfected with the KSHV-infectious bacterial artificial chromosome (KSHV-Bac36; An et al., 2006; Mutlu et al., 2007).
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IV. KSHV-MEDIATED ONCOGENESIS Due to the extensive association of KSHV with two different human malignancies (KS and PEL), KSHV is considered to be a human oncogenic virus (Brooks et al., 1997; Cathomas, 2003; Ensoli and Sirianni, 1998). Unlike other oncogenic viruses, KSHV is a complex DNA virus and infection not only leads to cell (endothelial) morphology changes, growth rate, and extended life span, but also provokes deregulated angiogenesis, inflammation, and modulation of immune system in favor of tumor growth (Fig. 3; Dagna et al., 2005; Ensoli and Sturzl, 1998). However, in most experimental systems in vitro infection of endothelial cells with KSHV did not fully result in neoplastic transformation. Moreover, although KSHV encodes oncogenic genes that could potentially induce all KS-related malignant phenotype, the evidences which link KSHV infection to the development of KS mostly occurs in AIDS or immunosuppressed patients, but rarely in general population. This indicates that the presence of KSHV DNA alone in healthy individuals is not sufficient to cause clinical KS, and that the existence of cofactors like HIV infection or drug-induced immunosuppression are important for KSHV-associated disease progression (Cathomas, 2003; Goedert, 2000). In view of the fact that the vast majority of KS spindle cells are latently infected with KSHV, it has been documented that latent infection plays an essential role in KSHV-induced malignancy and pathogenesis (Deng et al., 2004; Fakhari et al., 2006; Staudt and Dittmer, 2003). Nevertheless, a small percentage of infected cells were also found to undergo lytic replication leading to production of mature virus and cell lysis. This indicates that KSHV lytic replication may also be important for KS development (Fig. 3). This notion is further supported by the facts that some drugs targeting KSHV replication have been shown to be effective in inhibiting KS tumor growth in vivo (Mocroft et al., 1996; Robles et al., 1999), and that there is a strong correlation between viral load and progression of KS tumor (Brown et al., 2005; Duprez et al., 2005; Polstra et al., 2004).
A. Induction of cellular growth and survival Extensive studies have shown that KSHV targets multiple pathways to induce cell proliferation and survival for promoting tumor development. One line of evidence is that genetic instability is found to be commonly seen in KS tumors and PEL cells (Delli Bovi et al., 1986; Gaidano et al., 1997; Popescu et al., 1996), and that KSHV infection is sufficient to induce chromosome instability (Pan et al., 2004).This requires at least five KSHV genes—LANA-1 (or LANA; Cai et al., 2006b; Friborg et al., 1999; Radkov et al., 2000; Si and Robertson, 2006), RTA (Gwack et al., 2001b), k-ZIP,
Latently infected cell
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VEGF-R
Fas(CD95)
vGPCR
K1 KCP MIR1
vCCL PI3K FADD vIAP
LANA
IAPs
BAX BAD NoxA
p53
Cdk4/6
LANA
Rb
p27Kip1 E2F G1
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Complement Cellular G-protein activation AKT signaling Degradation of MHC I protein mTOR KSHV miRNAs
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p16 p21cip
vCyclin
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vCyclin
vIRF3
vBcl2
NF-kB
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VEGF Ang-2 bFGF TNF-b IL-6 IL-8 vIL-6
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Ras
AKT
LANA2
vFLIP
MIR2
Receptors
AP-1
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Cyclin A/E Proliferation
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Tumorigenesis
FIGURE 3 Putative mechanisms of KSHV-mediated regulation of angiogenesis, cell growth and survival, and immune evasion. Cell cycle progression in G1 is controlled by cyclin D/Cdk4/6 complexes which phophorylates Rb. Phosphorylation of Rb leads to Rb inactivation and release transcription factor E2F and transcription of S-phase genes, like Cyclin E and A (Malumbres and Barbacid, 2005). Additionally, p53mediated Cdk2 and Bax activation and Fas-mediated Caspase activation individually induces cell-cycle arrest and apoptosis. In the latently infected cells, KSHV-encoded latent antigens (green) like LANA and vCyclin drive cell proliferation by targeting two cell-cycle checkpoints: (1) Inactivate Rb to release E2F; and (2) Block p53/p27Kip1-mediated cell-cycle arrest (Cai et al., 2006a,b; Friborg et al., 1999; Jarviluoma et al., 2006; Mann et al., 1999). Meanwhile, LANA cooperates with vFLIP to block Caspase and Bax-mediated apoptosis (Sarid et al., 1999). However, in a small amount of lytically infected cells, KSHV encodes some early lytic proteins (orange) like vGPCR vIRF and MIR1/2 or miRNA which dysregulate immune system to produce certain cytokines and help viral infection by angiogenesis and inflammation (Wang et al., 2004b).
