Advances in Virus Research, Volume 29

Advances in

VIRUS RESEARCH VOLUME 29

CONTRIBUTORS TO THIS VOLUME

BASILM. ARIF J. G. ATABEKOV

MICHAEL J. CARTER S. N. CHATTERJEE

RALFG. DIETZGEN

Yu. L. DOROKHOV DAVIDKATZ ALEXANDER KOHN M. MAITI GABRIELE MERTES VOLKER TER MEULEN L. Nuss DONALD

STANLEY B. PRUSINER EVAMARIE SANDER

Advances in VIRUS RESEARCH Edited by

MAX A. LAUFFER

KARL MARAMOAOSCH

Andrew Mellon Professor of Biophysics University of Pittsburgh Pittsburgh, Pennsylvania

Robert L. Starkey Professor of Microbiology Waksman Institute of Microbiology Rutgers University Piscataway, New Jersey

VOLUME 29

1984

ACADEMIC PRESS, INC. (Harcourt Brace Jovanovich, Publishers) Orlando San Diego New York London Toronto Montreal Sydney Tokyo

COPYRIGHT @ 1984, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

Orlando, Flonda 32887

United KingdomEdition published by ACADEMIC PRESS. INC. (LONDON) LTD. 24/28 Oval Road, London'NWl 7DX

Library of Congress Cataloging in Publication Data

I S B N 0-12-039829-X PRINTED IN THE UNITED STATES OF AMERICA 84 85 86 87

9 8 1 6 5 4 3 2 1

53- 1 1 559

CONTENTS CONTRIBUTORS TO VOLUME29 ...............................................

ix

Prions: Novel Infectious Pathogens

STANLEY B . PRUSINER ........................ I. I1. Terminology ........................................................

111. Prion Diseases ...................................................... ................................. IV . Epidemiology ........... ................................. V. Assays for Scrapie Prions . VI . Pathogenesis ........................................................ VII . Host Genes Controlling Incubation Times .............................. VIII . Transmission of Prions .............................................. IX . Biophysical and Biochemical Properties of Scrapie Prions ................ X . Molecular Models for the Prion ....................................... XI . Concluding Remarks ................................................. References .......................................

2 3 5 11 12 15 19 21 23 44

47

Molecular Biology of Wound Tumor Virus

DONALD L . Nuss I . Introduction ........................................................ I1. TheVirion .......................................................... I11. Transcription .......................................................

IV . Translation ................................... ................... V. Transmission ..................................................... VI . Infection of the Plant Host ............................. VII . Concluding Remarks ................................................. References .............. ........................

57 58 63 67

72 85

89 90

The Application of Monoclonal Antibodies in the Study of Viruses

MICHAEL J . CARTER AND VOLKER TER MEULEN I. Introduction ........................................................

I1. Virus Identification .................................................. I11. Further Definition of Virus-Specific Protein Structure, Function, and ................................... Synthesis .....................

IV. Investigation of Virus Pathogene and Protection from Virus Infection ... V . Conclusions . . . . . . .............................................

...............................

V

95 99 105 119 122 124

vi

CONTENTS

Monoclonal Antibodies against Plant Viruses

EVAMARIE SANDER AND RALFG . DIETZCEN I. I1. I11. IV. V. VI .

Introduction ........................................................ Production of Monoclonal Antibodies against Plant Viruses .............. Characterization of Monoclonal Antibodies ............................. Monoclonal Antibody-Determined Antigenic Properties .................. Application of Monoclonal Antibodies for Virus Diagnosis ............... Conclusions ......................................................... References ..........................................................

131 133 146 150 161 163 165

lmmunosorbent Electron Microscopy for Detection of Viruses

DAVIDKATZAND ALEXANDER KOHN I. Introduction ........................................................ I1. ISEM Methods ...................................................... I11. Discussion .......................................................... References ..........................................................

169 170 188 193

The Entomopoxviruses

BASILM. ARIF I. I1. I11. IV. V. VI . VII.

Introduction ........................................................ Host Range ......................................................... Structural Features .................................................. Viral Components ................................................... Virus Infection and Multiplication ..................................... Is There a Potential for These Viruses in Pest Control? .................. Conclusion.......................................................... References ..........................................................

195 198 198 200 206 209 210 211

Use of Protoplasts and Separate Cells in Plant Virus Research I. I1. I11. IV. V. VI . VII. VIII .

EVAMARIE SANDER AND GABRIELE MERTES Introduction and Scope of the Review .................................. Isolation of Protoplasts from Leaves ................................... Isolation of Protoplasts from Cell Suspension Cultures ................... Inoculation of Protoplasts ............................................ Determination of Virus Replication .................................... Infection of Cells from Callus Tissue and Cell Suspension Cultures ........ Resistance and Antiviral Substances ................................... Concluding Remarks and Perspectives ................................. References ..........................................................

215 217 223 226 243 248 251 255 258

CONTENTS

vii

Vibriophages and Vibriocins: Physical. Chemical. and Biological Properties I. I1. 111. IV . V. VI . VII . VIII . IX .

S. N . CHATTERJEE AND M . MAITI Introduction ........................................................ Biological Properties of Vibriophages .................................. Sensitivity of Vibriophages to Physical and Chemical Agents ............. Morphology and Other Properties of Vibriophages ....................... Classification of Vibriophages ......................................... Practical Uses of Vibriophages ..................... ...... Lysogenic Vibriophages .............................................. Vibriocins ........................................... Concluding Comments ............................. ........ References ..........................................................

264 266 278 283 290 292 294 298 306 307

Plant Virus-Specific Transport Function and Resistanceof Plants to Viruses

J . G . ATABEKOV AND Yu . L . DOROKHOV I . Introduction ........................................................ I1. Transport of Virus Genome from Infected to Healthy Cells: An Active Virus-CodedFunction ................................................ I11. Resistance of Plants to Viruses as a Problem of Transport of the Virus Genome from Infected t o Healthy Cells ................................ IV. The Transport Form of Viral Infection ................................. V. Concluding Remarks ................................................. References .......................................................... AUTHORINDEX ............................................................. SUBJECT INDEX ............................................................. CONTENTSOFRECENTVOLUMES ..............................................

313 315 337 348 358 359 365 389 407

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CONTRIBUTORS T O V O L U M E 29 Numbers in parentheses indicate the pages on which the authors' contributions begin.

BASIL M. ARIF(195),Forest Pest Management Institute, CanadianForestry Service, Department of the Environment, Sault Ste. Marie, Ontario P6A 5M7, Canada J. G. ATABEKOV (313), Department of Virology and Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, MOSCOW 117234, U S S R MICHAEL J. CARTER' (95), fnstitut fur Virologie der Universitut Wiirzburg, 0-8700 Wurzburg, Federal Republic of Germany S . N. CHATTERJEE (263), Biophysics Division, Saha Institute of Nuclear Physics, Calcutta 700 037, India RALFG. DIETZGEN (131), Institut Biologie II, Uniuersitiit Tiibingen, 0-7400 Tiibingen, Federal Republic of Germany Yu. L. DOROKHOV (313), Department of Virology and Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State Uniuersity, Moscow 117234, U S S R DAVIDKATZ(169), Department of Virology, Israel Institute for Biological Research, Ness Ziona, Israel ALEXANDER KOHN(169), Department of Virology, Israel Institute forBiological Research, Ness Ziona, Israel M. MAITI(263), Indian Institute of Chemical Biology, Calcutta 700 032, India GABRIELE MERTES(215), fnstitut fur Biologie II, Universitat Tubingen, 0-7400 Tubingen, Federal Republic of Germany VOLKER TER MEULEN (95), Institut f u r Virologie der Universitat Wurzburg, 0-8700 Wurzburg, Federal Republic of Germany DONALD L. Nuss (57), Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201 STANLEY B. PRUSINER (l),Departments of Neurology and of Biochemistry and Biophysics, University of California, San Francisco, California 94143 EVAMARIE SANDER (131, 215), Institut Biologie II, Universitat Tiibingen, D-7400 Tiibingen, Federal Republic of Germany

Present address: Department of Virology, Royal Victoria Infirmary, Newcastle upon Tyne, England.

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ADVANCES IN VIRUS RESEARCH. VOL . 29

PRIONS: NOVEL INFECTIOUS PATHOGENS

.

Stanley B Prusiner Departments of Neurology and of Biochemistry and Biophysics University of California Son Francisco. California

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Prion Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Common Features . . . . . . . . . . . . . . . . . . . . . . . B. Scrapie . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Transmissible Mink Encephalopathy (TME). . . . . . . . . . . . D. Chronic Wasting Disease (CWD) . . . . . . . . . . . . . . . . . E . Kum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Creutzfeldt- Jakob Disease (CJD) . . . . . . . . . . . . . . . . G. Gerstmann-Straussler Syndrome (GSS) . . . . . . . . . . . . . IV. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Scrapie . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Kum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Creutzfeldt-Jakob Disease . . . . . . . . . . . . . . . . . . . V. Assays for Scrapie Prions . . . . . . . . . . . . . . . . . . . . . . A . Endpoint Titrations . . . . . . . . . . . . . . . . . . . . . . B. Incubation Time Interval Assay . . . . . . . . . . . . . . . . . C. Radiolabeling of Scrapie PrP . . . . . . . . . . . . . . . . . . . VI . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Lack of Immune Response . . . . . . . . . . . . . . . . . . . . B. AmyloidPlaques . . . . . . . . . . . . . . . . . . . . . . . . VII. Host Genes Controlling Incubation Times . . . . . . . . . . . . . . . A . SIP/LIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . B SINC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. PID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Transmission of Prions . . . . . . . . . . . . . . . . . . . . . . . A. Isolates or Strains of Prions . . . . . . . . . . . . . . . . . . . B . Adaptation of Prions upon Passage . . . . . . . . . . . . . . . . IX. Biophysical and Biochemical Properties of Scrapie Prions . . . . . . . . A . Purification and Hydrophobicity of Prions . . . . . . . . . . . . . B. Search for Nucleic Acid in the Prion . . . . . . . . . . . . . . . C. Molecular Size of the Prion . . . . . . . . . . . . . . . . . . . D. Scrapie Prion Contains a Protein (PrP) . . . . . . . . . . . . . . E . Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . X . Molecular Models for the Prion . . . . . . . . . . . . . . . . . . . A . Hypothetical Structures for the Prion . . . . . . . . . . . . . . . B . Current Models of the Prion Cannot Exclude a Nucleic Acid . . . . .

.

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Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-039829-X

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STANLEY B. PRUSINER

XI. Concluding Remarks References. . . . .

........................ ........................

47 47

I. INTRODUCTION The structure of the unusual infectious agent causing scrapie has defied elucidation for more than 4 decades. Sheep and goats develop scrapie many months or even years after inoculation or exposure. Scrapie is characterized by loss of coordination and difficulty walking which are typical of cerebellar dysfunction (Stockman, 1913). The unexpected results of experiments designed to probe the structure of the scrapie agent have continued to present an increasingly fascinating puzzle (Prusiner, 1982). The unusual biological properties of the scrapie agent were evident even before there was an interest in its chemical and physical structure: the infectious agent caused a devastating degeneration of the central nervous system (CNS) in the absence of an inflammatory response (Beck et al., 1964: Kasper etal., 1981; Zlotnik, 1962). Although the immune system remained intact, its surveillance system was unaware of a raging infection. Early studies showed that the scrapie agent was resistant to formalin and heat (Gordon, 1946). The agent achieved status as a scientific curiosity when its extreme resistance to ionizing and ultraviolet (UV) irradiation was discovered (Alper etal., 1966, 1967). Early attempts to separate or purify the scrapie agent away from tissue elements were greatly hampered by the necessity for bioassay of each sample in sheep or goats (Pattison and Millson, 1960). To establish the titer of a single sample, a whole herd of animals was needed. In addition, highly susceptible breeds of sheep gave inconsistent data. Goats were much better hosts for scrapie infection since they exhibited much more uniform susceptibility. A more hopeful bioassay was developed in 1961 based on the observation that scrapie could be transmitted to mice (Chandler, 1961). For nearly two decades, the accepted bioassay for a single sample containing the scrapie agent required that 50 to 60 inoculated mice be held for up to 12 months prior to final scoring of an endpoint titration. As early as 1963, an alternative bioassay using incubation period measurements was suggested (Eklund etal., 1963; Hunter etd., 1963). Although several investigators reported such measurements, the method remained unexploited for many years. In 1978,we began to develop a bioassay for the scrapie agent employing incubation period measurements (Prusiner etal., 1980d, 1982b). The

PRIONS

3

hamster was used since several years earlier it had been reported that these animals developed scrapie twice as fast as mice and the titers of the agent in their brains were 10- to 100-fold higher (Marsh and Kimberlin, 1975). By measuring both the time intervals from inoculation to onset of illness and from inoculation to death, an objective, manageable bioassay was devised. Although still cumbersome, extremely costly, and quite slow, the incubation time interval assay has permitted significant progress in the last 5 years. We now know that the scrapie agent contains aprotein which is required for its infectivity (Prusiner etal., 1981b). Hydrolytic degradation of the protein catalyzed by proteases, reversible chemical modification by diethylpyrocarbonate (DEP), and denaturation by four different reagents all lead to a diminution of scrapie infectivity (McKinley etal., 1981; Prusiner etal. 198lb; Prusiner, 1982). Recently, the protein has been identified by radioiodination and electrophoresis through sodium dodecyl sulfate (SDS) -polyacrylamide gels (Bolton etaL,1982;Prusiner etal., 1982a). It is an unusual protein (PrP) in that it exhibits microheterogeneity and is resistant to digestion by proteases. This resistance to hydrolysis catalyzed by proteases disappears when the native protein is denatured. All attempts to demonstrate a nucleic acid within the scrapie agent have been unsuccessful, to date (Prusiner, 1982). In addition, size estimates of the infectious agent suggest that it is probably too small to contain even a single gene. Even though the possibility of a small nucleic acid within the core of the scrapie agent cannot be eliminated at present, the unusual properties of the agent seem to distinguish it from both viruses and viroids (Diener etal., 1982; Prusiner, 1982). These unusual properties and the discovery of a protein within the infectious scrapie agent prompted me to introduce a new term for this class of infectious pathogens. They are called “prions.” Earlier studies by other investigators showed that humans develop two scrapie-like diseases: kuru and Creutzfeldt- Jakob disease (CJD) (Gajdusek, 1977). In this review, I discuss the properties of the scrapie prion and its relationship to the infectious agents causing other apparently similar diseases.

11. TERMINOLOGY More than two score years ago, Greig called attention to the unusual and elusive aspects of scrapie when writing about the various names that had been used to describe this disorder (Greig, 1940). He wrote:

4

STANLEY B. PRUSINER The nature of the disease to which the name ‘scrapie’ is usually applied has long remained obscure. It is perhaps for this reason that the names by which the disease is known are colloquialin character and refer to certain of its more outstandingsymptoms, particularly the persistent itch and the disturbances in gait; thus, in England it was known as rubbers, the goggles; in Scotland as scrapie, cuddy trot, and yeuky pine; in France it is named la tremblante(the trembles);and in Germany is referred to as gnubber krankheit (itching disease) and traber krankheit (trotting disease).

Studies over the last 20 years have shown that the molecular properties of the infectious agent causing scrapie are different from those of both viruses and viroids (Prusiner, 1982). Because of these differences, the term “prion” was introduced to denote this novel class of infectious pathogens. The word, prion, was derived from proteinaceous and infectious because the first macromolecule to be identified within the scrapie agent was a protein (Prusiner, 1982). The definition of a prion must remain operational until its entire structure is known: “prions are small proteinaceous infectious particles which are resistant to inactivation by most procedures that modify nucleic acids” (Prusiner, 1982). At present, we still do not know if the prion contains a nucleic acid. It is unlikely that prions contain genes coding for their proteins; however, the presence of a small nucleic acid or oligonucleotide within the interior of the prion has not been excluded. Certainly if prions are comprised of protein alone or a nucleoprotein complex with a polynucleotide too small to code for the prion protein, these features will distinguish them from viruses (Prusiner, 1982). The protein of M,27,000-30,000 is a structural macromolecule within the prion and is denoted by the symbol, PrP, fromprionprotein (McKinley etal., 1983b). This terminology is analogous to that used for structural viral proteins denoted by VP. Genetic loci controlling scrapie incubation periods in sheep and mice have been found. In sheep the alleles of this locus have been termed SIP from short incubation period and LIP from long incubation period (Dickinson, 1976). Two and perhaps three loci in mice have been discovered. The first was called SINC from scrapie incubation (Dickinson and Meikle, 1969). The second locus to be described has been found for both scrapie and CJD in mice; it is called PID-1 from prion incubation determinant 1983). At present, it is unclear whether or not a third (Kingsbury etal., locus exists which is sex linked. An additional note of clarification concerns the term “incubation time interval assay’’ (Prusiner etal., 1982b). Although the use of incubation period mesurements for determining viral or prion titers is not new, the methodology that we employed to determine the titer of the infectious agent was sufficiently different to require a new terminology. Kuru is a Fore word meaning tremor (Gajdusek, 1977; Gajdusek and

5

PRIONS

Zigas, 1957). The Fore people live in the eastern highlands of Papua New Guinea and were the tribe most afflicted by kuru. Creutzfeldt - Jakob disease (CJD) was first described by a German physician, H. G. Creutzfeldt, in 1920 (Creutzfeldt, 1920). Two cases similar to the one described by Creutzfeldt were described by A. Jakob 3 years later (Jakob, 1923). For many years the disease was commonly called JakobCreutzfeldt disease presumably because Jakob's report was more definitive. Gerstmann - Straussler syndrome (GSS) was first described by J. Gerstmann in 1928 (Gerstmann, 1928; Gerstmann et d.,1936). Like CJD, many decades passed before the transmissibility of GSS was demonstrated. Until the molecular structures of the slow infectious agents causing CJD and GSS are determined, it will remain unclear whether GSS represents a variant of CJD or it is a distinct disease.