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LANA-2 (Rivas et al., 2001), and vIRF-1 (Nakamura et al., 2001; Seo et al., 2001; Shin et al., 2006), which have been shown to interact with and suppress the function of tumor suppressor p53 and Rb resulting in suppression of their activities. Loss of p53 and Rb function leads to inhibition of the DNA damage repair, cell death, and cell-cycle checkpoint which contribute to KSHV-induced oncogenesis. Furthermore, to accelerate cellular proliferation, KSHV encodes vCyclin to promote cell-cycle progression from G1 to S phase by interaction with phosphorylated cyclindependent kinase 6 (Cdk6; Chang et al., 1996; Child and Mann, 2001; Godden-Kent et al., 1997; Li et al., 1997; Sarek et al., 2006). Due to constant selection pressure of favoring cell survival, viruses have evolved different strategies to avoid apoptosis to promote tumor growth and survival by dysregulating cellular signaling pathways. For instance, KSHV encodes vFLIP (K13/ORF71), which like its cellular homolog FLIP, contains the DED domain to inhibit apoptosis by blocking signaling through the death receptor (Belanger et al., 2001; Djerbi et al., 1999). Nevertheless, a recent report utilizing a transgenic mouse model questions the ability of vFLIP to inhibit Fas-mediated apoptosis (Chugh et al., 2005). Several studies have suggested that the antiapoptosis ability of vFLIP is primarily associated with the activation of NF-kB pathway which is essential for cell survival (Chaudhary et al., 1999; Field et al., 2003; Guasparri et al., 2006; Keller et al., 2000, 2006; Liu et al., 2002; Matta and Chaudhary, 2004; Matta et al., 2003;Sun et al., 2005, 2006). Consistently, NF-kB activation by vFLIP leads to cellular transformation and an increased incidence of lymphoma in the transgenic vFLIP mice (Chugh et al., 2005; Sun et al., 2003). It is also important to note that KSHV constitutively activates the NF-kB pathway by encoding vGPCR to produce several cytokines and chemokines (Bais et al., 1998; Couty et al., 2001, 2009; Grisotto et al., 2006; Montaner et al., 2001; Munshi et al., 1999; Schwarz and Murphy, 2001) and vIRF1–3 (Burysek et al., 1999;Flowers et al., 1998; Gao et al., 1997; Kirchhoff et al., 2002; Li et al., 1998; Lubyova and Pitha, 2000; Seo et al., 2002). Treatment with inhibitors of the NF-kB pathway has been shown to completely repress PEL tumors in a mouse model and in vitro tissue culture (Keller et al., 2006; Wang and Damania, 2008). In addition, KSHV was found to promote cell proliferation through autocrine and/or paracrine signaling by encoding or inducing the secretion of various growth factors such as vIL-6 (Molden et al., 1997; Nicholas et al., 1997), IL-6 (Xie et al., 2005), IL-8 (Cerimele et al., 2001), VEGF (Masood et al., 2002), and basic fibroblast growth factor (bFGF; Naranatt et al., 2004; Wang et al., 2004a). It is believed that a variety of cellular growth factors and cytokines regulated by KSHV play pivotal roles in the development and progression of KS. In addition, cell death is shown to be deregulated by KSHV-encoded antiapoptotic proteins. For instance, KSHV-encoded cellular Bcl-2 homolog vBcl-2 (ORF16) is able to protect
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cells from Bax-mediated apoptosis (Cheng et al., 1997; Polster et al., 2004; Sarid et al., 1997), and the viral homolog of human survivin also referred to as inhibitor of apoptosis protein (vIAP) encoded by KSHV ORF K7 was shown to inhibit caspase 3 activity and apoptosis by forming a bridge between cellular Bcl-2 and active caspase 3 (Mahotka et al., 1999; Wang et al., 2002). Another critical strategy is that KSHV encodes a large nuclear antigen called LANA (ORF73), which has no cellular homologs. It has been documented that LANA is essential for many viral functions including gene expression, DNA replication, and episomal maintenance (Ballestas et al., 1999; Barbera et al., 2006; Cotter and Robertson, 1999; Garber et al., 2002; Hu et al., 2002; Lim et al., 2002). In addition to tethering the KSHV DNA to host chromosome during mitosis, LANA not only plays a role in the maintenance of latency by repressing the transcriptional activity of RTA (a lytic reactivator of KSHV; Lan et al., 2004), but also induce oncogenesis by disrupting p53 and Rb function on cell-cycle checkpoint (Cai et al., 2006b; Friborg et al., 1999; Radkov et al., 2000; Si and Robertson, 2006). Our recent studies suggested that LANA is also able to directly induce the level of cellular IAP expression to enhance the life span and proliferation of KSHV-infected cells (Lu et al., 2009).
B. Regulation of angiogenesis The typical tumor cell in KS biopsies is a spindle-shaped cell expressing endothelial cell markers with some markers for smooth muscle cells, macrophages, and dentric cells (Flore, 2004). Recent findings have shown that KS is a highly angiogenic neoplasm with dense and irregular shaped blood vessels, and that KSHV infection is involved in angiogenesis and lymphangiogenesis (Carroll et al., 2004; Hong et al., 2004; Wang et al., 2004a). However, different from the angiogenesis in wound healing and female reproduction, pathological angiogenesis is correlated with tumor growth and metastasis (Carmeliet, 2005; Folkman, 2006). Although the mechanisms of angiogenesis in KS tumor development remain to be further elucidated, it has been demonstrated that KSHV-induced angiogenic factors and inflammatory cytokines play essential roles in driving the late stages of KS tumor development. For example, in an SCID mouse model with human skin grafts, neutralization of VEGF blocks the earlystage KS cells growing into KS-like tumors (Masood et al., 2002; Samaniego et al., 2002). Many other angiogenic cytokines including bFGF, IL-6, IL-8, TNF-b, and ephrin B2 have also been shown to be targeted by KSHV (Masood et al., 2005; Naranatt et al., 2004; Wang et al., 2004a; Xie et al., 2005). In clinical samples, higher levels of serum VEGF and mRNA levels of angiopoietins (Ang-1 and Ang-2) were also seen in the AIDS patients with KS than without KS (Brown et al., 2000; Weindel
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et al., 1992). Moreover, cyclooxygenase-2 (Cox-2) and heme oxygenase-1 induced by KSHV infection were also shown to play an important role in angiogenesis (McAllister et al., 2004; Sharma-Walia et al., 2006, 2010a,b). In addition to the host factor, a number of KSHV-encoded proteins like vIL-6, vGPCR, vCCL-1, and vCCL-II has been shown to act in concert with vCyclin, vFLIP, and vIRF1 to stimulate hematopoiesis and promote angiogenesis by regulating the paracine secretion of angiogenesis-related growth factors and proinflammatory molecules through different signaling pathways (Aoki et al., 1999; Lagos et al., 2007; Montaner et al., 2001, 2003, 2006; Sodhi et al., 2000; Stine et al., 2000). Moreover, recent studies have identified the small GTP-binding protein Rac1 as a key mediator of vGPCR-mediated paracine neoplasia. Prevention of the vGPCR-induced activation of Rac1 efficiently blocks the activation of a series of key transcription factors including NF-kB, AP-1, and NF-AT, and inhibition of cytokine secretion and sarcomagenesis in vitro and in vivo (Montaner et al., 2004).