111. PRION DISEASES Six diseases, three of animals and three of humans, are probably caused by prions (Table I). The slow infectious agents causing transmissible mink encephalopathy (TME),chronic wasting disease (CWD), kuru, CJD, and GSS are not well characterized; thus, further knowledge about the properties of these infectious agents must be obtained before they can be firmly classified as prions. For ease of discussion, all the diseases listed in Table I are referred to as prion diseases even though a prion etiology must be considered tentative until the molecular properties of each slow infectious agent are well defined. TABLE I PRION DISEASES" Disease

Natural host

Scrapieb Transmissible mink encephalopathy (TME) Chronic wasting disease (CWD) Kuru Creutzfeldt - Jakob disease (CJD) Gerstmann-Straussler syndrome (GSS)

Sheep and goats Mink Mule deer and elk Humans- fore Humans Humans ~~

Alternative terminologies include subacute transmissible spongiform encephalopathies and unconventional slow virus diseases. Prions have been shown to cause only scrapie; they are presumed to cause the other diseases listed.

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STANLEY B. PRUSINER

A . Common Features

All of the prion diseases share many features. These hallmarks are listed in Table 11. All known prion diseases are confined to the central nervous systems (CNS). Prolonged incubation periods ranging from 2 months to more than 2 decades have been observed (Table 111) (Sigurdsson, 1954). The clinical course in these diseases is usually rather stereotyped and progresses to death. The clinical phase of prion illnesses may last for periods ranging from a few weeks to a few years (Table IV). A reactive astrocytosis is found throughout the CNS in all these diseases (Beck et al., 1964;Zlotnik, 1962). Neuronal vacuolation is also found, but it is not a constant or obligatory feature. The infectious agents or prions causing these diseases possess unusual molecular properties that appear to distinguish them from both viruses and viroids (Prusiner, 1982). B. Scrapie Scrapie is a disease of sheep and goats. It is a neurological disorder characterized by pruritus, incoordination, and ataxia of gait (Stockman, 1913). The disease is progressive and invariably fatal, as are all other prion diseases. A prolonged incubation period precedes the onset of clinical illness. The pathology of scrapie is confined to the CNS (Beck et al., 1964; Zlotnik, 1962). A prominent astroglial reaction of basal ganglia, mid brain, cerebellum brain stem, and spinal cord is seen. Some proliferation of astrocytes in cerebral cortex is also observed. Vacuolation of neurons is rarely seen in natural scrapie, but is prominent in the experimental disorder transmitted to rodents. Scrapie has been transmitted to mice, hamsters, ferrets, mink, and monkeys (Gajdusek, 1977). Scrapie has not been transmitted to chimpanzees and rabbits (D. C. Gajdusek, personal communication; S. B. Prusiner, unpublished observations). In both hamsters and mice with scrapie, pathologic changes in the retina are prominent (Buyukmihci et al., 1980; Hogan et al., 1981;Kozlowski et al., 1982). The scrapie agent has been found to replicate to high titers in retina as well as throughout the brain. Regional differences in scrapie agent titer found throughout the goat brain were not observed in hamsters (Baringer et al., 1981; Hadlow et al., 1974). C. Transmissible M i n k Encephalopathy ( T M E ) Early observations of TME were reported by Hartsough and Burger (1965)as well as Hadlow and Karstad (1968). Mink develop a neurological disorder characterized by diminished grooming, difficulty eating and swal-

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PRIONS TABLE I1 O F PRION DISEASES CHARACTERISTICS

Diseases are confined to the nervous system Prolonged incubation period of months to decades precedes the onset of clinical illness Progressive clinical course of weeks to years invariably leads to death All diseases exhibit a reactive astrocytosis and many show vacuolation of neurons The infectious agents (prions) causing these diseases exhibit properties which distinguish them from both viruses and viroids

TABLE I11 FOR PRION DISEASES INCUBATION PERIODS IN NATURAL AND EXPERIMENTAL HOSTS ~~

CJD Host Natural Humans Sheep and goats Mink Experimental Apes Monkeys Sheep Goats Ferrets Mink Domestic cats Guinea pigs Hamsters Mice

Kuru

Scrapie

TME

(incubation period in months)' 18-360

60-360 24 - 60 7-12

11-71 4-73

10-82 8-92

36 - 48

39 18-71 45

19-30 7-16 5-18 3-20

8-72 8-36 8-36

8-33 49-88 12-39

12-21

12-21

2-6 5-15

5-7

Data complied from Alpers (1979); Dickinson and Fraser (1977, 1979); Dickinson etal., (1968); Eklund etal., (1967); Gibbs and Gajdusek (1973, 1978); Gordon (1966a,b); W. J. Hadlow (unpublished observations); Hourrigan etal., (1979); (1976, Kimberlin and Walker (1977,197813);Manuelidis etal., 1978a); Marsh and Hanson (1977); Marsh and Kimberlin (1975); Masters etal., (1979); Pattison (1966); Pattison and Millson (1960, 1961); Zlotnik and Rennie (1965).

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STANLEY B. PRUSINER TABLE IV DURATIONS OF ILLNESS FOR P R f O N DISEASES IN NATURAL AND EXPERIMENTAL HOSTS CJD Host Natural Humans Sheep and goats Mink Experimental Apes Monkeys Sheep Goats Mink Domestic cats Guinea pigs Hamsters Mice

Kuru

Scrapie

TME

(duration of illness in months)" 1-55

3-12

2-6 1-2

1-6 1-27 2-6

1-15 1-23

1-22

1-8

2-6 2-6 1-2

1-2 1-5 1-2

1 1-2

1-2

2-6 1

1-2

Data compiled from Alpers (1979); Dickinson and Fraser (1977,1979);Dickinson et al., (1968);Eklundet al. (1967); Gibbs and Gajdusek (1973, 1978); Gordon (1966a,b); W. J. Hadlow (unpublished observations); Hourrigan et al., (1979); Kimberlin and Walker (1977, 197813);Manuelidis et al. (1976,1978a); Marsh and Hanson (1977); Marsh and Kimberlin (1975); Masters et al. (1978); Pattison (1966); Pattison and Millson (1960, 1961);Zlotnik and Rennie (1965).

lowing, hyperexcitability, and signs of incoordination. Progressive debility and bradykinesia ensue, leading to death. The pathological changes in their brains are similar to those found in scrapie. In fact, mink inoculated with scrapie agent develop a disease indistinguishable both clinically and pathologically from TME (Marsh and Hanson, 1979). Laboratory studies have shown that the TME agent is transmissible to mink, goats, hamsters, and monkeys. In contrast to the scrapie agent, the TME agent is not transmissible to mice (Marsh and Kimberlin, 1975). Studies on the biophysical properties of the TME agent indicate that it is similar to the scrapie prion (Marsh etal., 1974).

D. ChronicWasting Disease (CW D ) CWD has been recently described (Williams and Young, 1980). The disease has been found in both mule deer and elk (Williams and Young,

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1982). Like TME, the pathology of CWD is similar to that of scrapie. Laboratory studies have shown that CWD is transmissible to mule deer, ferrets, and squirrel monkeys (Williams etal., 1982).

E. Kuru Kuru was the first slow infectious disease of humans to be identified (Gajdusek etal., 1966). Typically kuru begins with a prodromal phase characterized by headache and/or joint pains (Gajdusek and Zigas, 1959). Within a few months the victims develop difficulty walking. Incoordination of both the upper and lower extremities ensues. In the more advanced stages of the disease, truncal and limb ataxia become so pronounced that the victims are unable to stand or sit without assistance. In the early stages of the disease, kuru is primarily a cerebellar disorder; however, late in the course of the illness intellectual function diminishes (Hornabrook, 1968; Prusiner etaL, 1982~).Dementia and its attendant signs have been observed in advanced cases of kuru. Kuru is confined to the eastern highlands of Papua New Guinea (Gajdusek, 1977;Gajdusek and Zigas, 1959). The Fore and surrcunding tribes are the victims of kuru. Two years after the clinical and epidemiological features of the disease were first reported by Gajdusek and Zigas (1957), Hadlow drew attention to the similarities between kuru and scrapie (Hadlow, 1959); later, the transmissibility of kuru to apes was demonstrated (Gajdusek etal., 1966). Kuru has not been transmitted to laboratory rodents (Gibbs etal., 1979); thus, studies on the properties of the kuru agent are not extensive (Gibbs et al., 1978). Published data on the kuru agent indicate it is similar to that causing scrapie. The pathology of kuru is characterized by the widespread vacuolation of neurons and proliferation of astroglial cells (Beck and Daniel, 1979). Most, but not all, of the cases examined have shown the presence of “kuru plaques.” These appear to be accumulation of amyloid and degenerating neurites as evidenced by their staining properties.

F. Creutzfeldt-JakobDisease (CJD) Early studies on the pathology of kuru suggested that the microscopic changes in this disorder were similar to those found in CJD (Klatzo etaL, 1959). This analogy, like that described by Hadlow for kuru and scrapie, also lead to successful transmission studies. In 1968, the transmission of CJD to apes was reported by Gibbs etal. (1968) and Gibbs and Gajdusek (1969). These studies were of landmark importance for they removed CJD from a wastebasket of degenerative neurological disorders of unknown etiology. CJD typically presents as a dementia in the sixth decade

10

STANLEY B. PRUSINER

of life (Kirschbaum, 1968; Roos etal., 1973). It afflicts males and females with an equal preponderance (Masters etal., 1978). The pathology of the disease is confined to the brain and is characterized by variable degrees of neuronal vacuolation and astrocyte proliferation (Beck and Daniel, 1979). Some cases of CJD show numerous senile plaques similar to those found in Alzheimer’s disease. About 10% of the cases of CJD begin with ataxia much like kuru, but dementia occurs at a much earlier stage than is seen in kuru (Prusiner etal., 1982~).Ten to 20% of CJD cases are familial. In fact, some cases of familial CJD have been found in families with familial Alzheimer’s disease (Masters etal., 1981a). The relationship between these disorders is unknown, but the observations are of great interest especially in view of the reported possible transmission of familial Alzheimer’s disease to apes (Gibbs and Gajdusek, 1978). Unfortunately, attempts to repeat these transmission studies have failed (Goudsmit etal., 1980). In contrast to Alzheimer’s disease, CJD is readily transmissible to apes. A few cases of CJD have been transmitted to rodents such as guinea pigs, hamsters, and mice (Gibbs etal., 1979; Manuelidis and Manuelidis, 1979; Manuelidis etal., 1976,1978a; Sat0 etal., 1980; Tateishi etal., l979,1980b, 1981). Numerous epidemiological studies have tried to link scrapie and CJD, but without success. Studies in collaboration with William Hadlow have shown that goats inoculated with brain extracts prepared from patients dying of CJD cause a disease indistinguishable from natural scrapie (Hadlow etal., 1980b). The incubation periods varied from 3 to 4 years. Both the clinical signs and the pathologic findings of experimental caprine CJD are similar to those of natural scrapie. These observations indicate that the infectious pathogens causing scrapie and CJD must be quite similar. Experimental scrapie and CJD are indistinguishable both clinically and neuropathologically when transmitted to either monkeys (Gibbs and Gajdusek, 1972) or mice (Manuelidis and Manuelidis, 1979; Sat0 etal., 1980; Tateishi etal., 1979,1983), again emphasizing the similarities between the infectious agents (Prusiner and Kingsbury, 1984). Interestingly, chimpanzees, which are the best hosts for experimental CJD and kuru, do not develop scrapie (Gajdusek, personal communication). Chimpanzees inoculated intracerebrally with the scrapie agent more than 13 years ago are still healthy. Pathologic changes in the retina of mice with experimental CJD were found to be more extensive than those $een in hamsters and mice with scrapie (Hogan etal., 1983). Extensive loss of cells throughout the retina was observed in mice with CJD showing only early signs of neurological dysfunction. Patients with CJD infrequently present with visual com-

PRIONS

11

plaints, but a few cases in which optic atrophy was a prominent feature of the illness have been reported (Kitagawa etal., 1983; Lesser etal., 1979; Motomura etal., 1977). Ionizing irradiation studies on the kuru and CJD agents indicate that they resemble the scrapie agent in their extreme resistance to inactivation (Gibbs etal., 1978). Chemical inactivation studies on the murine CJD agent also indicate that it is similar to that causing scrapie (Prusiner and Kingsbury, 1988; Tateishi etal., 1980a). All available evidence indicates that both CJD and kuru are caused by prions.

G. Gerstmann - Straussler Syndrome(GSS) Recent studies by Tatieshi and his colleagues have shown that Japanese cases of CJD are generally transmissible to rodents (Tateishi etal., 1979, 1983). Pathologic examination of these CJD cases has shown numerous kuru plaques. It has been suggested that these cases and others like them represent a distinct variant of CJD called GSS (Masters and Gajdusek, 1982;Masters etal., 1981b). Patients with GSS present with ataxia nearly a decade earlier compared to individuals with CJD (Kuzuhara etal., 1983; Masters etal., 1981b; Seitelberger, 1962, 1981). In contrast to GSS, the amyotrophic form of CJD is clearly not transmissible to apes and probably should be classified as amyotrophic lateral sclerosis (ALS) with dementia (Gibbs and Gajdusek, 1982; Masters and Gajdusek, 1982; Salazar etal., 1983).

Iv.

EPIDEMIOLOGY

Scrapie and CJD are found throughout the world with a few exceptions (Masters etal., 1978). For example, Australia and New Zealand claim to have eradicated scrapie in sheep and goats. In contrast, kuru is confined to a small region of the Eastern Highlands Province of Papua New Guinea as noted above (Gajdusek and Zigas, 1959). The center of the kuru region is occupied by the Fore people. Kuru has also been found in all of the tribes which border on the Fore.

A. Scrapie Many countries have scrapie surveillance programs. Since sheep with scrapie cannot be treated, they are destroyed. Policies designed to control the spread of scrapie generally require sacrifice of the entire flock; however, sacrifice of only “blood lines” is sometimes used in an attempt to

12

STANLEY B. PRUSINER

increase compliance. How scrapie is spread among sheep is unknown 1972). Epide(Hadlow etal., 1980a; Hourrigan etal., 1979;Pattison etal., miologic studies in Iceland emphasize the mysteries surrounding natural scrapie infection of sheep (Phlsson, 1979). Even though pastures once used by scrapie sheep were left vacant for several years, healthy sheep subsequently occupying these pastures eventually developed scrapie.

B. Kuru Continuing epidemiologic surveillance of the kuru region indicates the disease is disappearing (Alpers, 1979;Gajdusek, 1977). In 1957,more than 350 kuru victims were reported amongst a population numbering 10,000. Since that time, there has been a steady decline in the incidence of the disease. Currently, less than 30 patients with kuru are being found. Each year the youngest patient with kuru is older than the youngest the year before. Only people born before 1960 are developing kuru. These observation support the notion that kuru was transmitted by ritualistic cannibalism and that patients currently developing kuru have incubation periods exceeding two decades (Alpers, 1979;Prusiner etal., 1982~).There is no evidence for an animal, plant or insect reservoir in kuru. Indeed, kuru appears to be transmitted only through cannibalism.

C. Creutzfeldt - JakobDisease Although CJD is found throughout the world, it is a rare disease. The incidence of CJD seems to be relatively constant at one per lo6 population 1978). While small clusters of CJD have been reported, no (Masters etal., epidemics of this infectious disorder have been found. In contrast to CJD, Alzheimer’s disease is 5000 times more common (Katzman, 1981). A considerable effort has been made to link CJD in humans with consumption of scrapie-infected sheep meat (Gajdusek, 1977). However, no conclusive evidence to date has linked consumption of scrapie-infected meat with development of CJD. On the other hand, inoculation of goats with brain tissue from patients dying of CJD does cause a disease indistinguishable from natural scrapie (Hadlow etal., 1980b). How an infectious neurological disorder like CJD can maintain its incidence at about one per million population throughout the world remains unknown.

V. ASSAYS FOR SCRAPIE PRIONS At present, the only methods for measuring scrapie prion infectivity remain the incubation time interval assay and the endpoint titration.

PRIONS

13

Both methods are extremely slow because they require waiting for the onset of clinical neurological dysfunction followinga prolonged incubation period. The length of the incubation period varies greatly with the animal species, route of inoculation, and dose of prions. A comparison of the incubation periods for different species is given in Table 111. Clearly, the hamster inoculated intracerebrally with a high dose (lo7ID, units) has the shortest incubation period. Thus, it is the preferred animal for scrapie research. A. Endpoint Titrations An endpoint titration for the scrapie prion is performed by serially diluting a sample at 10-fold increments (Prusiner etal., 1978a). Each dilution is typically inoculated intracerebrally into six mice and the waiting process ensues. Since the highest dilutions at which scrapie develops are the only observations of interest, typically 12 months must be allowed to pass before the titration may be scored. From the score a t the highest positive dilutions, a titer or concentration of infectious agent in the original sample can be calculated. Not only are the resources great with respect to animals and time, but pipetting 10 serial 10-fold dilutions can create additional problems with the accuracy of the measurements. Because of the increased cost, limited numbers of endpoint titrations have been performed in hamsters. Five to six months are required to pass before the titration in hamsters may be scored.

B. Incubation TimeInterval Assay The incubation time interval assay reduces the number of animals, the time required for bioassay and potential pipetting errors compared to the 1980d, 1982b). With hamsters, studies endpoint titration (Prusiner etal., on the scrapie agent have been dramatically accelerated by development of a bioassay based on measurements of incubation time (Prusiner etal., 1980d, 1982b). It is now possible to assay samples with the use of four animals in 60 to 70 days if the titers of the scrapie agent in the sample are high. As shown in Fig. 1,the interval from inoculation to onset of illness ( y) was inversely proportional to the dose injected intracerebrally into random bred weanling Syrian hamsters. The logarithm of the mean interval (?) in days minus a time factor of 40 is a function of the logarithm of the dose over a wide range; the time factor was determined by maximizing the linear relation between the time interval and dose. With a factor of 40, the regression coefficient of the line is 0.87. A similar analysis was performed for the time interval from inoculation to death ( 2 ) . With a time factor of 61, the regression coefficient of the line is 0.86 (Fig. 1A).