C. Immune evasion It has been demonstrated that the immune evasion strategies exploited by KSHV leads to uncontrolled cell proliferation and thereby promote tumorigenesis (Choi et al., 2001; Moore and Chang, 2003; Ploegh, 1998). With the exception of modulation of immune response, KSHV encodes multiple viral encoded proteins which are directly involved in the inhibition of host innate and adaptive immunity (Choi et al., 2001; Means et al., 2002). These include the viral proteins that interfere with interferon signaling, complement system, cytokine secretion, and antigen processing and presentation (Fig. 3).
1. Interference of interferon signaling Interferon response is the first line of host immune response against viral infection (Fenner et al., 2006). Host cells start to produce and secrete interferon a/b upon virus infection. It has been shown that the interferon response is regulated by cellular interferon factors (cIRFs) at the transcriptional level. To interfere with this response, KSHV encodes four viral homologs of IRF (vIRF1–4; Means et al., 2002; Moore and Chang, 2003). Among these, vIRF1 functions as a repressor of cellular IFN-mediated signal transduction by directly binding to the IFN-stimulated response DNA element. Sequestration of p300/CBP provides another strategy for vIRF1 to broadly inhibit IFN-mediated gene expression (Li et al., 2000). The observation of vIRF1-induced cell transformation and tumorigenesis in nude mice, suggests that vIRF1 plays a potential role in oncogenesis (Gao et al., 1997; Li et al., 1998). Another IRF encoded by KSHV is vIRF3 also called LANA-2 (Esteban et al., 2003; Rivas et al., 2001). It has been
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demonstrated that vIRF3 is a B cell-specific viral latent protein without DNA-binding ability and is able to inhibit dsRNA-activated protein PKR and p53-dependent apoptosis (Esteban et al., 2003; Rivas et al., 2001). Another mechanism for KSHV to evade the effect of interferon response is expressing viral IL-6. By using the different binding receptor from the cellular homolog, vIL-6 directly binds gp130 independent of gp80, and activates STAT1 phosphorylation and MAPK serine/threonine kinase pathways (Chatterjee et al., 2002; Miles et al., 1990; Molden et al., 1997).
2. Dysregulation of complement system Another strategy in the first defense mechanism against virus attack is to deregulate the complement system. Like many other viruses, KSHV also interferes with complement and this is targeted by the KSHV-encoded ORF4 also called complement control protein (KCP) which has homology to human complement regulators (Mark et al., 2004, 2006). Through four conserved element termed short consensus repeats (SCRs), KCP disrupts the progression of the complement cascade (Mark et al., 2004, 2007; Mullick et al., 2003; Spiller et al., 2003, 2006). The disruption of complement activation in a mouse model can lead to both acute viral infection and establishment of latency elucidating the importance of complement inhibition during virus infection (Kapadia et al., 2002).
3. Viral induction of cytokine secretion Cytokines are the signaling molecules that are used extensively in cellular communication for immune response. Normally, cytokines are unstable and short-lived, and this property is used to prevent too strong and detrimental a response from the host defense system. To promote cytokine stabilization, KSHV expresses the latent protein Kaposin B which activates the p38-MK2 pathway, and leads to increased expression of cytokines including cellular IL-6 and granulocyte-macrophage colonystimulating factor (GM-CSF; McCormick and Ganem, 2005, 2006; Wang et al., 2004a). Another strategy of KSHV modulation of cytokine signaling is by directly expressing signaling ligands and receptors. For instance, the transmembrane receptor KIS encoded by KSHV K1 gene is constitutively activated through its cytoplasmic immunoreceptor tyrosine-based activation motif (IATM; Lagunoff et al., 1999; Lee et al., 1998a). Upon stimulation, K1 IATM is tyrosine phosphorylated. The K1 signaling activates the PI3K/Akt pathway and in turn a series of downstream cellular transcription factors including AP-1, NF-AT, and NF-kB which lead to expression of a number of inflammatory cytokines, such as IL-6, IL-8, IL-10, and VEGF (Lee et al., 1998b; Samaniego et al., 2001; Wang et al., 2004b). Further studies have confirmed that the K1 protein is an oncogenic protein which is able to transform primary HUVEC (Lee et al., 1998b; Wang and Damania, 2008; Wang et al., 2006). Besides Kaposin B and KIS, the latent
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protein vFLIP has also been shown to induce IL-8 and IL-6 expression via NF-kB and JNK/AP-1 pathways (An et al., 2003; Sun et al., 2006). Taken together, in the latently infected cells, KSHV-induced cytokine secretion can function in both autocrine and paracrine fashion, and may contribute to the development of KSHV-associated neoplasms.
4. Viral disruption of antigen processing and presentation Antigen processing and presentation through the major histocompatibility complex (MHC) is a critical step in initiating effective cell-mediated adaptive immune response against pathogens. Downregulation of the cell surface MHC class I molecules is a key viral immune evasion strategy. KSHV encodes two zinc finger membrane proteins MIR1 and MIR2 (also called K3 and K5) which are E3 ubiquitin ligases for modulation by ubiquitylation of MHC I molecules on the infected cell surfaces (Coscoy and Ganem, 2000, 2001). The ubiquitylated MHC I molecules then undergoes endocytosis and is degraded in the lysosome (Coscoy et al., 2001). Interestingly, it has also been demonstrated that MIR1 is able to act as an E3 ubiquitin ligase on ubiquitylation of lysineless molecules (Cadwell and Coscoy, 2005, 2008; Coscoy and Ganem, 2003). Additionally, MIR2 dowregulates ICAM-1 and B7-2 which are ligands for NK cell-mediated cytotoxicity receptors (Ishido et al., 2000). The second strategy for KSHV to evade the adaptive immune system is to encoding several viral chemokines (vCCL). At least three vCCLs have been identified and known to inhibit Th1 cell-mediated immune responses by binding with cellular chemokine receptors on Th1 helper cells which result in blocking signal transduction of G-proteins (Means et al., 2002; Moore and Chang, 2003). Additionally, KSHV also encodes its own versions of the chemokine receptors like vGPCR (Sodhi et al., 2004a). vGPCR is a relative of cellular IL-8 receptors CXCR1 and CXCR2 (Sodhi et al., 2004a). Similar to K1, vGPCR is also constitutively active and induces an array of proinflammatory cytokines and growth factors such as IL-1b, IL-6, IL-8, TNF-a, VEGF, and bFGF through AP-1, NF-kB, and HIF1 pathways (Montaner et al., 2004; Schwarz and Murphy, 2001; Sodhi et al., 2000). However, vGPCR activates several downstream kinases including Lyn, JNK, Akt, and p38 not only by constitutive activation but also by certain induction of chemokines like IL-8 (Montaner et al., 2001; Sodhi et al., 2000, 2004b).
D. Response to microenvironmental stress As the tumor progresses, the cancer cells and its surrounding tissues form a microenvironment with characterizations of both hypoxia and oxidative. Many observations have showed that premalignant cells progress differently in different microenvironments, and hinder the effectiveness of antitumor treatments such as radiation therapy and chemotherapy
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(Bissell and Radisky, 2001; Liotta and Kohn, 2001). This hostile microenvironmental stress not only promotes tumor growth and protects it from immune attack, but also affect the host’s susceptibility to pathogens (Bissell and Radisky, 2001; Liotta and Kohn, 2001). KSHV-associated KS lesions are usually found in the lower extremity of the human body like the feet and hands where there is lower oxygen supply (hypoxia). Investigation of the relationship between the host microenvironment and viral tumor cells will provide new insights into the mechanisms of tumorigenesis and will be a great value in development of therapeutic strategies against clinically relevant viral diseases (Fig. 4).