14

STANLEY B. PRUSINER

.015

->N \ \

.005

I

1

1

2

3

4

5

6

2

8

9

L O G DOSE

FIG.1. Bioassay of the scrapie prion by incubation time interval measurements. Linear relationships were obtained by plotting (A) logarithm of time intervals minus a time factor as function of logarithm of dose and (B) reciprocal of time intervals as a function of logarithm of dose. The time interval for inoculation to onset of illness is denoted by Y and from inoculation to death by Z.

Linear relationships were also obtained when the reciprocals of the time intervals were plotted as a function of the logarithm of the dose (Fig. 1B). A comparison of the endpoint titration and incubation time interval assay is shown in Table V. The economies of both time and resources

15

PRIONS

TABLE V

COMPARISON OF BIOASSAYS FOR THE SCRAPIE PRION Endpoint titration Rodent species Time (days) Animals (no.) Sample dilution

Mice 360 60 lo-' to

Incubation time interval Hamsters 60-70 4

lo-'"

lo-'

afforded by the incubation time interval assay are highly significant. I estimate that our research has been accelerated more than 100-foldby the incubation time interval assay. It is doubtful that the purification and characterization studies described below could have been performed using the endpoint titration method to assay the samples.

C. Radiolabeling of Scrapie PrP While the incubation time interval assay has been the cornerstone of our work to date, we are beginning to use radiolabeling of PrP as an indicator for the presence of the scrapie agent. As described in Section XD, evidence that PrP is part of the scrapie prion is now compelling. A simple procedure for determining the presence and amount of PrP involves radiolabeling the purified native protein (PrP) with lZ5I-labeledBolton - Hunter reagent, digesting it with proteinase K for 30 to 60 minutes at 37"C, and then denaturing it prior to electrophoresis on a 15% polyacrylamide gel. The recent successful production of antibodies to PrP will allow the development of a rapid immunoassay which will replace measurements of P r P using gel electrophoresis (Bendhein etal., 1984). VI. PATHOGENESIS The pathogenesis of scrapie in mice, goats, and sheep has been studied extensively. In general, the highest titers of the scrapie prion early during the course of infection are found in lymphoid tissue (Eklund etal., 1967; 1974,1980a). The concentration of the prion in lymphoid Hadlow etal., tissues such as spleen and lymph nodes plateaus relatively early at a level 10- to 100-foldbelow that eventually reached in the brain. Hadlow and his colleagues demonstrated that initial infection occurs through the lymphoid system with subsequent spread to the CNS when a peripheral route of

16

STANLEY B. PRUSINER

inoculation is used. While all of the data obtained by animal bioassays are consistent with this view, further technological developments in the assay of prions are required before firm knowledge about the spread of scrapie can be ascertained. We do not know if replication of prions actually occurs in lymphoid tissue or they simply accumulate there after export from another tissue. We have virtually no information about which lymphoid cell types contain prions. Several investigators have tried to study the spread of the scrapie and CJD agents in experimental rodent models. Evidence for spread of scrapie prions along peripheral nerves and CNS fiber tracts has been reported (Fraser, 1982; Kimberlin and Walker, 1979,1980; Kimberlin etal., 1983). However, the lack of sensitive histological techniques for detecting prions makes the interpretation of these data difficult. After intravenous inoculation of mice with the scrapie agent ( 106.3ID,, units), the prion concentration decreased rapidly with a tl,2of 5.2 minutes (Hotchin et al.,1983). The CJD agent has been detected in the buffy coat of guinea pigs indicating a blood-borne route for the spread of the agent (Manuelidis et aL, 197813). The brain, spinal cord, and eye are the only tissues in which high titers of the prion are found and pathological changes are observed (Baringer et al., 1981). Both scrapie and CJD are pure neurological disorders -no symptoms referrable to organs outside the CNS are observed. Thus, the clinical presentation of these illnesses is consistent with their pathologies. Why the scrapie and CJD agents accumulate most extensively in the CNS is unknown. How prions cause vacuolation of neurons and stimulate astrocytic proliferation is also unknown. We do not even know in which cells of the CNS prions multiply. Recent studies on the pathogenesis of scrapie in hamsters show that after intracerebral inoculation with lo7 ID,, units, the animals are free of symptoms for more than 50 days (Baringer et al., 1981). However, by 48 days the titers of the scrapie prion in all regions of brain and spinal cord are maximal. At this time, virtually no pathological changes are evident. By 60 days after inoculation, the animals showed clear signs of neurological dysfunction. During the ensuing 10 to 15 days, the animals demonstrated a progression of their neurological symptoms followed by death. Animals sacrificed at 71 days showed extensive pathological changes, but titers of the prion remained the same as those found at 48 days. From these results, we are forced to conclude that prion replication precedes the pathologic process since the prion titer reaches a maximum before clinical signs and pathologic changes become evident. Thus, the trigger for development of neuronal dysfunction is unknown. Recently, the 2-[14C]deoxyglycose(2-DG) autoradiographic technique for measuring regional cerebral metabolism (Sokoloff et aL,1977)has been

PRIONS

17

applied to studies on the pathogenesis of scrapie. Hamsters were inoculated in the striatum or substantia nig-ra with -lo4 ID,, units using a stereotactic apparatus. Fifty to sixty days after inoculation and 3 to 4 weeks prior to the onset of clinical signs, autoradiographs showed diminished glucose metabolism in the thalamus, medial geniculate bodies and 1983a,b). The authors suggest that teminferior colliculi (GrBgoire etal., poral changes in 2-DG metabolism reflect the spread of the scrapie agent within the brain; however, they present no infectivity data to support this hypothesis. A. Lackof Immune Response

One of the fascinating aspects of scrapie and CJD pathogenesis is the lack of an immune response throughout the courses of these illnesses. The infectious scrapie prion apparently replicates in both the lymphoid system and the CNS without any host inflammatory response. The infection is so devastating that it causes widespread destruction of the CNS and kills the animal. Why there is no inflammatory response is unknown. Numerous attempts to demonstrate antiscrapie antibodies have all been unsuccessful, until recently (Table VI). Immunization of rabbits with purified PrP or scrapie prion preparations has produced high titer antisera (R. A. Barry, M. P. McKinley, P. E. Bendheim, G. K. Lewis, and S. B. Prusiner, in preparation; Bendheim etal., 1984). These antibodies specifically react with PrP and scrapie prion rods as determined by immunoblots and immunoelectron microscopy, respectively. Production of prion antibodies required immunizing the rabbits with 80 to 100 pg of PrP suspended in Freund’s adjuvant. Earlier attempts to produce antibodies in mice and rabbits using 10- 20 p g of PrP per immunization were unsuccessful. To date, neutralization of scrapie prion infectivity with antibodies raised against PrP or infectious particles has not been accomplished. Recent observations show that a gene within the major histocompatibility complex (MHC) of the mouse controls the length of incubation period in experimental CJD and probably in scrapie (Kingsbury etal., 1983) (Section V1II.C). Modulation of prion diseases by a gene in the MHC was unexpected since prions replicate and cause disease in the absence of any detectable immune response. It is well documented that the courses of many viral illnesses are modified by the MHC (Klein, 1975).

B. AmyloidPlaques There continues to be considerable interest about the amyloid plaques found in prion diseases. The CNS of animals with scrapie, CWD, and

18

STANLEY B. PRUSINER TABLE VI

ATTEMPTSTO DEMONSTRATE ANTI-SCRAPIE ANTIBODIES ~~

Serological technique Neutralization

Precipitation

Complement fixation

Immunofluorescence

Antinuclear antibody Passive hemmagglutination Passive hemolysis Passive anaphylaxis Immunoconglutinin Anticardiolipin ELISA

Antigen preparation Brain suspension

Freon-clarified brain Brain cerebrospinal fluid Cerebrospinal fluid Spleen homogenate Brain suspension Spleen extract Brain extract Brain suspension Brain section Brain suspension Monolayer from brain explant cultures DNA (E.coli) RNA (virus) Cerebrospinal fluid Brain extract Brain extract Cerebrospinal fluid Brain or spleen extracts Cardiolipin Fraction E,

Reference Pattison etal. (1964): Clarke and Haig (1966); Gibbs etal. (1965); Gibbs (1967); Gardash’yan etal. (1971) Porter etal. (1973) Chandler (1959) Moulton and Palmer (1959) Gardiner (1965) Gibbs etal. (1965) Chandler (1959) Chandler (1959) Gibbs etal. (1965) Gardash’yan etal. (1971) Moulton and Palmer (1959) Gibbs etal. (1965) Porter etal. (1973) Cunnington etal. (1976) Chandler (1959) Chandler (1959) Chandler (1959) Chandler (1959) Chandler (1959) Kasper etal. (1981, 1982)

experimental CJD, as well as humans with kuru, CJD, and GSS, have been observed to contain collections of amyloid (S.Bahamanjar, E. S. Williams, F. Johnson, S. Young, and D. C. Gajdusek, personal communication; Beck and Daniel, 1979; Beck et al., 1964; Bruce and Fraser, 1975,1982; Field et al., 1967a; Fraser and Bruce, 1973; Moretz etal., 1983; Wisniewski etal., 1975, 1981). These amyloid plaques stain with Congo red or trichrome. Isolates of the scrapie agent (87A and 87V) injected in VM mice have been reported to produce large numbers of amyloid plaques (Bruce and Fraser, 1982). Such plaques have been reported in the brains of sheep and goats with scrapie as well as in the brains of NIH-Swiss Webster mice after primary transmission of scrapie from sheep (Fukatsu etal., 1983). Recent studies show that the majority of cases with CWD exhibit significant numbers of amyloid plaques (S. Bahmanjar, E. S. Williams, F. Johnson, S.

PRIONS

19

Young, and D. C. Gajdusek, personal communication). Mice with experimental CJD exhibit amyloid plaques, but the plaques disappear upon second passage of the agent in mice (Tateishi etal., 1984). The majority of cases of kuru show plaques which are presumably comprised of amyloid within their cores (Beck and Daniel, 1979). The kuru plaques differ from senile plaques in that senile plaques have a collection of amorphous material, presumably degenerating dendrites, around their amyloid core. The kuru plaques do not possess such a large halo. Both kuru and senile plaques have been reported in CJD, but they are not a constant feature of the disease (Chou and Martin, 1971; Yagshita, 1981). Kuru plaques do seem to be a constant feature of GSS (Masters etal., 1981b; Seitelberger, 1981). Recent studies have given rather unexpected results with respect to amyloid plaques in prion diseases (Prusiner, 1984). Purification of scrapie prions to near homogeneity has shown that these particles aggregate to form amyloid-like birefringent rods (Prusiner etal., 1983). Purified preparations of infectious scrapie prions were found to contain one major protein and to exhibit green birefringence under polarized light after staining with Congo red dye. These observations raised the possibility that amyloid plaques might be composed of paracrystalline arrays of prions analogous to inclusion bodies which are usually composed of virions or aggregates of viral proteins. As described above, antibodies to PrP or scrapie prions stain amyloid plaques in the brain sections from scrapie-infected hamsters (Bendheim etal., 1984). When sections stained with immunoperoxidase were counterstained with Congo red dye, the immunoreactive and green birefringent structures were found to be coincident. Indeed, amyloid plaques in scrapie appear to be causative rather than a consequence of the disease. The senile plaques seen in CJD are similar to those found in aged animals and humans, as well as in patients with the presenile and senile forms of Alzheimer’s disease (Divry, 1934; Terry and Wisniewski, 1970; Wilcock and Esiri, 1982; Wisniewski etal., 1983). The role of senile plaques in the pathogenesis of Alzeheimer’s disease remains to be established (Prusiner, 1984).

VII. HOSTGENESCONTROLLING INCUBATION TIMES

A. SIPILIP In early studies on scrapie in sheep, the influence of host background was appreciated. Certain breeds of sheep were found to be much more susceptible to scrapie infection than others (Gordon, 1966a) (Table VII). The

20

STANLEY B. PRUSINER TABLE VII COMPARATIVE SUSCEPTIBILITY OF DIFFERENT BREEDS OF SHEEP TO SCRAPIE"

Breedb

Number"

Percentaged

Breedb

Number"

Percentaged

Herdwick Dalesbred Swaledale S. S.Cheviot Derby. Gritstone Exmoor Horn Border Leicester Scot. Blackface South Devon Romney Marsh Welsh Cheviot Ryeland

28/36 31/43 25/46 16/45 16/46 14/41 11/42 8/44 6/35 7/43 6/40 5/34

78 72 54 36 35 34 26 18 17 16 15 15

Dorset Horn Suffolk Leicester Welsh Mountain Hampshire Down N. S. Cheviot Southdown Wiltshire Horn Shropshire Kerry Hill Clun Forest Dorset Down

6/45 6/51 5/42 4/42 3/30 4/45 3/38 4/57 2/41 1/41 1/52 0148

13 12 12 10 10 9 8 7 5 2 2 0

Data from Gordon (1966a); incubation periods ranged from 3.5 to 24 months. Sheep inoculated subcutaneously with 5 ml of 10% suspension of scrapieinfected brain tissue in saline. Number with scrapie after 2 years of observation over the number tested. Percentage developing scrapie a

alleles of a gene labeled SIP and LIP have been suggested to control the length of the incubation period in sheep (Dickinson, 1976). Studies on sheep were difficult because even susceptible breeds lacked uniform response to the scrapie agent. Subsequent studies with goats showed that these animals are more uniformly susceptible to scrapie (Pattison and Millson, 1960).

B. SINC The lack of highly inbred strains of sheep and goats as well as their long scrapie incubation periods made them far less useful than mice in the study of host genetic influences on the development of scrapie. Early studies on scrapie in mice reported the existence of a SINC gene which controls the length of the incubation period. Two alleles have been defined by Dickinson and his colleagues who reported that most mice exhibited short incubation periods when inoculated with the ME-7 agent isolate (S7,S7) (Dickinson and Meikle, 1971; Outram, 1976). In contrast, the VM mouse showed a prolonged incubation period (P7,P7). The F, generation of crosses between VM and other mice exhibited three different responses: (1)partial dominance with incubation periods falling between those of the parents, (2) overdominance with incubation periods longer than either

21

PRIONS

parent, and (3) dominance. Different agent isolates have been reported to alter the response of the mice.

C. PID Recent studies indicate that among mice classified as S7,S7 by Dickinson, one and possibly two genes are operative in controlling the scrapie and CJD incubation periods (Kingsbury etal., 1983). Alleles of one of the genes coding for longer incubation times appear to be autosomal dominant. Using congenic mice inoculated with the CJD agent, the PID-1gene was found to be located in the D subregion of the major histocompatibility complex (H-2) on chromosome 17. The q allele codes for short incubation times while the d allele codes for longer ones. Thep, s, b,and k alleles code for intermediate times. The sex of the animal was also observed to have a profound effect on the length of the incubation time in some strains of mice. Whether or not this phenomenon is sex linked remains to be established. A comparison of the SINC and PID-1 genes is given in Table VIII. OF PRIONS VIII. TRANSMISSION

The mechanisms whereby prions are transmitted in nature are largely unknown. Oral transmission of scrapie to sheep and goats has been shown experimentally, but no information is available about the natural means of spread (Pattison and Millson, 1961). TME is presumed to occur after mink ingest scrapie-infected meat, but there is no firm evidence to support this hypothesis. The means by which CJD spreads is unknown, but the TABLE VIII OF PID AND SINC GENES COMPARISON PRION INCUBATION TIMES CONTROLLING

PID Control of incubation times Location Fine structure Alleles coding for longer incubation times

SINC

CJD, scrapie

Scrapie

Chromosome 17 D subregion of H-2 complex Autosomal dominant

Unknown Unknown Partial or overdominant

22

STANLEY B. PRUSINER

transmission of kuru by ritualistic cannibalism seems well documented (Section V,A). Experimental transmission of prion diseases to laboratory animals has been extensively studied over the past 3 decades. Variation among animal species in their susceptibility to prion diseases implies that prions possess diverse molecular structures. Differences in incubation periods for various isolates or “strains” also suggest multiple structures for prions (Dickinson andFraser, 1977;Prusiner, 1982). These observations on the apparent diversity of prions as well as the ability of prions to adapt upon repeated passage in a given host are of importance in understanding how prions replicate (Prusiner, 1982). However, such findings cannot substitute for biophysical and biochemcial studies in determining the molecular structure of prions.

A. Isolates or Strains of Prions Dickinson co-workers have isolated numerous “strains” of the scrapie agent by passage in mice at limiting dilution (Dickinson and Fraser, 1977). These strains appear to be quite stable once isolated. There is no evidence for the mutation of one strain into another. The various prion strains have been characterized by their incubation periods and neuropathological profiles. The purity of these isolates is unknown since plaque purification methods are unavailable; the scrapie agent does not replicate well in cell culture. Kimberlin and Walker (1978a) claimed t o have separated prions causing scrapie in mice from those causing scrapie in hamsters. Again, the purity of these isolates is unknown; this caveat is exemplified by recent studies showing that mice can be infected with hamster scrapie prions after a prolonged incubation period (R. Carp, unpublished observations). If a large number of strains of the scrapie agent exist as suggested from the foregoing summary, the most plausible model for the prion would seem to require a nucleic acid. As discussed in Section X,B, there is no evidence to date for such a structure. Whether prions contain a small polynucleotide within their core remains to be established (Prusiner, 1982).

B. Adaptation of Prions upon Passage Adaptation of prions has been observed upon repeated passage in the same host species as evidenced by reduction in the lengths of incubation periods (Gibbs et al., 1979; Kimberlin and Walker, 1977; Kingsbury et al., 1982; Manuelidis and Manuelidis, 1979; Prusiner et al., 1980a; Tateishi et al., 1979). This adaptation process has been widely observed in both

PRIONS

23

experimental scrapie and CJD. Frequently, a reduction of the incubation periodby as much as 50% is seen by repeated passage in a given species. As discussed above, the genetic background of the host may also be of importance in determining the length of the incubation period. Hadlow observed that the scrapie agent when passaged in mink retained its ability to infect goats, but lost its ability to infect mice (W. J. Hadlow, unpublished observation). Interestingly, the infectious agent causing mink encephalopathy has a similar host range (Marsh and Kimberlin, 1975). The TME agent can be passaged in goats but not mice. The nature of the molecular changes that distinguish the scrapie agent propagated in mink from that found in goats and mice will be important. IX. BIOPHYSICAL AND BIOCHEMICAL PROPERTIES OF SCRAPIE PRIONS

A. Purification and Hydrophobicity of Prions Purification of the scrapie agent has been crucial for studies defining the properties of the infectious particle. The need to purify the agent away from cellular molecules prior to meaningful characterization studies is no different from investigations on many other biological macromolecules. Several investigators have mounted major efforts to purify and characterize the scrapie agent over the past 2 decades (Hunter, 1979; Kimberlin, 1976; Prusiner et al., 1979). Early studies suggested 1976; Millson et al., that the scrapie agent was distributed throughout virtually all subcellular fractions (Hunter, 1979; Kimberlin, 1976). The interpretation of those observations was complicated by the imprecision of the endpoint titrations of the agent. Nevertheless, the scrapie agent was reported to be intimately associated with cellular membranes, and from this association the “membrane hypothesis” evolved (Gibbons and Hunter, 1967; Hunter et al., 1968). When various extraction procedures failed to release the agent from membrane fractions, it was concluded that the agent is a replicating membrane fragment that cannot be separated from cellular membranes. Several different purification procedures have been reported. One involved copurification of the scrapie agent and microsomes (Semancik et al., 1976). Another involved isolation of a “membrane-free” fraction after prolonged ultracentrifugation (Malone et al., 1978). This fraction contained 1to 10% of the scrapie agent as was precipitated with ammonium sulfate prior to SDS -gel electrophoresis. After electrophoresis, the agent was eluted from the gel in order to obtain a further purification (Malone et al., 1979). Although the results of these studies seemed encouraging initially, subsequent work has been disappointing (Prusiner et al., 1980~).

24

STANLEY B. PRUSINER

Using equilibrium sucrose and sodium chloride density gradients, Siakotos and co-workers (1976) attempted to purify the scrapie agent from murine brain. They suggested that there was a peak of infectivity at a sucrose density of 1.19 g/cm3. However, multiple peaks of infectivity were found throughout the gradients, indicating considerable heterogeneity of the agent with respect to density. These results showed that density gradient centrifugation, when applied to crude suspensions of membranous material from brain, is probably not useful in isolating the scrapie agent. Other studies from the laboratory of Gajdusek demonstrated considerable heterogeneity of the agent in metrizamide and cesium chloride density gradients (Brown etal., 1978). Since the initial purification of many biological macromolecules involves a series of differential centrifugations (Prusiner, 1978), we began our studies on the scrapie agent by defining its sedimentation properties in fixed-angle rotors in order to develop a preparative protocol. These studies showed that the agent from both murine spleen and brain sedimented over a range of particle sizes from 60 S to 1000 S (Prusiner etal., 1978a). On the basis of the information derived from these sedimentation profiles, a partial purification scheme for the murine scrapie agent from spleen was derived (Prusiner etal., 1978b). The preparation was devoid of cellular membranes and enriched for the scrapie agent 20- to 30-fold with respect to protein and DNA. Studies on the agent by rate-zonal sucrose gradient centrifugation gave sedimentation coefficients for the agent ranging from 40 S to >500 S. Sucrose density gradient centrifugation revealed a particle density ranging from 1.08 to more than 1.30 g/cm3, an indication that some forms of the agent might be associated with lipids. Further sedimentation studies showed that the agent aggregated with cellular elements on heating the agent in a partially purified fraction (Prusiner etal., 1980b). The agent was stable in nonionic and nondenaturing anionic detergents, but was inactivated by SDS. Free-flow electrophoresis showed that most of the agent has a net negative charge, but significant charge heterogeneity was found (Prusiner etal., 1980b). Heterogeneity of the scrapie agent with respect to size, density, and charge suggested that hydrophobic domains on its surface might be responsible for these phenomena (Prusiner etal., 1978b, 1980e). Such domains are usually formed by the juxtaposition of nonpolar side chains of amino acids within a protein. These initial studies on the murine agent from spleen revealed the complexities of scrapie agent purification. We then developed an improved assay based on measurements of the incubation time (Prusiner etal., 1982b). With this new bioassay, we created a purification scheme for the agent from hamster brain, where the titers are highest (Kimberlin and

PRIONS

25

Walker, 1977). The initial steps of the purification were similar to those for the murine agent (Prusiner etal., 1978b). Deoxycholate extracts (P4) were digested sequentially with micrococcal nuclease and proteinase K. The digestions were performed at 4"C to prevent aggregation of the agent, which is observed at elevated temperatures (Prusiner etal., 1978b, 1980e). The digested preparations were then subjected to cholate-sodium dodecyl sarcosinate (Sarkosyl) extraction followed by ammonium sulfate precipitation (P5). Most of the remaining digested proteins and nucleic acids were separated from the scrapie agent by Sarkosyl agarose gel electrophoresis at 4 ° C (Prusiner etal., 1981b). Such preparations of the eluted scrapie agent (E,) were approximately 100-fold purified with respect to cellular protein (Prusiner et al., 1981b). With these enriched preparations we demonstrated that a protein within the agent is required for infectivity ID,, units of (Prusiner etal., 1981b). Fraction E, contained 106.5to agent, 20 to 50pglrnl of protein, < 1pg/ml of DNA, and < lOpg/ml of RNA. More recently we have developed an improved purification protocol for the scrapie agent (Prusiner etal., 1982a). This protocol has several advantages over the earlier ones noted above. In the earlier protocols, the sedimentation of the agent in a microsomal membrane fraction required prolonged ultracentrifugation and thus severely limited the size of the preparations. In addition, the preparative Sarkosyl electrophoresis of the agent was slow, tedious, and of limited capacity. In our new purification protocol, differential ultracentrifugation was supplanted by polyethylene glycol 8000 (PEG-8000) precipitation, and preparative gel electrophoresis was replaced by rate-zonal gradient centrifugation. This protocol allowed us to purify the agent from 100- to 1000-foldwith respect to protein. The protocol includes Triton X-lOO/sodium deoxycholate extraction and PEG-8000 precipitation, nuclease and protease digestion, cholate and Sarkosyl extraction, ammonium sulfate precipitation, Triton X-lOO/SDS extraction, and rate-zonal sedimentation through a discontinuous sucrose gradient (Fig. 2). The highest degree of purification was found in a fraction from the 25/60% sucrose interface near the bottom of the gradient (fraction 2). Typically, the titer in fraction 2 was ID,, units/ml and the protein concentration was 40 pg/ml. When Triton X-100 or octylglucoside extractions were used in place of the Triton X-lOO/SDS extraction, the distribution of the scrapie agent in the discontinuous sucrose gradients was altered and no substantial purification was obtained. Since sufficient quantities of prions needed for further purification could not be obtained by the procedure described above (Prusiner etal., 1982a), we developed a large scale purification protocol using the same detergent extractions, precipitations, and enzyme digestions noted above, but employing continuous flow and zonal rotor centrifugation (Prusiner et

26

I:

STANLEY B. PRUSINER

.22

.14

- .10 -z_ .08 ;- .06 s 0 rg

Ly

I-

L

FRACTION NUMBER

FIG.2. Rate-zonal discontinuous sucrose gradient centrifiguration for purification of the scrapie prion. Fraction P, (4 ml) suspended in 2% (v/v) Triton X-l00/0.8% (w/v) SDS was layered onto a sucrose step gradient containing 4 ml of 60% (w/v) sucrose at the bottom and 32 ml of 25% sucrose. The sucrose solutions contained 20 mM Tris-OAc, pH 8.3 1 mM EDTA, and no detergent. The gradients were centrifugated at 50,000rpm for 120 minutes in a vertical VTi5O rotor at 4°C and fractionated from the bottom. Fractions were stored at -70°C prior to assay for the scrapie agent (O), sucrose (A), protein (01,and Azso(0).

al., 1983). This large scale protocol has yielded preparations enriched 3,000- to 10,000-fold for scrapie prions with respect to protein. These extensively purified preparations were found to contain one major protein which polymerizes to form rod-shaped particles (Prusiner et al., 1983). The availability of these purified preparations has allowed us (1)to show that scrapie prions aggregate to form amyloid-like rods that stain with Congo red dye and exhibit green birefringence (Prusiner et al., 1983), (2) to produce antibodies to both prions and PrP molecules as well as show that these antibodies decorate prion rods and stain amyloid plaques (R. A. Barry, M. P. McKinley, P. E. Benheim, G. K. Lewis, and S. B. Prusiner, in preparation; Benheim et al., 1984), (3) to determine the N-terminal amino acid sequence of P r P from which corresponding oligonucleotide probes have been synthesized (Prusiner etal., 1984), and (4) to demonstrate that PrP is a sialoglycoprotein (D. C. Bolton, R. Meyer, and S. B. Prusiner, in preparation).

27

PRIONS

B. SearchforNucleic Acidin thePrion Multiple studies have shown that the scrapie agent in crude preparations was resistant to nuclease digestion (Hunter, 1979; Millson etal., 1976; Prusiner etal., 1980e) and to UV irradiation at 254 nm (Alper etal., 1967;Latarjet, 1979). The objection to these studies was that aprotective coat prevented nucleases from penetrating the agent, as well as shielding it from radiation (Table IX). At several different stages of purification we have searched for susceptibility of the agent to nuclease digestion. No decrease in scrapie infectivity has been observed following digestion with micrococcal nuclease, nuclease P, deoxyribonucleases I and 11, ribonucleases A and T,, and phosphodiesterases I and I1 at 10,100, and 500pg/ml for 3 to 30 hours at 37°C. Ribonucleases I11 and H at 1and 10 units/ml also showed no effect upon scrapie prion titer. Although nuclease sensitivity has been described for the scrapie agent (Marsh etal., 1978), we have been unable to confirm this observation (Prusiner etal., 1980~). The complete lack of scrapie agent sensitivity to nucleases in view of inactivation by proteases is of interest. Numerous viruses are resistant to nuclease; presumably, these enzymes do not penetrate the viral protein TABLE IX

RESISTANCE OF THE SCRAPIE PRION TO PROCEDURES THAT ATTACKNUCLEIC ACIDS Procedure

Resistant

UV irradiation

Ribonucleases, deoxyribonucleases 254 nm

Divalent cation hydrolysis

Zn2+

Psoralen photoreaction

AMT, HEP, HMT, MNT, TMP”

Chemical modification

Hydroxylamine

Nucleases

~~~~

~

~~~

~~

Possible explanations Enzymes unable to penetrate protein shell Shielded by protein shell or no critical nucleotide dimers formed Ions unable to penetrate protein shell Monoadducts of single-stranded genome do not inactivate or psoralens unable to penetrate protein shell Nucleophiles react only with surface protein and are unable to penetrate the shell or react minimally with double-stranded genome

~~

AMT, 4 Aminomethyl-4,5 ,8-trimethylpsoralen; HEP, 1-a-4 hydroxyethylpsoralen; HMT, 4 -hydroxymethyl-4,5 ,8-trimethylpsoralen; MMT, 4 -methoxymethyl-4,5 ,8-trimethylpsoralen; TMP, 4,5’&trimethylpsoralen. a

28

STANLEY B. PRUSINER

coats (Rose, 1974; Schaffer and Schwerdt, 1959). Addition of ribonuclease A at 0.1 ,ug/ml to a crude nucleic acid extract containing potato spindle tuber viroid (PSTV)decreased the PSTV titer by a factor of >106in1hour at 25°C (Diener and Raymer, 1969). Hydrolysis of a single phosphodiester bond within a viroid probably inactivates it (Diener, 1979; Sanger etd., 1979). In contrast, there are many examples of proteins that retain their biological activities after limited proteolysis (Mihalyi, 1978). We do not know in the case of the scrapie agent how many peptide bonds must be cleaved to cause inactivation, Studies with the optically clear fraction E, as well as sucrose gradient fraction 2 have confirmed the resistance of the scrapie agent to UV inactivation (Alper etal., 1967; Latarjet, 1979). Fraction S,, P,, and E, as well as gradient fraction 2 were irradiated a t 254 nm with increasing doses. Although no inactivation of the agent in fraction S, was observed, a minimal but probably significant decrease was found in fractions P5and E6as well as gradient fraction 2 (S. B. Prusiner, J. Cleaver, and D. F. Groth, unpublished observations). The kinetics of inactivation by irradiation at 254 nm suggest a single-hit process. The resistance of the scrapie agent to inactivation by irradiation a t 254 nm is much greater than that observed for viruses and viroids (Prusiner, 1982). Clearly, the inactivation of the scrapie agent at extreme energy levels indicates a photochemistry of a far different nature from that observed for virus and viroid inactivation through the formation of thymine or uracil dimers. Proteins are relatively resistant to irradiation at 254 nm (McLaren and Shugar, 1964) and are probably the target within the scrapie agent in these irradiation studies. Observations on the resistance of the scrapie agent to procedures attacking nucleic acids have been extended by means of three other techniques (Table IX). The agent has been incubated at pH 7 in the presence of 2 mM Zn(NO,), a t 65°C for periods as long as 24 hours without loss of infectivity (Table X). Under these conditions polymers of RNA are completely reduced to mononucleotides, and polymers of DNA undergo considerable hydrolysis (Butzow and Eichorn, 1965, 1975). Photochemical inactivation of the scrapie agent with psoralens was attempted with samples at several levels of purification, both from murine spleen and hamster brain. Five different psoralens of varying degrees of hydrophobicity were used (Isaacs etal., 1977). It was expected that the most hydrophobic psoralens would readily partition into the scrapie agent. No inactivation of the scrapie agent was observed with any of these psoralens over a wide range of dosages (McKinley etal., 1983a). Psoralens may form diadducts upon photoactivation within base-paired regions of nucleic acids and 1978; Hearst monoadducts within single-stranded regions (Hanson etal., and Thiry, 1977). Psoralens have several advantages in searching for a

29

PRIONS

TABLE X

RESISTANCE OF THE SCRAPIE PRION TO Zn2+CATALYZED HYDROLYSIS AT 65°C" Zn(NO,), concentration (mM) Time (hours) 2 4

8 24

0.2

0

2

[log titer (ID6ounits/ml)] 7.3 k 0.03 8.0 k 0.26 7.3 k 0.37 7.1 k 0.16 7.1 k 0.23

7.6 k 0.35 7.2 k 0.16 7.5 -+ 0.20 7.0 k 0.07

7.0 It_ 0.16 7.1 zk 0.16 7.5 k 0.26 7.1 k 0.13

Fraction E, was dialyzed for 24 hours a t 4°C against 10 mM NaNO,, pH 7.4, containing 2% (w/v) Sarkosyl (DG 232).

nucleic acid genome: (1)low reactivity with proteins, (2) penetration of viral protein and lipid coats, and (3) formation of stable covalent linkages on photoactivation. Psoralens have been found to inactivate numerous viruses, but not, for example, picornaviruses (C. Hanson, personal communication). Psoralens, like acridine orange and neutral red dyes (Crowther and Melnick, 1961), do not penetrate the protein coat of poliovirus. Photoadducts with viral RNA were formed when psoralens or the above tricyclic dyes were added to cultured cells replicating the poliovirus. In contrast to psoralens, hydroxylamine readily inactivates poliovirus a t neutral p H (Borgert etal., 1971). Hydroxylamine does not generally react with proteins at neutral pH, but it does decarbethoxylate modified proteins and it does modify cytosine bases (Bornstein and Balian, 1970). At concentrations up to 0.5 M a t neutral pH hydroxylamine failed to alter scrapie agent infectivity (McKinley etal., 1981). Under these conditions, most animal and plant viruses as well as bacteriophage are inactivated by hydroxylamine except for the paramyxoviruses which are resistant (Franklin and Wecker, 1959; Freese etaL, 1961; Phillips and Brown, 1967; Schuster and Wittman, 1963; Tessman, 1968). In contrast, inactivation of the scrapie agent by carbethoxylation upon treatment with DEP was found to be reversible with NH,OH (McKinley etal., 1981). The extreme resistance of the scrapie agent to inactivation by procedures that modify nucleic acids suggest that its structure is different from that of both viruses and viroids. While there are examples of viruses that are resistant to inactivation by two or even three of the five procedures in Table IX, we are unaware of any viruses which, like the scrapie agent, are

30

STANLEY B. PRUSINER

resistant by all of these procedures. However, the possibility must be considered that the putative genome of the scrapie agent is buried within a tightly packed protein shell which excludes nucleases, UV irradiation, ZnZf,psoralens, and NH,OH. Also, we cannot exclude an unusual nucleic acid with a different base structure or polymer packing that might exhibit the resistant characteristics described for the scrapie agent. In a comparative study of the scrapie agent and PSTV, we found that the two pathogens had virtually opposite properties. The viroid resisted inactivation by procedures that modify or hydrolyze proteins while the prion was inactivated by these methods. In contrast, the prion resisted inactivation by procedures that modify or hydrolyze nucleic acids while the viroid was inactivated by these methods. From these observations we concluded that except for the small size of these infectious pathogens, viroids and prions seem to be antithetical (Diener etal., 1982). Of interest are studies showing a large oxygen effect upon exposure of the scrapie agent to ionizing radiation (Alper etal., 1978). Viruses and nucleic acids characteristically show a small oxygen effect. Biological membranes and probably lipoproteins show large oxygen effects. The increased sensitivity of the scrapie agent to ionizing radiation in the presence of oxygen presumably reflects the hydrophobic protein with bound lipids that is required for infectivity (Prusiner et al., 1981b). These data do not eliminate the possibility that the agent also contains a nucleic acid.