1. Hypoxia stress The rapid progression of the primary tumor usually generates a hypoxic microenvironment inside the tumor lesion. To investigate the effect of hypoxic stress on KSHV-infected cells, we and others have demonstrated that KSHV is able to mimic hypoxic stress to establish latent infection in these cells (Cai et al., 2007; Carroll et al., 2006). Our studies further showed that the EC5S (Elongin BC-Cul5-SOSC-box) E3 ubiquitin complex is recruited by KSHV latent antigen LANA to degrade the HIF1a negative regulators p53 and VHL (Cai et al., 2006b, 2007). A recent study also indicated that another latent antigen vIRF3 can play a role in stabilization of HIF1a and production of VEGF (Shin et al., 2008). Interestingly, several studies have focused on the life cycle of KSHV and discovered that the KSHV genome contains multiple HIF1a-binding DNA elements, and that hypoxic stress induces KSHV lytic replication (Cai et al., 2006a; Davis et al., 2001; Haque et al., 2003, 2006). In addition, hypoxia was also shown to increase the cell toxicity of ganciclovir and azidothymidine in PEL cells in vitro (Davis et al., 2007; Long et al., 2009), as well as inactivate the function of tumor suppressor VHL and so contribute to viral infection (Cai et al., 2010b).
2. Oxidative stress Oxidative stress represents an imbalance between the production of reactive oxygen species (ROS) and the cellular ability to remove ROS and repair cellular damage. When the concentrations of ROS exceed the ability of the cell to turn over these species, oxidative stress will result and lead to the widespread oxidation and damage of biomolecules including DNA and proteins (Berlett and Stadtman, 1997; Finkel and Holbrook, 2000). There is accumulating evidence which shows that oxidative stress can affect the interaction between the host and viral pathogens, and hence viral pathogenesis (Beck et al., 2000). Recent studies have indicated that herpesvirus infections like HSV-1 and RRV can induce oxidative stress in cells and in tissues through induction of oxidized proteins which are enriched in the VICE (virus-induced chaperone enriched domains) foci
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A Hypoxia
KSHV Tumor cell Blood vessel
Reoxygenation,
Hypoxic stress selection
C
B
ROS, H 2O2 Oxidative stress
Apotopsis
Hypoxia facilitates viral infection
Increase HIFa, VEGF Malignant cell survival
E
Virus lytic replication
D
Angiogenesis and proliferation
KSHV viron
FIGURE 4 Microenvironmental stress promotes the development of KSHV-mediated malignancy. (A) Proliferation of KSHV-infected tumor cells initiates the formation of hypoxia microenvironment; (B) Hypoxic stress induces cell selection by apoptosis and mutation and increases HIFa and VEGF levels; meanwhile, (C) Oxidative stress caused by reoxygenation or overproduction of ROS also induces similar response as hypoxic stress, and thereby (D) selects the survival of malignant tumor cells to promote angiogenesis and tumor progression. On the other hand, (E) the microenvironmental stresses reactivate KSHV from latent infection to produce new virion particles and help viral primary infection.
in the nucleus (Mathew et al., 2010). However, the biological consequences of virus-induced oxidative stress have not been fully characterized. For KSHV, it is speculated that the redox status may affect replication and pathogenicity of KSHV (Wang et al., 2004d). Most recently, in transgenic Rac1 mice, it has been showed that constitutive activation of Rac1 is sufficient to cause KS-like tumors, and the treatment of antioxidants contributes to inhibition of KS-like tumor growth (Ma et al., 2009). Additionally, it has been reported that KSHV-encoded miRNA upregulates xCT (the inducible subunit of a membrane-bound
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amino acid transporter) which facilitates KSHV dissemination and persistence in the environment of oxidative stress (Qin et al., 2010b). This indicates that oxidative stress can play a causal role in carcinogenesis mediated by chronic viral infection and inflammation and might be a potential target for therapeutic interventions.