C. Molecular Sizeof thePrion One explanation for the extreme resistance of the scrapie agent to inactivation by irradiation at 254 nm is that the putative nucleic acid within the agent is quite small (Alper etal., 1966, 1967; Latarjet, 1979; Prusiner, 1982). The resistance of the agent to inactivation by irradiation at 254 nm by pyrimidine dimer formation could be explained by a putative nucleic acid of 50 bases or less (S.B. Prusiner, J. Cleaver, and D. F. Groth, unpublished observations). This estimate assumes that the pyrimidines are randomly distributed within the nucleic acid and that one dimer is sufficient to inactivate the agent. Alternatively, the protein(s) of the scrapie agent might be modified by irradiation at 254 nm. Interestingly, a similar size for the putative nucleic acid of the scrapie agent can be calculated from the psoralen experiments. Assuming that the psoralen was able to freely penetrate the protein exterior of the agent, then only a scrapie nucleic acid of 40 bases or less could have eluded psoralen photoaddwt formation under the conditions of our experiments (McKinley etal., 1983a). These data are consistent with other lines of evidence indicating that the scrapie particle is quite small, as described below.

PRIONS

31

The extreme resistance of the scrapie agent to inactivation by ionizing radiation raised the possibility that the agent is quite small (Alper etal., 1966). Target calculations have given minimum M , ranging from 64,000 to 150,000 (Alper etal., 1966; Latarjet, 1979). However, two important factors could not be taken into account in these calculations. The first is the possibility that multiple copies of the agent might exist within a single infectious particle as would occur with aggregation. We have good evidence that the agent readily associates with cellular elements and probably aggregates with itself in purified preparations (Prusiner etal., 1978b, 1979, 1980b). The second is the efficiency of the cellular repair processes. For example, polyoma virus dsDNA (3 X lo6 daltons) has been found to be almost as resistant to ionizing radiation as either viroids or the scrapie agent (Latarjet etal., 1967; Semancik etal., 1973). The extreme efficiency of the cellular repair processes for the polyoma virus dsDNA genome accounts for its apparent resistance to damage by ionizing radiation (Latarjet, 1979). Studies on the scrapie agent in murine spleen have shown a continuum of sizes ranging from 40 S or less to more than 500 S by rate-zonal sucrose gradients (Prusiner et.al., 1978b, 1979). Parvoviruses are among the smallest viruses identified and they have sedimentation coefficients of 100 S to 110 S (Rose, 1974;Schaffer and Schwerdt, 1959). In heated preparations extracted with sodium deoxycholate, the scrapie agent associated with cellular elements to form large infectious particles of > 10,000 (Prusiner etal., 1979, 1980b). Such particles are the size of mitochondria. Sedimentation studies of CJD agents adapted to both guinea pigs and mice suggest that the sizes of these agents are similar to that observed for the scrapie agent (Prusiner and Kingsbury, 1984). Gel electrophoresis has shown that the scrapie agent exists as a succes1980c,e). Sarkosyl agarose sion of particles of varying size (Prusiner etal., gel electrophoresis of partially purified fractions showed that some forms migrated more slowly than DNA restriction endonuclease fragments of 15 X lo6 dalton DNA fragments. Digestion of crude preparations with nucleases and proteases facilitated the entry of the agent into these gels. One report showed that most of the scrapie agent migrated with 5 S RNA molecules in the presence of SDS (Malone etal., 1979). We were unable to confirm these findings since SDS inactivated the agent (Prusiner etal., 1980c,d). Gel filtration studies with anionic detergents and chaotropic ions have given results similar to those described for early rate-zonal sucrose gradients and gel electrophoresis. Typically most of the agent eluted in the void volume followed by a continuum of particles apparently of decreasing size (Prusiner etaL, 1980e, 1981a). In contrast, incubation of the scrapie

32

STANLEY B. PRUSINER

agent overnight with 10%(weight to volume) sulfobetaine 3-14, a zwitterionic detergent, appears to have dissociated the agent (S.B. Prusiner, and D. F. Groth, unpublished observations). Under these conditions the scrapie agent eluted as a peak behind bovine serum albumin (BSA), but slightly ahead of ovalbumin. If the agent has a globular shape in sulfobetaine 3-14, then it may have an M , of 50,000 or less. How much detergent is bound to the agent and how the detergent influences the apparent M , of the agent remains to be determined (Reynolds, 1981). Similar observations have been recorded with another detergent, 1-dodecyl propanediol3-phosphorylcholine, which is a synthetic derivative of lysolecithin. Confirmation of these findings by other techniques is mandatory since anomalous behavior of proteins during gel filtration is well known (Andrews, 1971; Nozaki et al., 1976). Rate-zonal gradient centrifugation studies have indicated that the scrapie agent may have an observed sedimentation coefficientas low as 2 S. Since we do not know the density of the scrapie agent in detergent solutions, it is possible that the agent was floating in these rate-zonal gradients and that this value is artifactually low. We have also found that the scrapie agent, after disaggregation in zwitterionic detergents, passes through nucleopore filters with 15 nm pores (S. B. Prusiner and D. F. Groth, unpublished observations). If the scrapie agent does have an MI of 50,000 or less, then a nucleic acid within such a globular structure will be too small to code for a protein. A spherical scrapie agent of MI 50,000 would have a diameter of 4 to 6 nm (Nozth and Rich, 1961;Tanford, 1961). Let us assume that the agent has a protective protein which is 1nm (10 A) thick. The volume of the core will be 14.1 nm3. From measurements of DNA packing in crystals and bacteriophage (Earnshaw and Casjens, 1980; Giannoni et al., 1969; Langridge et al., 1960), there is space for a 12-nucleotide polymer consiting of six base pairs. Dehydration of the polymer would permit 32 nucleotides to be encapsidated. Indeed, if such oligonucleotides exist within the agent, they must have a function other than that of a template directing the synthesis of scrapie coat proteins.

-

D. Scrapie Prion Contains a Protein (PrP) Convincing evidence that scrapie agent infectivity depends upon a protein(s) was provided by experiments reported in 1981 (Prusiner et al., 1981b). Those studies depended upon the development of a purification scheme for the agent from hamster brain using Sarkosyl gel electrophoresis. The infectivity of the scrapie agent was shown to be susceptible to degradation by proteinase K. Diminution of infectivity was shown to be

PRIONS

33

dependent upon the time of digestion, the concentration of proteinase K, and the activity of the enzyme. Similar results were also obtained with trypsin (Prusiner, 1982). As shown in Fig. 3, the titer of the scrapie agent progressively declined as the time of trypsin digestion increased. The kinetics of scrapie agent degradation were altered by increasing the amount of trypsin. Higher concentrations of trypsin accelerated the decline in scrapie agent titer. A recent report has confirmed our findings in partially purified fractions from murine brain (Lax etal., 1983). Proteinase K was found to consistently reduce the titer of these fractions. Although earlier studies with proteases showed occasional reductions of scrapie infectivity (Cho, 1980; Hunter and Millson, 1967; Hunter etal., 1969), the inconsistency and paucity of the experimental data left considerable doubt about the proper interpretation of the results. In addition to the protease studies, we showed that the scrapie agent could be inactivated by chemical modification with DEP (McKinley etal., 1981) (Fig. 4). The chemical modification was found to be reversible using hydroxylamine and was accompanied by a restoration of scrapie agent infectivity. The specificity of reversible chemical modification by DEP provided another important line of evidence showing a protein(s) was required for infectivity. 8-

E

7-

\ 0 In

0

v

k

6-

.Im

. I -

0

2

5-

Time of Digestion (h)

FIG.3. Inactivation of the scrapie prion by digestion with trypsin. Fraction E, was digested with trypsin, 0 pg/ml 100pg/ml (A), or 500 pg/ml (0)for increasing periods of time up to 30 hours. Aliquots were taken for bioassay in animals after the digestion was terminated with 10 mM phenylmethylsufonylfluoride (PMSF) and freezing with dry ice followed by storage at -70°C.

(a),

34

STANLEY B. PRUSINER

I

L 0)

t

c .-

t 0 0

2

4.0

-

3.0~

0

10 20 Diethylpyrocarbonote (mM)

FIG.4. Dose-dependent inactivation of the scrapie prion with increasing concentration of DEP. Standard errors of titers are denoted by bars. Fraction E, containing the scrapie agent and 75 pg/ml of protein was dialyzed overnight a t 4°C against 20 mMTris-HC1, pH 7.4, containing 1 mM EDTA and 0.2%Sarkosyl. Freshly prepared aqueous DEP was added to give the desired final concentration of DEP in a 0.1-ml incubation mixture. All samples were mixed well and allowedto incubate at room temperature for 30 minutes. Reactions were terminated by plunging the vials into a dry ice-ethanol bath. Vials were then stored a t - 20°C for 3 to 4 weeks prior to bioassay.

Once convincing studies showed the presence of a protein(s) within the scrapie agent, the results of earlier studies with denaturation reagents could be accurately interpreted. SDS, urea, guanidinium thiocyanate, and phenol had all been found to inactivate the scrapie agent, but the actions of these reagents were not sufficiently specific to establish that a protein(s) was required for infectivity. For example, Hunter and co-workers (1969) showed that exposure of the scrapie agent to 6.0 Murea decreased the titer by a factor of 100. This high concentration of urea could have denatured protein or nucleic acid. We have found that exposure of the scrapie agent in partially purified fractions to 3 Murea a t 4°C decreases the titer by a factor of 50 (Fig. 5). Removal of the urea after 2 hours was accompanied by

35

PRIONS

2 0

1

2

3

4

5

6

7

8

Urea (M)

FIG.5 . Inactivation of the scrapie prion by urea. Fraction P, was dispersed in 2.0%(v/v) Triton X-100 for 16 hours a t 4 ° C and then exposed to urea concentrations up to 8 M for 2 hours. After 2 hours an aliquot was frozen a t - 70°C (0)and another was dialyzed (A) a t 4 ° C against 20 m M Tris-OAc, p H 8.3, 1.0 m M EDTA, and 2.0%Triton X-100. Spectropore dialysis tubing with pores retaining molecules of M,> 12- 14K was used. Typically 0.4 ml of sample was dialyzed against 400 ml of buffer for 1-2 hours. Three changes of dialysate were made. Upon completing the dialysis, samples were stored at -70°C prior to bioassay. Enzyme grade urea purchased from Schwartz/Mann was freshly prepared immediately prior to the experiment in order to minimize the concentration of cyanate.

a return of infectivity. This observation is similar to our findings with KSCN where removal of the KSCN was accompanied by an apparent return of infectivity (Prusiner etal., 1981a). Whether urea or cyanate ions (Stark etal., 1960) are responsible for the loss of scrapie infectivity in these experiments is not known. From our data the most likely target within the scrapie agent for denaturation by urea is a protein. Evidence that scrapie infectivity depends upon a protein(s) is summarized in Table XI. Both proteinase K and trypsin, which catalyze the hydrolysis of peptide bonds, destroy scrapie infectivity in fractions prepared by Sarkosyl gel electrophoresis. As noted above, nucleases and phosphodiesterases do not alter the infectivity of the scrapie agent. Chemical modification of free amino groups by iodinated 3-(4-hydroxyphenyl) propionic acid N-hydroxylsuccinimide ester (Bolton-Hunter reagent) has no effect on scrapie infectivity in contrast to modification by

36

STANLEY B. PRUSINER TABLE XI

THAT SCRAPIE PRION INFECTIVITY DEPENDS ON PROTEIN^ EVIDENCE ~

~

~ ~ _ _ _ _ _ _ _

Procedure

Stable

Protease digestion Chemical modification

Hydroxyphenylpropionamide

Denaturing regents Detergents Salts Organic solvents Denaturants

TX-100, NP40, OGS, SB-314, ET-12H, CHAPS, cholate, Sarkoyl Na+,K+, C-,S04-2,P04-3EDTA-4 Methanol, ethanol, propanol

Labile Proteinase K, trypsin DEP, butanedione, PMSF

SDS, LDS, SDeS Gdn+,TCA-,SCNPhenol, 2-chloroethanol Urea

DEP, Diethylpyrocarbonate; PMSF, phenylmethylsolfonylfluoride; TX-100, Triton X-100; NP40, Nonidet P-40; OGS, octylglucoside; SB 3-14, sulfobetaine 3-14; ET-12H, l-dodecyl propanediol-3-phosphorylcholine;CHAPS, 3-[(3-cholamidoproSarkosyl, sodium dodecyl sarcosinate; pyl) dimethylammonio]-l-propanesulfonate; SDS, sodium dodecyl sulfate; LDS, lithium dodecyl sulfate; SDeS, sodium decyl sulfate; EDTA, ethylenediaminetetraacetic acid; Gdn, guanidinium; TCA,-trichloroacetic acid; SCN, thiocyanate.

DEP which reversibly inactivates the agent (McKinley etal., 1984). These results suggest that PrP has an active site which is required for maintenance of agent infectivity. The catalytic activities of numerous enzymes have been shown to be reduced after chemical modification of histidine residues with DEP and to be restored upon treatment with hydroxylamine (Miles, 1977). Inactivation of the scrapie agent or prion was found after exposure to denaturing detergents, strong chaotropic salts, harsh organic solvents, and denaturants like urea. Nondenaturing detergents, salts, and solvents were found not to alter prion infectivity. When the cumulative evidence for a protein within the scrapie agent became compelling, we began to search for scrapie specific proteins. In our initial studies, we radioiodinated fractions purified by the Sarkosyl gel electrophoresis procedure. The radioiodinated proteins were then separated by SDS polyacrylamide gel electrophoresis; scrapie and control samples were compared by this method in search of a polypeptide which was unique to fractions from scrapie-infected brain. Although we observed a polypeptide of M , 29,000that was present in some scrapie preparations and absent in controls, our results were inconsistent. This inconsistency may have been due to insufficient amounts of the scrapie protein in many of our

PRIONS

37

purified preparations. With the development of an improved purification scheme, the scrapie protein could be found in every preparation. Subsequent studies showed that this protein could be readily iodinated (Bolton et al., 1982) with lz5I-1abeled3-(4-hydroxyphenol) propionic acid N-hydroxysuccinimide ester which reacts with free amino groups of proteins (Bolton and Hunter, 1973). The unreacted ester was removed by reacting it with glycine and separating the proteins from the iodinated glycine by precipitation with quinine hemisulfate. The radioiodinated proteins were then separated from each other by SDS-polyacrylamide gel electrophoresis shown in Fig. 6. The radioiodinated polypeptide migrating with an apparent M, of 27,000-30,000 is the scrapie prion protein (PrP). PrP can be distinguished from normal brain proteins by (1)its microheterogeneity ( M , 27,000 - 30,000) observed during SDS polyacrylamide gel electrophoresis and (2) its resistance to protease digestion in the native or nondenatured state. Figure 6 illustrates a comparison between purified scrapie and normal brain fractions from Triton X- lOO/SDS discontinuous sucrose gradients after treatment with proteinase K. Though both samples originally contained similar amounts of protein, protease digestion under nondenaturing conditions hydrolyzed all proteins in the normal brain fraction, but left P r P intact in the scrapie fraction. Having found a protein which was present only in fractions purified from scrapie-infected brains, we asked if (1)this protein is a structural component of the prion or (2) it arises as a pathologic by-product during infection. To answer this question multiple experimental approaches were employed. The very diversity of the methods used coupled with the sameness and consistency of the answers indicates that this scrapie protein (PrP) must be a structural component of the prion (Table XII). TABLE XI1

SCRAPIE PROTEIN AND PRION -EVIDENCE THAT PRP Is A STRUCTURAL COMPONENT OF THE PRION PrP is found only in animals infected with the prion Scrapie prion and PrP copurify by two different procedures Concentration of PrP is directly related to the titer of prions Several properties of PrP are similar to those of the prion (poor immunogenicity, resistance to proteases, chemical modification by DEP) Changing the properties of PrP results in a corresponding alteration in the prion

38

STANLEY B. PRUSINER

S

FIG. 6. Scrapie prion protein (PrP) radioiodinated and separated by SDSpolyacrylamide gel electrophoresis. Fraction 2 from a Triton X-lOO/SDS discontinuous sucrose gradient containing the scrapie agent (S), or an analogous fraction purified from normal hamster brain (N), was concentrated by precipitation with SDS and quinine hemisulfate. The proteins were radioiodinated with N-succinimidyl 3-(4-hydro~y-5-[’~~1]iodophenyl) propionate and then reprecipitated as above. The pellets were suspended in 10 mM Tris buffer, pH 7.4,containing 0.2% Sarkosyl and proteinase K (100 pg/ml). The samples were incubated at 37 C for 30 minutes and the digestions stopped by boiling in electrophoresis buffer containing 2% SDS and 5% 2-mercaptoethanol. The digestedproteins were analyzed by electrophoresis in a 15% polyacrylamide gel. An autoradiogram of the gel is shown. PrP is denoted by arrow.