V. POTENTIAL THERAPIES AGAINST KSHV-ASSOCIATED MALIGNANCES Since the discovery of the virus, understanding of the molecular biology, pathogenesis, and tumorigenesis of KSHV have been increasing and lead to the development of rational therapeutic trials and drug design (Dittmer and Krown, 2007). Although the introduction of HAART (Highly active antiretroviral therapy) has effectively reduced the incidence of AIDS-KS in the USA (Nguyen et al., 2008), it still remains a clinical problem with only half of patients achieving resolution (Vanni et al., 2006). In addition to HAART, the current treatment regiments include radiotherapy and conventional chemotherapy (like liposomal daunorubicin and taxanes) are used to treat the symptoms (Vanni et al., 2006). However, these therapies do not prevent new KS lesions from developing (Sullivan et al., 2006, 2009). Several promising approaches are ongoing with preclinical or clinical trials. These aim to interrupt the angiogenesis process (VEGF/ VEGFR, NF-kB/vFLIP, angiopoietin-2, DLL4, and MMP), cell proliferation (cKit/PDGFR, PI3K/AKT/mTOR, and Notch), or both (Rac/Ros; see reviewed elsewhere Dezube et al., 2006; Dittmer and Krown, 2007; Mesri et al., 2010; Sullivan et al., 2006). Moreover, through targeting KSHV lytic replication, interferon-a is now used as an approved treatment for KS (Grundhoff and Ganem, 2004). Using lentivirus-mediated RNA interference to inhibit viral latent gene expression, has provided another feasible approach for treatment of established lymphomas in murine model (Godfrey et al., 2005). Along with the discovery of the unique mechanisms used by viral pathogen, we believe that the use of antiviral agents and small molecules that specifically target the signaling pathways of KSHVinfected tumor cells will be effective therapies with fewer side effects in the treatment of KSHV-associated malignancies.
ACKNOWLEDGMENTS Due to space restrictions, we regret that we had to omit many important references. The work in authors’ laboratory is support from the National Cancer Institute, including NCI CA072510 and CA091792, NIDCR DE014136, NIAID AI067037, and DE17338 (E. S. R.). E. S. R. is a scholar of the Leukemia and Lymphoma Society of America. S. C. V. is supported by the NIH pathways to independence award, CA126182.
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INDEX A Adaptive immunity. See Hepatitis C virus (HCV) Alkaline exonucleases, 25 Anti-inflammatory cytokines, 18 B Basic leucine zipper (bZIP) proteins, 5–6 C CD21 and CD23a immune receptors, 20 CD81 receptor, 49–50 Cellular immunity, HCV evading and silencing mechanisms inhibitory receptor signaling, 62–64 mutational escape, 60–62 regulatory CD8þ Tcell activity, 64–66 HCV-specific T cell immunity, 55–56 T cell responses, acute phase CD4þ cells, 59–60 CD8þ cells, 56–59 Cytokine anti-inflammatory, 18 proinflammatory, 19–20 D Delayed early (DE) genes, 4 E EBV SM protein, 23 Epstein-Barr virus (EBV) latency, 3 G Gammaherpesvirus lytic infection. See also Host mRNA manipulation MHV-68, 4 phylogenetic relationship, 2–3 Gobal repression, of host gene expression cellular mRNA degradation, 25–28 evasion, of host shutoff, 30 host shutoff, 25–26
hyperadenylation and nuclear retention, 28–29 poly(A) binding protein relocalization, 29 H HCVpp protein, 47–48 HCV-specific T cell immunity, 55–56 Hepatitis C virus (HCV) cellular immunity evading and silencing mechanisms, 60–66 HCV-specific T cell immunity, 55–56 T cell responses, acute phase, 56–60 discovery, 44 existence, 44 humoral immunity antibody responses and infection outcome, 52–53 attachment and entry, 48–51 attenuation and evasion, 53–54 entry and neutralization, 47–48 neutralization epitopes mapping, 51–52 patterns of replication, 45–47 vaccination, 66–67 Host mRNA manipulation cell cycle manipulation control, 7–11 gene expression and mRNA accumulation, 6–8 progression inhibition, 11–12 cell proliferation, differentiation, and tumorigenesis, 20–23 cellular mRNA alteration EBV SM protein, 23 STAT1 and IFN-stimulated genes induction, 23–25 global repression cellular mRNA degradation, 25–28 evasion, of host shutoff, 30 host shutoff, 25–26 hyperadenylation and nuclear retention, 28–29 poly(A) binding protein relocalization, 29