PRIONS

39

Samples purified from age-matched hamsters either uninoculated or inoculated with normal brain have failed to show evidence of PrP (Bolton etal., 1982). If PrP is found in normals, then it is present at levels <0.1% of that found in scrapie-infected brains (Bolton etal., 1984). PrP has been found using two different purification schemes: one employing Sarkosyl gel electrophoresis and the other with sedimentation through discontinuous sucrose gradient (Bolton, etal., 1984). In both purification schemes the scrapie prion and PrP have been found to copurify. This copurification indicates that the molecular properties of both the infectious prion and P r P must be similar. Further evidence that P r P is a structural component of the scrapie prion come from studies showing that the amount of this protein correlates with the number of infectious particles. A clear relationship between PrP concentration and the scrapie agent titer was observed. The kinetics for degradation of both the infectious prion and native P r P by proteinase K were similar (McKinley etal., 1983b). More than 2 hours of digestion at 37°C with 100 pgjml or proteinase K were required to demonstrate a decrease in prion titer and PrP concentration. In contrast, digestions of fraction 2 up to 30 hours with trypsin or SV-8 protease failed to cause a decrease in either prion titer or PrP concentration. One possible explanation for these results lies in the specificity of these proteases. Proteinase K is a nonspecific protease while trypsin and SV-8protease are both amino acid specific. Presumably, no surface Lys or Arg within the prion is accessible for trypsin catalyzed cleavage; similarly, no surface Glu is accessible for SV-8 protease cleavage. The correlation between the infectious prion and native PrP provides additional support for the hypothesis that the protein is a component of the scrapie agent. It is noteworthy that trypsin catalyzed the hydrolysis of peptide bonds within prions in fraction E,prepared by Sarkosyl gel electrophoresis, while trypsin did not alter the infectivity of prions in fraction 2 prepared by discontinous sucrose gradients. One possible explanation for this differential sensitivity to proteases is the difference in detergents used in purification of the two fractions. E, was prepared using only anionic detergent throughout the entire procedure (Prusiner etal., 1980c), while F, was purified using combinations of anionic and nonionic detergents (Prusiner etal., 1982a). Further studies are clearly required to elucidate the mechanism responsible for this difference in sensitivity of prions to digestion by trypsin. In addition to the parallel resistances of the infectious prion and native P r P to digestion by proteases, other properties of the prion and P r P have also been found to be similar. P r P can be radiolabeled with [14C]DEPand DEP reversibly inactivates the prion by chemical modification (Bolton et

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al., 1982;McKinley etal., 1981). Both PrP and the prion appear to be poor immunogens, as discussed in Section VII,A. Denaturation of prions by boiling for 2 minutes in the presence of SDS caused a reduction of infectivity by a factor of 10,000 and a significant enhancement in proteolytic degradation of P rP (Bolton etal., 1984).While degradation of native PrP by proteinase K required > 2 hours at 37"C, cleavage of denatured PrP was accomplished in <0.5 hours at 25°C. Denaturation also rendered PrP susceptible to proteolytic cleavage by SV-8 protease; whereas, native P r P is completely resistant to SV-8 catalyzed hydrolysis. The resistance of native P rP to degradation by proteases resembles that reported for some other proteins including avidin (Green, 1975), DNA binding protein (Krauth and Werner, 1979),laminin (Ott etal., 1982),and RNA 3'-terminal phosphate cyclase (Filipowicz etal., 1983), acetylcholinesterase (Vigny etal., 1979), a A lens crystallin (Bloemendal etal., 1982), amyloid (Cohen and Calkins, 1964; Emerson etal., 1966; Kim etal., 1969; Pras etal., 1969; Ruinen etal., 1968; Sorenson and Binington, 1964), hemoglobin (Kimura etal., 1978), transformation-sensitive membrane glycoprotein (Carter and Hakomori, 1978), peptidyltransferase (Cox and Kotecha, 1980), and chloroplast pigment protein (Suss etal., 1976). The mechanisms of protease resistance appear to include (1)conformation of the protein, (2) aggregation of the protein, and (3) binding of lipids as well as oligosaccharides t o the protein (Mihalyi, 1978; Rupley, 1967). In the case of PrP, all three of these mechanisms may be of importance in maintaining PrP in a protease-resistant state. The discovery of PrP is already having a profound effect upon scrapie research. The protein can be detected and quantitated within 1day after radioisotopic labeling. This represents a substantial decrease in the time required to gain information about the structure of the prion. Using two-dimensional gel electrophoresis, we have found that PrP is composed of at least 8 charge isomers (D. C. Bolton and S. B. Prusiner, in preparation). The isoelectric points of these isomers range from pH 4-8.4. Digestion of PrP with alkaline phosphatases failed to alter its migration during nonequilibrium pH gradient electrophoresis (NEPHGE) while digestion with neuriminidase did (D. C. Bolton and S. B. Prusiner, in preparation). We have also found that P rP stains with periodic acid-Schiff (PAS) reagent after electrophoresis into SDS polyacrylamide gels (R. Meyer and S. B. Prusiner, in preparation). Our finding that PrP is a sialoglycoprotein is consistent with its size heterogeneity (Bolton etal., 1982) and retarded migration in SDS polyacrylamide gels. By SDS polyacrylamide gel1 electrophoresis, P rP has an apparent M, of 27,000 - 30,000 (Prusiner etal., 1982a) while SDS HPLC size exclusion chromatography has given a M, value of 19,500 (Prusiner etal., 1984).

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Amino acid sequencing studies have shown that PrP has a single amino terminus but does possess “ragged” ends (Prusiner etal., 1984). The sequence is N-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-LysPro-Ser-Lys. This sequence data has allowed us to synthesize polypeptides which are being used as immunogens as well as to construct mixtures of oligonucleotides which encode portions of the amino acid sequence. Using the oligonucleotides and antibodies to PrP, we are attempting to clone and identify cDNAs encoding PrP. From such studies, knowledge about the origin and replication of scrapie prions should emerge.

E. Electron Microscopy Numerous attempts to identify the scrapie agent using electron microscopy have been made over the last two decades. Progress in purification has allowed us (Prusiner etal., 1982a, 1983) and subsequently others (Diringer etal., 1983) to demonstrate the presence of rod-shaped particles in preparations substantially enriched for scrapie infectivity with respect to protein. The rods measure 10-20 nm in diameter and 100-200 nm in length by negative staining. By rotary shadowing, the rods were found to have a diameter of 25 nm (Fig. 7). Some of the rods have a twisted and flattened appearance while a few rods seem to have a subfilamentous structure; however, no morphologically definable unit structure has been found. Copurification of the rods and scrapie infectivity by two different procedures, one utilizing Sarkosyl gel electrophoresis (Prusiner etal., 1980c, e, 1981b) and the other sucrose gradient sedimentation, prompted us to suggest that the rods might be either a pathologic product of infection or an aggregated form of the prion (Prusiner etal., 1982a). Subsequent studies showed the latter to be the case -extensively purified preparations of scrapie prions were found to contain ID,, units/ml, one major protein (PrP) and rod-shaped particles. The high degree of purity of our preparations allowed us to establish that the rods must be composed of P r P molecules. Having already shown that P r P is a component of the scrapie prion (Table XII), we concluded that rods must be a form of the prion (Prusiner etal., 1983). Recent immunoelectron microscopic studies have provided direct evidence that the rods are composed of PrP molecules by showing that antibodies to PrP decorate the prion rods (R. A. Barry, M. P. McKinley, P. E. Bendheim, G. K. Lewis, and S. B. Prusiner, in preparation). Sonication of the scrapie prion rods reduced their length to < 100 nm, but did not alter infectivity (M. P. McKinley, M. B. Braunfeld, and S. B. Prusiner, in preparation). While prion infectivity was unaltered by sonication for as long as 16 min, sonication of M13 filamentous bacteriophage

-

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STANLEY B. PRUSINER

PRIONS

43

for 15 sec reduced the titer by a factor of > lolo. These observations are consistent with our hypothesis that the rod-shaped particles are aggregates of prions. However, currently available data do not allow us to exclude the possibility, though unlikely, that the scrapie prion is a small rod-shaped or filamentous virus. In contrast to virtually all viruses including those with cylindrical shapes, a morphologic description of the unit prion structure has not been possible. The ultrastructural appearance of the rod-shaped particles in purified preparations of prions suggested the possibility that these rods are amyloid (Cohen etal., 1982; Diringer etal., 1983; Prusiner etal., 1983). Since the brains of some animals infected with the scrapie prion had been reported t o contain amyloid plaques (Section VII, B), we stained purified preparations of scrapie prions with Congo red dye (Prusiner etal., 1983). By bright field microscopy, the stained preparations appeared red and by polarization microscopy green birefringence was observed. It is generally accepted that amyloid is composed of proteins which aggregate to form rod-shaped or fibrillar structures and which exhibit green birefringence after staining with Congo red dye. Using the purification protocols developed for scrapie prions, rodshaped particles have been found in sucrose gradient fractions prepared from both murine and human brains infected with CJD prions (J. M. Bockman, D. T. Kingsbury, M. P. McKinley, and S. B. Prusiner, in preparation). These same fractions contain protease-resistant proteins which cross-react with antibodies produced against scrapie PrP. By analogy with scrapie, we believe that CJD prions aggregate to form amyloid-like rods and that amyloid plaques in the brains of humans and animals with CJD are probably composed of paracrystalline arrays of prions. Several early studies reported abnormal tubular-like structures in scrapie-infected rodent brain (Field etal., 1976b; Field and Narang, 1972; Narang, 1974a,b,c;Raine and Field, 1967). The studies were inconclusive because scrapie agent infectivity was never correlated with these ultrastructural findings. More recently, one report has called attention to filamentous structures measuring 4-6 nm in diameter which can be dis1981). Two or four filaments form tinguished from amyloid (Merz etal., FIG.7. Electron micrographs of rod-like structures in purified fractions containing the scrapie prion. Fraction 2 from a Triton X-lOO/SDS discontinuous sucrose gradient was applied to Formvar-coated grids which had been freshly carbon shadowed, glow discharged, and treated with 1 pg/ml poly-L-lysine. Solid bars are 100 nm. (A) Sample was rotary shadowed with tungsten at an angle of incidence of 8". Dimensions of individual rods vary from 100 to 200 nm in length and have a diameter of 25 nm. (B) Sample was negatively stained with freshly prepared 1%uranyl formate. Dimensions of individual rods vary from 150to 200 nm in length and have a diameter of 10 to 20 nm. All micrographs were taken using a JEOL lOOB electron microscope at 60 keV.

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STANLEY B. PRUSINER

helical fibrils measuring up to 1000 nm in length which were found in preparations from brains of rodents with scrapie and brains of humans 1981,1983). The fibrils have also been found in dying of CJD (Merz etal., 1983). preparations from scrapie-infected mouse spleen (Merz et al., Some investigators have suggested on the basis of ultrastructural morphology alone that the rod-shaped particles found in purified preparations are related to these long fibrils (Diringer etal., 1983). Their purification protocols which are similar to those developed by us (Section X,A) utilize detergent extractions, enzyme digestions, differential centrifugation, and sucrose gradient sedimentation. Since antibodies to PrP molecules have been shown to decorate the prion rods found in purified preparations (R. A. Barry etal., in preparation), it should be possible to determine by immunoelectron microscopy whether or not the long fibrils found in crude extracts as well as the tubular-like structures in brain sections are composed of immunologically related molecules. Elongated structures have also been described in the brains of patients dying of CJD, and based on morphology alone, some investigators have suggested that the CJD agent is a spiroplasma (Bastian, 1979; Bastian et 1981). Attempts to detect spiroplasma DNA from CJD and scrapie al., brain tissue using radiolabeled cDNA probes have been negative (C. J. Gibbs, Jr., personal communication). A recent study reports that no spiroplasma or other mycoplasma were cultivated from brain tissue of 18 CJD cases and no antibodies to several spiroplasma were detected in sera from 15 patients (Leach etal., 1983). Furthermore, the unusual molecular properties of the scrapie agent make it much different from spiroplasma. Curious arrays of spherical particles about 25 nm in diameter have been found within postsynaptic evaginations in the brains of scrapie-infected mice (Baringer and Prusiner, 1978; Bignami and Parry, 1971,1972; David1968; Lamar etal., 1974; Lampert etal., 1971). Similar Ferreira etal., structures in brains of scrapie-infected sheep and of humans with CJD have raised the possibility that the scrapie and CJD agents are composed of such particles. The conspicuous absence of these spherical particles from hamster brains, which have the highest known titers of the prion, make such particles unlikely candidates for the prion (Baringer etal., 1979).

X. MOLECULAR MODELSFOR THE PRION A. Hypothetical Structures forthePrion

Investigators have been aware of the unusual properties of the scrapie agent for many years. Hypotheses on the chemical structure of the scrapie

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agent have included Sarcosporidia parasite (M’Fadyean, 1918; MGowan, 1914), “filterable” virus (Cho, 1976; Cuille and Chelle, 1939; Eklund etal., 1963;Wilson etal., 1950), small DNA virus (Kimberlin and Hunter, 1967), replicating protein (Griffith, 1967; Lewin, 1972,1981; Pattison and Jones, 1967), replicating abnormal polysaccharide within membranes (Gibbons and Hunter, 1967; Hunter etal., 1968), DNA subvirus controlled by a transmissible linkage substance (Adams, 1970; Adams and Field, 1968), provirus consisting of recessive genes generating RNA particles (Dadington, 1969; Parry, 1962, 1969), naked nucleic acid similar to plant viroids (Diener, 1972,1973), unconventional virus (Adams, 1973; Gajdusek, 1977, 1978; Gajdusek and Gibbs, 1978; Hunter, 1972; Pattison, 1965; Stamp, 1967),aggregated conventional virus with unusual properties (Rohwer and Gajdusek, 1980), replicating polysaccharide (Field, 1966, 1967), nucleoprotein complex (Latarjet etal., 1970), nucleic acid surrounded by a polysaccharide coat (Adams and Caspary, 1967; Narang, 197413;Siakotos etal., 1979),spiroplasma-like organism (Bastian, 1979; Bastian etal., 1981; Gray etal., 1980), multicomponent system with one component quite small (Hunter et al., 1973; Somerville et al., 1976), membrane-bound DNA (Hunter etal., 1973; Marsh etal., 1978; Somerville etal., 1976), and a viroid-like nucleic acid surrounded by cellular proteins (Kimberlin, 1982a,b). The unusual properties of the scrapie agent coupled with the slow, tedious, and imprecise bioassays have served to stimulate this enlarging array of hypotheses. Only purification of the scrapie agent to homogeneity and analysis of its macromolecular structure will determine which of these hypotheses, if any, are correct.

B. Current Modelsof thePrionCannotExcludea Nucleic Acid Experimental data indicate that the molecular properties of the scrapie prion distinguish it from viruses, viroids, and plasmids. The prion contains a single major protein (PrP) of apparent Mr 27,000 to 30,000.

TABLE XI11 CURRENTMOLECULAR MODELS FOR THE PRION Prions contain undetected nucleic acids Genome codes for prion protein(s) Oligonucleotide Prions are devoid of nucleic acids

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Whether or not the prion contains minor protein components as well as a nucleic acid core remains to be established (Table XIII). P r P is required for infectivity. Denaturation, chemical modification, or hydrolysis of PrP was accompanied by a diminution in the infectivity of the prion. The resistance of the prion to procedures that modify or hydrolyze nucleic acids and its apparent small size suggest that the prion is a novel infectious pathogen. While the possibility that the prion contains a nucleic acid core surrounded by a tightly packed protein coat seems the most plausible, there is no experimental evidence for a polynucleotide within the prion. The second possibility is that the prion contains only protein and is devoid of nucleic acid. The latter model is consistent with the experimental data, but is clearly heretical. Skepticism of the second model is certainly justified. Only purification of the scrapie agent to homogeneity and determination of its chemical structure will allow a rigorous conclusion as to which of these two models is correct. If a currently undetected nucleic acid is found within the prion, its size and function will be of great interest. The results of many studies on the scrapie agent make it unlikely that the prion contains a nucleic acid of sufficient size to code for PrP. On the other hand, we cannot exclude the possibility that the prion contains an oligonucleotide. Should that prove to be the case, this would be a major feature distinguishing prions from viruses. There seems to be little advantage in championing one model over another; however, several previously postulated structures for the scrapie agent can now be discarded. The requirement of a protein for infectivity eliminates the possibilities that the scrapie agent is composed entirely of polysaccharide or nucleic acid. Thus, the replicating polysaccharide and naked nucleic acid-viroid hypotheses are no longer viable. The hypothetical nucleic acid surrounded by a polysaccharide coat can also be eliminated. The recent identification of a structural protein (PrP) within the prion and its absence in preparations from uninfected controls allows us to discard hypotheses suggesting that the putative scrapie nucleic acid is surrounded by nonspecific cellular proteins. Studies demonstrating the small size of the scrapie agent distinguish it from conventional viruses, spiroplasma-like organisms, and parasites such as Sarcosporidia. Rigid categorization of the scrapie agent at this time remains premature. Determination of its entire molecular structure will be requiredprior to deciding whether prions represent a distinct subgroup of extraordinarily small and unusual viruses or a pathogen which is clearly distinguishable from viruses.

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XI. CONCLUDING REMARKS Although further studies on the structure of prions are necessary, a considerable body of evidence suggests that prions are subviral pathogens. As described above and elsewhere, the molecular properties of the prion are antithetical to those of the viroid, another subviral pathogen. It is of interest to ask whether additional subviral pathogens with structures fundamentally different from those of prions and viroids will be found. Since the causes of many chronic degenerative diseases of humans remain unknown, it is important to ask whether any of these disorders are caused by viroids, prions, or another unidentified subviral pathogen (Diener, 1979;Gajdusek, 1977;Gross, 1979). Diseases where investigators have entertained the possibility of a subviral pathogen etiology include Alzheimer’s senile dementia, multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, diabetes mellitus, rheumatoid arthritis, lupus erythematosus, and a variety of neoplastic disorders (Gajdusek, 1977; Gross, 1979). Recent progress in the purification and characterization of the scrapie prion suggests that an optimistic view for further advances in the field is justified. Pessimistic hypotheses that predict purification of the scrapie agent is not possible and that the molecular properties of the prion are not a consequence of its highly specific structure, no longer need to be considered. Over the last 5 years, scrapie research has been transformed from an intriguing yet forbidding problem into an exciting and productive area of investigation.

ACKNOWLEDGMENTS The author thanks Drs. M. P. McKinley, D. C. Bolton, P. E. Bendheim, R. A. Barry, C . Bellinger, R. Meyer, D. E. Garfin, F. R. Masiarz, and K. C. Kasper as well as Mss. S. P. Cochran, D. F. Groth, K. A. Bowman, F. Elvin, E. Hennessey, R. Mead, and L. Gallagher for their invaluable help throughout many phases of this work. The continuing support and encouragement of Drs. F. Seitz, J. R. Krevans, R. Schmid, R. C. Morris, Jr., R. A. Fishman, and I. F. Diamond is gratefully acknowledged. Collaborative efforts of Drs. W. J. Hadlow, C. M. Eklund, J. R. Baringer, T. 0. Diener, D. P. Stites, D. T. Kingsbury, J. E. Cleaver, J. E. Hearst, and R. C. Williams have been much appreciated. Support for this work has been pmvided by researeh grants from the National Institutes of Health, NS14069 and AG02132, as well as by gifts from R. J. Reynolds Industries, Inc., Sherman Fairchild Foundation, and the W. M. Keck Foundation.

REFERENCES Adams, D. H. (1970). Pathol. Biol. 18, 559-577. Adams, D. H. (1973). Biochem. SOC.Trans. 1, 1061-1064.

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Adams, D. H., and Caspary, E. A. (1967). Br.Med. J.3, 173. Adams, D. H., and Field, E. J. (1968). Lancet 2, 714-716. Alper, T., Haig, D. A., and Clarke, M. C. (1966). Biochem. Biophys. Res. Commun. 22, 278- 284. Alper, T., Cramp. W. A., Haig, D. A., and Clarke, M. C. (1967). Nature (London) 214, 764- 766. Alper, T., Haig, D. A., and Clarke, M. C. (1978). J . Gen. Virol. 41, 503-516. Alpers, M. P. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 1,pp. 67-92. Academic Press, New York. Andrews, P. (1971). Methods Biochem. Anal. 18, 1-53. Baringer, J. R., and Prusiner, S. B. (1978). Ann. Neurol. 4, 205-211. Baringer, J. R., Wong, J., Klassen, T., and Prusiner, S. B. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2, pp. 111-121. Academic Press, New York. Baringer, J. R., Bowman, K. A., and Prusiner, S. B (1981). J.Neuropathol. Exp.Neurol. 40, 329. Bastian, F. 0. (1979). Arch. Pathol. Lab. Med. 103,665-669. Bastian, F. O.,Hart, M. N., and Cancilla, P. A. (1981). Lancet 1, 660. Beck, E., and Daniel, P. M. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S.B. Prusiner and W. J. Hadlow, eds.), Vol. 1,pp. 253-270. Academic Press, New York. Beck, E., Daniel, P. M., and Parry, H. B. (1964). Brain 87, 153-176. Bendheim, P. E., Barry, R. A,, DeArmond, S. J., Stites, D. P., and Prusiner, S. B. (1984). Nature (London) 310,418-421. Bignami, A., and Parry, H. B. (1971). Science 171, 389-390. Bignami, A., and Parry, H. B. (1972). Brain 95, 319-326. Bloemendal, H., Hermsen, T., Dunia, I., and Benedetti, E. L. (1982). Exp.Eye Res.35, 61-67. Bolton, A. E., and Hunter, W. M. (1973). Biochem. J. 133,529-539. Bolton, D. C., McKinley, M. P., and Prusiner, S. B. (1982). Science 218, 1309-1311. Bolton, D. C., McKinley, M. P., and Prusiner, S. B. (1984). Biochemistry (in press). Borgert, K., Koschel, K., Tauber, H., and Wecker, E. (1971). J.Virol. 8, 1-6. Bornstein, P., and Balian, G. (1970). J.Biol. Chem. 245, 4854-4856. Brown, P., Green, E. M., and Gajdusek, D. C. (1978). Proc.SOC. Exp.Biol. Med. 158, 513-516. Bruce, M. E., and Fraser, H. (1975). Neuropathol. Appl. Neurbiol. 1,189-202. Bruce, M. E., and Fraser, H. (1982). Neuropathol. Appl. Neurobiol. 8, 71-74. Butzow, J. J., and Eichorn, G. L. (1965). Biopolymers 3,95-107. Butzow, J. J., and Eichorn, G. L. (1975). Nature (London) 254, 358-359. 77, Buyukmihci, N., Rorvik, M., and Marsh, R. F. (1980). Proc. Natl. Acad. Sci. U.S.A. 1169- 1171. Carter, W. G., and Hakomori, S. (1978). J. Biol. Chem. 253,2867-2874. Chandler, R. L. (1959). Vet. Rec. 71, 58-59. Chandler, R. L. (1961). Lancet 1,1378-1379. Cho, H. J. (1976). Nature (London) 262,411-412. Cho, H. J. (1980). Intervirology 14, 213-216. Chou, S. M., and Martin, J. D. (1971). Acta Neuropathol. 17, 150-155. Clarke, M. C., and Haig, D. A. (1966). Vet. Rec. 78, 647-649. Cohen, A. S., and Calkins, E. (1964). J. Cell Biol. 21, 481-486. Cohen, A. S., Shirahama, T., and Skinner, M. (1982). I n “Electron Microscopy of Proteins” (J. R. Harris, ed.), Vol. 3, pp. 165-206. Academic Press, New York.

PRIONS

49

Collis, S. C., and Kimberlin, R. H. (1983). J . Comp. Pathol. 93, 331-338. Cox, R. A., and Kotecha, S. (1980). Biochem. J. 190, 199-214. Creutzfeldt, H. G. (1920). Z. Gesamte Neurol. Psychiatr. 57, 1-18. Crowther, D., and Melnick, J. L. (1961). Virology 14, 11-21. Cuille, J., and Chelle, P. L. (1939). C. R. Hebd.Seances Acad. Sci. 208, 1058- 1060. Cunnington, P. G., Kimberlin, R. H., Hunter, G. D., and Newsome, P. M. (1976). IRCS Med. Sci.: Libr. Compend. 4, 250. Darlington, C. D. (1969). I n “Virus Diseases and the Nervous System” (C. W. M. Whitty, J. T. Hughes, and F. 0. McCallum, eds), pp. 133- 138. Blackwell, Oxford. David-Ferreira, J. F., David-Ferreira, K. L., Gibbs, C. J., Jr., and Morris, J. A. (1968). Proc. SOC.Exp. Biol. Med. 127, 313-320. Dickinson, A. G. (1976). I n “Slow Virus Diseases of Animals and Man” (R. H. Kimberlin, ed.), pp. 209-241. Am. Elsevier, New York. Dickinson, A. G., and Fraser, H. (1977). I n “Slow Virus Infections of the Central Nervous System” (V. ter Meulen and M. Katz, eds.), pp. 3-14. Springer Verlag, Berlin and New York. Dickinson, A. G.,and Fraser, H. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 1,pp. 367-386. Academic Press, New York. Dickinson, A. G., and Meikle, V. M. (1969). Genet. Res.13,213-225. Dickinson, A. G., and Meikle, V. M. (1971). Mol Gen. Genet. 112, 73-79. Dickinson, A. G., and Outram, G. W. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2, pp. 13-31. Academic Press, New York. Dickinson, A. G., Meikle, V., and Fraser, H. (1968). J. Comp. Pathol. 78, 293-299. Diener, T. 0. (1972). Nature (London) 253,218-219. Diener, T. 0. (1973). Ann. Clin. Res.5,268-278. Diener, T. 0. (1979). “Viroids and Viroid Diseases,” pp. 1-252. Wiley, New York. Diener, T. O., and Raymer, W. B. (1969). Virology 37, 351-366. Diener, T. O., McKinley, M. P., andprusiner, S. B. (1982). Proc. Nutl. Acad.Sci. U.S.A. 79, 5220- 5224. Diringer, H., Gelderblom, H., Hilmert, H., Ozel, M., Edelbluth, C., and Kimberlin, R. H. (1983). Nature (London) 306, 476-478. Divry, P. (1934). J. Belg. Neurol. Psychiat. 34, 197-201. Earnshaw, W. C., and Casjens, S. R. (1980). Cell 21, 319-331. Eklund, C. M., Hadlow, W. J., and Kennedy, R. C. (1963). Proc. SOC.Exp. Biol. Med. 112, 974-979. Eklund, C. M., Kennedy, R. C., and Hadlow, W. J. (1967). J. Infect. Dis. 117, 15-22. Emerson, E. E., Kikkawa, Y., and Gueft, B. (1966). J. Cell Biol. 28, 570-577. Field, E. J. (1966). Br. Med.J . 2, 564-565. Field, E. J. (1967). Dtsch. Z. Neruenheilkd. 192, 265-274. Field, E. J., and Narang, H. K. (1972). J . Neurol. Sci. 17, 347-364. Field, E. J., Raine, C. S., and Joyce, G. (1967a). Acta Neuropathol. 8 , 47-56. Field, E. J., Raine, C. S., and Joyce, G. (1967b). Acta Neuropathol. 9, 305-315. Filipowicz, W., Konarska, M., Gross, H. J., and Shatkin, A. J. (1983). Nucleic Acids Res.11, 1405-1418. Franklin, R. M., and Wecker, E. (1959). Nature (London) 184,343-345. Fraser, H. (1982). Nature(London) 295,149- 150. Fraser, H., and Bruce, M. E. (1973). Lancet 1, 617. Freese, E., Bautz-Freese, E., and Bautz, E. (1961). J. Mol. Biol. 3, 133-143.

50

STANLEY B. PRUSINER

Fukatsu, R., Amyx, H. L., Gibbs, C. J., Jr., and Gajdusek, D. C. (1983). J. Neuropathol. Exp. Neurol. 42, 348. 197,943-960. Gajdusek, D. C. (1977). Science Gajdusek, D. C. (1978). In“Human Diseases Caused by Viruses” (H. Rothschild, F. Allison, Jr., and C. Howe, eds.), pp. 231-258. Oxford Univ. Press, London and New York. Gajdusek, D. C., and Gibbs, C. J., Jr. (1978). In“Viruses andEnvironment” (E. Kurstak and K. Maramorosch, eds.), pp. 79-98. Academic Press, New York. J. Med.257,974-978. Gajdusek, D. C., and Zigas, V. (1957). N .Engl. Gajdusek, D. C., and Zigas, V. (1959). Am. J. Med.26, 442-469. 209,794-796. Gajdusek, D. C., Gibbs, C. J., Jr., and Alpers, M. (1966). Nature (London) Gardash’yan, A. M., Nartississov, N. V., andBobkova, 0. V. (1971). Bull. Exp.Biol. Med.6, 67 - 69. Gardiner, A. C. (1965). Res.Vet. Sci. 7 ,190-195. 78, 906-908. Gerstmann, J. (1928). Wien.Med. Wochenschr. Gerstmann, J., Straussler, E., and Scheinker, I. (1936). 2. Neurol. 154, 736-762. Giannoni, G., Padden, F. J., Jr., and Keith, H. D. (1969). Proc. Natl. Acad.Sci. U.S.A. 62, 964-971. Gibbons, R. A., and Hunter, G. D. (1967). Nature(London) 215,1041-1043. Gibbs, C. J., Jr. (1967). Curr. Top. Microbiol. Immunol. 40, 44-58. Gibbs, C. J., Jr., and Gajdusek, D. C .(1969). Science 165, 1023-1025. Gibbs, C. J., Jr., and Gajdusek, D. C. (1972). Nature236,73-74. Gibbs, C .J., Jr., and Gajdusek, D. C. (1973). Science 182,67-68. Gibbs, C .J., Jr., and Gajdusek, D. C (1978). Aging(N.Y.) 7,559-577. Gibbs, C. J., Jr., and Gajdusek, D. C. (1982). In “Human Motor Neuron Diseases” (L. Rowland, ed.), pp. 343-353. Raven Press, New York. Gibbs, C. J., Jr., Gajdusek, D. C., andMorris, J. A. (1965). In“Slow, Latent andTemperature Virus Infections” (D. C. Gajdusek, C. J. Gibbs, Jr., and M. Alpers, eds.), Publ. No. 1378, NINB Managr. 2, pp. 195-202. National Institutes of Health, Bethesda, Maryland. Gibbs, C. J. Jr., Gajdusek, D. C., Asher, D. M., Alpers, M. P., Beck, E., Daniel, P. M., and 161,388-389. Matthews, W. B. (1968). Science Gibbs, C. J., Jr., Gajdusek, D. C., andLatarjet, R. (1978). Proc. Natl. Acad.Sci. U.S.A. 75, 6268-6270. Gibbs, C .J., Jr., Gajdusek, D. C., and Amyx, H. (1979). In“Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2, pp. 87-110. Academic Press, New York. Gibbs, C. J., Jr., Amyx, H. L., Bacote, A., Masters, C. L., and Gajdusek, D.C. (1980). J. Infect. Dk. 142,205-208. Gordon, W. S. (1946). Vet. Rec.68,516-520. Gordon, W. S. (1966a). In“Report of Scrapie Seminar,” ARS 91-53, pp. 19-36. U.S. Dept. of Agriculture, Washington, D.C. Gordon, W. S. (196613). In“Report of Scrapie Seminar,”ARS91-53, pp. 53-67. U.S. Dept. of Agriculture, Washington, D.C. Goudsmit, J., Morrow, C. H., Asher, D. M., Yanagihara, R. T.,Masters, C. L., Gibbs, C. J., Jr., and Gajdusek, D. C. (1980). Neurology 30,945-950. Gray, A,, Francis, R. T., and Scholtz, C. L. (1980). Lancet2, 152. Green, N. M. (1975). Adu.Protein Chem.29,85-133. BloodFlow Grkgoire, N., Gorde, J. -M., Nezri, C., Salamon, G., andBert, J. (1983a). J . Cereb. Metab.3, Suppl. 1, S500-S501. GrBgoire, N., Gorde-Durand, J. M., Bouras, C., Salamon, G., and Bert, J. (198313). Neurosci. Lett. 36, 181-187.

PRIONS

51

Greig. J. R. (1940). Trans. High. Agric. SOC.Scotl. [5] 5 2 , 71-90. Griffith, J. S. (1967). Nature (London)2 1 5 , 1043-1044. Gross, L. (1979). New Engl. J. Med. 3 0 1 , 432-434. Hadlow, W. J. (1959). Lancet 2 , 289-290. Hadlow, W. J., and Karstad, L. (1968). Can. Vet. J. 9, 193- 196. Hadlow, W. J., Eklund, C. M., Kennedy, R. C., Jackson, T. A., Whitford, H. W., andBoyle, C. C. (1974). J . Infect. Dis. 129, 559-567. Hadlow, W. J., Kennedy, R. C., Race, R. E., and Eklund, C. M. (1980a). Vet. Pathol. 17, 187- 199.

Hadlow, W. J., Prusiner, S. B., Kennedy, R. C., and Race, R. E. (1980b). Ann. Neurol. 8, 628- 631.

Hanson, C. V., Riggs, J. L., and Lennette, E. H. (1978). J. Gen. Virol. 40, 345-358. Hartsough, G .R., and Burger, D. (1965). J. Infect. Dis. 116,387-392. Hearst, J. E., and Thiry, L. (1977). Nucleic Acids Res. 4 , 1339-1348. Hogan, R. N., Baringer, J. R., and Prusiner, S. B. (1981). Lab. Invest. 4 4 , 34-42. Hogan, R. N., Kingsbury, D. T., Baringer, J. R., and Prusiner, S. B. (1983). Lab. Invest. 4 9 , 708-715.

Hornabrook, R. W. (1968). Brain 91,53-74. Hotchin, J., Sikora, E., and Baker, F. (1983). Intervirology 1 9 , 205-212. Hourrigan J., Klingsporn, A,, Clark, W. W., anddecamp, M. (1979). In “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 1,pp. 331-356. Academic Press, New York. Hunter, G. D. (1972). J. Infect Dis. 125,427-440. Hunter, G. D. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2, pp. 365-385. Academic Press, New York. Hunter, G. D., and Millson, G. C. (1967). J. Comp. Pathol. 77,301-307. Hunter, G. D., Millson, G. C., and Chandler, R. L. (1963). Res. Vet. Sci. 4 , 543-549. Hunter, G. D., Kimberlin, R. H., and Gibbons, R. A. (1968). J. Theor. Biol. 20, 355-357. Hunter, G. D., Gibbons, R. A,, Kimberlin, R. H., and Millson, G. C. (1969). J. Comp.Pathol. 79,101-108.

Hunter, G. D., Kimberlin, R. H., Collis, S., and Millson, G. C. (1973). Ann. Clin. Res. 5 , 262- 267.

Isaacs, S. T., Shen, C. J., Hearst, J. E., and Rapoport, H. (1977). Biochemistry 1 6 , 10581064.

Jakoh, A. (1923). “Die Extrapyramidalen Erkrankungen,” pp. 215- 245. Springer-Verlag, Berlin and New York. Kasper, K. C., Bowman, K., Stites, D. P., and Prusiner, S. B. (1981). I n “Hamster Immune Responses in Infectious and Oncologic Diseases” (J. W. Streilein, D. A. Hart, J. SteinStreilein, W. R. Duncan, and R. E. Billingham, eds.), pp. 401-413. Plenum, New York. Kasper, K. C., Stites, D. P. Bowman, K. A., Panitch, H., and Prusiner, S. B. (1982). J . Neuroimmunol. 3 , 187-201. Katzman, R. (1981). Curr. Neurol. 3 , 138-158. Kim, I. C., Franzblan, C., Shirahama, T., and Cohen, A. S. (1969). Biochim. Biophys. Acta. 181,465-467.

Kimberlin, R. H. (1976). “Scrapie in the Mouse,” pp. 1-77. Meadowfield Press, Durham. Kimberlin, R. H. (1982a). TIBS 7, 392-394. Kimberlin, R. H. (1982b). Nature (London)297,107-108. Kimberlin, R. H., and Hunter, G. D. (1967). J . Gen. Virol. 1, 115-124. Kimberlin, R. H., and Walker, C. A. (1977). J. Gen. Virol. 34,295-304. Kimberlin, R. H., and Walker, C. A. (1978a). J . Gen. Virol. 39,487-496.

52

STANLEY B. PRUSINER

Kimberlin, R. H., and Walker, C. A. (1978b). J. Comp. Pathol. 88, 39-47. Kimberlin, R. H., and Walker, C. A. (1979). J . Comp. Pathol. 89,551-562. Kimberlin, R. H., and Walker, C. A. (1980). J. Gen. Virol. 51,183-187. Kimberlin, R. H., Field, H. J., and Walker, C. A. (1983). J. Gen. Virol. 64, 713-716. Kimura, H., Yamato, S., and Murachi, T. (1978). J. Biochem. (Tokyo)84,205-211. Kingsbury, D. T., Smeltzer, D. A., Amyx, H. L., Gibbs, G. J., Jr., and Gajdusek, D. C. (1982). Infect. Immun. 3 7 , 1050-1053. Kingsbury, D. T., Kasper, K. C., Stites, D. P., Watson, J. D., Hogan, R. N., andPrusiner, S. B. (1983). J. Immunol. 1 3 1 , 491-496. Kirschbaum, W. R. (1968). “Jakob-Creutzfeldt Disease,” pp. 1-251. Elsevier, New York. Kitagawa, Y., Gotoh, F., Koto, A., Ebihara, S., Okayasu, H., Ishii, T., and Matsuyama, H. (1983). J. Neurol. 229,97-101. Klatzo, I., Gajdusek, D. C., and Zigas, V. (1959). Lab. Inuest. 8, 799-847. Klein, J. (1975). “Biology of the Mouse Histocompatibility-2 Complex,” pp. 1-620. Springer-Verlag, Berlin and New York. Kozlowski, P. B., Moretz, R. C., Carp, R. A., and Wisniewski, H. M. (1982). Acta Neuropathol. 56,9-12.