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144
Index
Host mRNA manipulation (cont.) immune response modulation anti-inflammatory cytokines, 18 CD21 and CD23a, 20 interferons and interferon-stimulated genes, 15–17 major histocompatibility complex (MHC), 13–14 NF-kB, 15 proinflammatory cytokines, 19–20 tumor growth factor beta (TGFb), 17–18 tumor necrosis factor alpha (TNFa), 14–15 mechanism, 2 transcription modulation EBV and KSHV bZIP proteins, 5–6 RTA proteins, 5–6 Humoral immunity, HCV antibody responses and infection outcome, 52–53 attachment and entry, 48–51 attenuation and evasion, 53–54 entry and neutralization, 47–48 neutralization epitopes mapping, 51–52 HVR-1 epitope, 53–54 Hypoxia-inducible factor (HIF) signaling, 107–108 Hypoxia stress, 117 I Immediate early (IE) genes, 4 Immune evasion antigen processing and presentation, 116 complement system dysregulation, 115 cytokine secretion, viral induction, 115–116 interferon signaling, 114–115 Immune response modulation anti-inflammatory cytokines, 18 CD21 and CD23a, 20 interferons and interferon-stimulated genes, 15–17 major histocompatibility complex (MHC), 13–14 NF-kB, 15 proinflammatory cytokines, 19–20 tumor growth factor beta (TGFb), 17–18 tumor necrosis factor alpha (TNFa), 14–15 Inhibitory receptor signaling, 62–64 Interferons (IFNs), 15–17
Interferon signaling, 114–115 Interleukin 10 (IL-10), 18 J JAK-STAT signaling, 106–107 Janus tyrosine kinases ( JAKs), 106 K Kaposi’s sarcoma-associated herpesvirus (KSHV), 3–4 animal and virus models, 109 discovery, 88–89 diseases associated Kaposi’s sarcoma, 89–90 multicentric Castleman’s disease, 90 primary effusion lymphoma (PEL), 90 epidemiology, 91–92 life cycle latent infection, 93–98 lytic replication, 98–102 oncogenesis angiogenesis regulation, 113–114 cellular growth induction and survival, 110–113 immune evasion, 114–116 microenvironmental stress response, 116–119 potential therapies, 119 primary infection animal and virus models, 109 cell signaling pathways, 102–104 entry and internalization, 104–106 interaction with cellular signaling pathways, 106–108 KSHV. See Kaposi’s sarcoma-associated herpesvirus (KSHV) L Large extracellular loop (LEL), 49–50 Latent nuclear antigen (LANA), 92 Life cycle, KSHV latent infection genes/transcripts, 95–96 genome, 92–94 replication and persistence, 96–98 lytic cycle lytic genes, 98–100 reactivation, 100–102 Low-density lipoprotein (LDL), 49 Lytic genes, 98–100
Index
M Microenvironment stress hypoxia, 117 oxidative, 117–119 Mitogen-activated protein kinase (MAPK) pathways, 106 Multicentric Castleman’s disease, 90 N Neutralization epitopes mapping, 51–52 NF-kB pathway, 15 Notch signal transduction pathway, 22–23, 107 O Oncogenesis angiogenesis regulation, 113–114 cellular growth induction and survival, 110–113 immune evasion antigen processing and presentation, 116 complement system dysregulation, 115 cytokine secretion, viral induction, 115–116 interferon signaling interference, 114–115 microenvironmental stress response hypoxia stress, 117 malignancy development, 117–118 oxidative stress, 117–119 P PABPC protein, 29 Primary effusion lymphoma (PEL), 90 Primary infection, KSHV
145
animal and virus models, 109 cell signaling pathways HIF, 107–108 JAK-STAT, 106–107 MAPK, 106 miRNA, 108 notch, 107 Wnt, 108 entry and internalization, 104–106 interaction with cellular signaling pathways, 106–108 Proinflammatory cytokines, 19–20 R Regulatory T cell (Treg), 64–66 Replication patterns, of HCV, 45–47 S SR-B1 receptor, 49–50 T T cell responses, acute phase CD4þ cells, 59–60 CD8þ cells, 56–59 frequencies, 57 intrahepatic, 58 Tumor growth factor beta (TGFb), 17–18 Tumor necrosis factor alpha (TNFa), 14–15 V Vaccination, HCV, 66–67 Viremia pattern, 45–46 W Wnt signaling, 108