Krauth, W., and Werner, D. (1979). Biochem, Biophys. Acta 664,390-401. Kuzuhara, S., Kanazawa, I., Sasaki, H., Nakanishi, T., and Shimamura, K. (1983). Ann. Neurol. 1 4 , 216-225. Lamar, C. H., Gustafson, D. P., Krasovich, M., and Hinsman, E. J. (1974). Vet. Pathol. 1 1 , 13-19.

Lampert, P., Hooks, J., Gibbs, C. J., Jr., and Gajdusek, D. C. (1971). Acta Neuropathol. 19, 81-93.

Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F., and Hamilton, L. D. (1960). J. Mol. Biol. 2 , 19-37. Latarjet, R. (1979). In “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2 pp. 387-408. Academic Press, New York. Latarjet, R., Cramer, R., and Montagnier, L. (1967). Virology 3 3 , 104-111. Latarjet, R., Muel, B., Haig, D. A., Clarke, M. C., and Alper, T. (1970). Nature (London) 227,1341 -1343.

Lax, A. J., Millson, G. C., and Manning, E. J. (1983). Res. Vet. Sci. 3 4 , 155-158. Leach, R. H., Matthews, W. B., and Will, R. (1983). J.Neurol. Sci. 5 9 , 349-353. Lesser, R. L., Albert, D. M., Bobowick, A. R., and O’Brien, F. H. (1979). Am. J. Ophthulmol. 87,317-321.

Lewin, P. (1972). Lancet 1, 748. Lewin, P. (1981). Can. Med. Assoc. J/ 124,1436-1437. McKinley, M. P., Masiarz, F. R., and Prusiner, S. B. (1981). Science 2 1 4 , 1259-1261. McKinley, M. P., Masiarz, F. R., Isaacs, S. T., Hearst, J. E., and Prusiner, S. B. (1983a). Photochem. Photobiol. 3 7 , 539-545. McKinley, M. P., Bolton, D. C., and Prusiner, S. B. (1983b). Cell35,57-62. McLaren, A. D., and Shugar, D. (1964). “Photochemistry of Proteins and Nucleic Acids.” Pergamon, Oxford. Malone, T. G., Marsh, R. F., Hanson, R. P., and Semancik, J. S. (1978). J. Virol. 2 5 , 933-935.

Malone, T. G., Marsh, R. F., Hanson, R. P., and Semancik, J. S. (1979). Nature (London) 278,575-576.

Manuelidis, E. E., and Manuelidis, L. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusinerand W. J. Hadlow, eds.), Vol. 2, pp. 147- 173. Academic Press, New York.

PRIONS

53

Manuelidis, E. E., Kim, J., Angelo, J.. and Manuelidis, L. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 223-227. Manuelidis, E. E., Gorgacz, E. J., and Manuelidis, L. (1978a). Proc. Natl. Acad. Sci. U.S.A. 75,3422-3436. Manuelidis, E. E., Gorgacz, E. J., and Manuelidis, L. (197815). Science 200, 1069- 1071. Marsh, R. F., andHanson, R. P. (1977). Fed. Proc. Fed.Am. Soc. Exp.Biol. 37,2076-2078. Marsh, R. F., and Hanson, R. P. (1979). In “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2, pp. 451-460. Academic Press, New York. Marsh, R. F., and Kimberlin, R. H. (1975). J. Infect. Dis. 131,104-110. Marsh, R. F., Semancik, J. S., Medappa, K. C., Hanson, R. P., andRueckert, R. R. (1974). J. Virol. 13, 993 - 996. Marsh, R. F., Malone, T. G., Semancik, J. S., Lancaster, W. D., and Hanson, R. P. (1978). Nature (London)275, 146-147. Masters, C. L., and Gajdusek, D. C. (1982). Recent Adu. Neuropathol. 2, 139-163. Masters, C. L., Harris, J. O.,Gajdusek, D. C., Gibbs, C. J., Jr., Bernoulli, C., and Asher, D. M. (1978). Ann. Neurol. 5, 177-188. Masters, C. L., Gajdusek, D. C., and Gibbs, C. J., Jr. (1981a). Brain 104,535-558. Masters, C. L., Gajdusek, D. C., and Gibbs, C .J., Jr. (1981b). Brain 104, 559-588. Merz, P. A., Somerville, R. A., Wisniewski, H. M., and Iqbal, K. (1981). Acta Neuropathol. 54, 63-74. Merz, P. A., Somerville, R. A,, Wisniewski, H. M., Manuelidis, L., and Manuelidis, E. E. (1983). Nature (London)306, 474-476. M’Fadyean, J. (1918). J. Comp. Pathot. 31, 102-131. MGowan, J. P. (1914). “Investigation into the Disease of Sheep Called ‘Scrapie’,” pp. 1- 114. Blackwood, Edinburgh. Mihalyi, E. (1978). “Application of Proteolytic Enzymes to Protein Structure Studies,” 2nd ed., pp. 152-193. CRC Press, Cleveland, Ohio. Miles, E. W. (1977). In “Methods in Enzymology” (C. H. W. Hirs and S. N. Timasheff, eds.), Vol. 47, pp. 431-442. Academic Press, New York. Millson, G. C., Hunter, G. D., and Kimberlin, R. H. (1976). In “Slow Virus Diseases of Animals and Man” (R. H. Kimberlin, ed.), pp. 243-266. Elsevier, New York. Moretz, R. C., Wisniewski, H. M., and Lossinsky, A. S. (1983). In “Aging of the Brain” (D. Samuel, ed.), pp. 61-79. Raven Press, New York. Motomura, S., Yamashita, Y., Kawanami, S., Ohta, M., and Kuriowa, Y. (1977). Clin. Neurol. (Tokyo) 1 7 , 758-764. Moulton, J. E., and Palmer, A. C. (1959). Cornell Vet. 49, 349-359. Narang, H. K. (1974a). Acta Neuropathol. 28, 317-329. Narang, H. K. (1974b). Acta Neuropathol. 29, 37-43. Narang, H. K. (1974~). Neurobiology 4, 349-363. Schechter, N. M., Reynolds, J. A., and Tanford, C. (1976). Biochemistry 15, Nozaki, Y., 3884-3890. Nozth, A. C. T., and Rich, A. (1961). Nature (London)191,1242-1245. Ott, U., Odermatt, E., Engel, J.,Furthmayr,H.,andTimpl, R. (1982). Eur.J.Biochem. 123, 63-72. Outram, G. W. (1976). I n “Slow Virus Diseasesof Animals and Man” (R. H. Kimberlin,ed.), pp. 325-357. Am. Elsevier, Amsterdam. Palsson, P. A. (1979). In “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 1, pp. 357-366. Academic Press, New York. Parry, H. B. (1962). Heredity 1 7 , 75-105.

54

STANLEY B. PRUSINER

Parry, H. B. (1969). I n “Virus Diseases and the Nervous System” (C. W. M. Whitty, J. T. Hughes, and F. 0. McCallum, eds.), pp. 99-105. Blackwell, Oxford. Pattison, I. H. (1965). J. Comp. Pathol. 75,159-164. Pattison, I. H. (1966). In “Slow, Latent and Temperate Virus Infections” (D. C. Gajdusek, C. J. Gihhs, Jr. and M. Alpers, eds.), Publ. No. 1378, NINDB Monog. 2 pp. 249-257. National Institute of Health, Bethesda, Maryland. Pattison, I. H., and Jones, K. M. (1967). Vet. Rec 8 0 , 1-8. Pattison, I. H., and Millson, G. C. (1960). J. Comp. Pathol. 70, 182-193. Pattison, I. H., and Millson, G. C. (1961). J. Comp. Pathol. 71, 171-176. Pattison, I. H., Millson, G. C., and Smith, K. (1964). Res.Vet. Sci. 5, 116-121. Pattison, I. H., Hoare, M. N., Jebbett, J. N., and Watson, W. A. (1972). Vet. Rec. 90, 465-468. Phillips, J. H., and Brown, D. M. (1967). Prog. Nucleic Acid Res.Mol. Biol. 7,349-368. Porter, D. D., Porter, H. G., and Cox, N. A. (1973). J. Immunol. 111,1407-1410. Pras, M., Zucker-Franklin, D., Rimon A., and Franklin, E. C. (1969). J. Exp. Med.130, 777- 791. Prusiner, S. B. (1978). J . Biol. Chem. 253,916-921. Pmsiner, S. B. (1982). Science 216, 136-144. Prusiner, S.B. (1984). N.Engl. J. Med. 310,661-663. Prusiner, S . B., and Kingshury, D. T. (1984). CRC Crit. Reu. Clin. Neurobiol. (in press). Prusiner, S. B., Hadlow, W. J., Eklund, C. M., Race, R. E., and Cochran, S. P., (1978a). Biochemistry 17,4987-4992. Prusiner, S . B., Hadlow, W. J., Garfin, D. E., Cochran, S. P., Baringer, J. R., Race, R. E., and Eklund, C. M. (1978h). Biochemistry 17,4993-4999. Prusiner, S. B., Garfin, D. E., Baringer, J. R., Cochran, S.P., Hadlow, W. J., Race, R. E., and Eklund, C. M. (1979). I n “Slow Transmissible Diseases of the Nervous System” (S. B. Prusiner and W. J. Hadlow, eds.), Vol. 2, pp. 425-464. Academic Press, New York. Prusiner, S. B., Cochran S. P., Groth, D. F., Hadley, D., Martinez, H. M., and Hadlow, W. J. (1980a). I n “Aging of the Brain and Dementia” (L. Amaducci, A. N. Davison, and P. Antuono, eds.), pp. 205-216. Raven Press, New York. Prusiner, S. B., Garfin, D. E., Cochran, S.P., McKinley, M. P., andGroth, D. F. (1980b). J. Neurochem. 35, 574-582. Prusiner, S. B., Groth, D. G., Bildstein, C., Masiarz, F. R., McKinley, M. P., and Cochran, S. P. (1980~).Proc. Natl. Acad. Sci. U.S.A. 77,2984-2988. Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P., and Martinez, H. M. (1980d). Biochemistry 19,4883-4891. Prusiner, S. B., Groth, D. F., Cochran, S.P., McKinley, M. P., and Masiarz, F. R. (1980e). Biochemistry 1 9 , 4892-4898. Prusiner, S.B., Groth, D. F., McKinley, M. P., Cochran, S.P., Bowman, K. A., and Kasper, K. C. (1981a). Proc. Nutl. Acad. Sci. U.S.A. 78,4606-4610. Prusiner, S. B., McKinley, M. P., Groth, D. F., Bowman, K. A., Mock, N. I., Cochran, S. P., and Masiarz, F. R. (1981h). Proc. Natl. Acad. Sci U.S.A. 78, 6675-6679. Prusiner, S. B., Bolton, D. C., Groth, D. F., Bowman, K. A., Cochran, S. P., and McKinley, M. P. (1982a). Biochemistry 21, 6942-6950. Prusiner, S. B., Cochran, S. P., Groth, D. F., Downey, D. E., Bowman, K. A., and Martinez, H. M. (1982b). Ann. Neurol. 11,353-358. Prusiner, S. B., Gajdusek, D. C., and Alpers, M. P. (1982~).Ann. Neurol. 1 2 , l - 9 . Prusiner, S.B., McKinley, M. P., Bowman, K. A., Bolton, D. C., Bendheim, P. E., Groth, D. F., and Glenner, G. G. (1983). Cell35,349-358.

PRIONS

55

Prusiner, S. B., Groth, D. F., Bolton, D. C., Kent, S. B., and Hood, L. E. (1984). Cell38, 127- 134. Raine, C. S., and Field, E. J. (1967). Acta Neuropathol. 9, 298-304. Reynolds, J. A. (1981). Recept. Recognit., Ser. B 11,33-60. Rohwer, R. G., and Gajdusek, D. C. (1980). In “Search for the Cause of Multiple Sclerosis and Other Chronic Diseases of the Central Nervous System” (A. Boese, ed.), pp. 333355. Verlag Chemie, Weinheim. Roos, R., Gajdusek, D. C., and Gihbs, C. J., Jr. (1973). Brain 96,1-20. Rose, J. A. (1974). In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), pp. 1-61. Plenum, New York. Ruinen, L., Van Bruggen, E. F. J., Scholten, J. H., Gruber, M., and Mandema, E. (1968). In “Amyloidosis” (E. Mandema, L. Ruinen, J. H. Scholten, and A. S. Cohen, eds.), pp. 194-205. Excerpta Medica, Amsterdam. Rupley, J. A. (1967). In “Methods in Enzymology” (C. H. W. Hirs, ed.), Vol. 11, pp. 905917. Academic Press, New York. Salazar, A., Masters, C. L., Gajdusek, D. C., and Gibbs, C. J., Jr. (1983). Ann.Neurol. 14, 17-26. Sanger, H. L., Ramm, K., Domdey, H., Gross, H. J., Henco, K., and Riesner, D. (1979). FEBS Lett. 99, 117-122. Sato, Y., Ohta, M., and Tateishi, J. (1980). Acta Neuropathol. 51, 135-140. Schaffer, F. L., and Schwerdt, C. E. (1959). Adv. Virus Res. 6, 159-204. Schuster, H., and Wittman, H. -G. (1963). Virology 19, 421-430. Seitelberger, F. (1962). Wien. Klin. Wochenschr. 4 1/42,687 -691. Seitelberger, F. (1981). Acta Neuropathol., Suppl. 7,341 -343. Semancik, J. S., Morris, T. J., and Weathers, L. G. (1973). Virology 53, 448-456. Semancik, J. S., Marsh, R. F., Geelen, J. L., andHanson, R. P. (1976).5. Virol. 18,693-700. Siakotos, A. N., Gajdusek, D. C., Gibbs, C. J., Jr., Traub, R. D., and Bucana, C. (1976). Virology 7 0 , 230-237. Siakotos, A. N., Raveed, D., and Longa, G .(1979). J. Gen. Virol. 43,417-422. Sigurdsson, B. (1954). Br. Vet. J. 110, 341-354. Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M. H., Patlak, C. S., Pettigren, K. D., Sakurada, O., and Shinohara, M. (1977). J. Neurochem. 28,897-916. Somerville, R. A,, Millson, G. C., and Hunter, G .D. (1976). Biochem. SOC.Trans. 4,11121114. Sorenson, G. D., and Binington, H. B. (1964). Fed. Proc., Fed. Am. SOC.Exp.Biol. 23,550. Stamp, J. T. (1967). Br.Med. Bull. 23, 133-137. Stark, G. R., Stein, W. H., and Moore, S. (1960). J. Biol. Chem.235,3177-3181. Stockman, S. (1913). J . Comp. Pathol. 26, 317-327. Suss, K. H., Schmidt, O., and Machold, 0. (1976). Biochem. Biophys. Acta 448, 103-113. Tanford, C. (1961). “Physical Chemistry of Macromolecules,” pp. 317-456. Wiley, New York. Tateishi, J., Ohta, M., Koga, M., Sato, Y., andKuroiwa, Y. (1979). Ann. Neurol. 5,581-584. Tateishi, J., Koga, M., Sato, Y., and Mori, R. (1980a). Ann. Neurol. 7,390-391. Tateishi, J., Sato, Y., Koga, M., Doi, H., and Ohta, M. (198Ob). Acta Neuropathol. 51, 127-134. Tateishi, J., Doi, H., Sato, Y., Suetsugu, M., Ishii, K., and Kuroiwa, Y. (1981). Acta Neuropethol. 53, 161- 163. Tateishi, J., Sato, Y., and Ohta, M. (1983). Prog.Neuropathol. 5, 195-222. 15, 278-280. Tatieshi, J., Nigara, H., Hikita, K., and Sato, Y. (1984). Ann. Neurol.

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Terry, R. D., and Wisniewski, H. (1970). In “Alzheimer’s Disease and Related Disorders” (G.E. W. Wolstenholme and M. O’Connor, eds.), Ciba Found Symp., pp. 145-168. Churchill-Livingstone, Edinburgh and London. Tessman, I. (1968). Virology 35,330-333. Vigny, M., Bon, S., Massoulie, J., and Gisiger, V. (1979). J. Neurockem. 33,559-565. Wilcock, G. K., and Esiri, M. M. (1982). J. Neurol. Sci5 6 , 343-356. Williams, E. S., and Young, S. (1980). J. Wild Dis. 1 6 , 89-98. Williams, E. S., and Young, S. (1982). J. Wildl. Dis. 18,465-471. Williams, E. S., Young, S., and Marsh, R. F. (1982). Wildl. Assoc. Annu. Conf. Abstract. Wilson, D. R., Anderson, R. D., and Smith, W. (1950). J.Comp.Pathol. 60,267-282. Wisniewski, H. M., Bruce, M. E., and Fraser, H. (1975). Science 190,1108-1110. Wisniewski, H. M., Mortez, R. C., andLossinsky, A. S. (1981). Ann. Neurol. 10,517-522. Wisniewski, H. M., Merz, G . S., Merz, P. A., Wen, G . Y., and Iqbal, K. (1983). Prog. Neuroputhol. 5,139-150. Yagishita, S. (1981). Acta Pathol. Jpn. 31,923-942. Zlotnik, I. (1962). ActaNeuropathol., Suppl. 1, 61-70. Zlotnik, I., and Rennie, J. C. (1965). J. Comp.Pathol. 75,147-157.

ADVANCES I N VIRUS RESEARCH, VOL. 29

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS Donald 1. Nuss Center for Laboratories and Research New York State Department of Health Albany, New York

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Virion. . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Physical Properties and Morphology. . . . . . . . . . . . . . . B. T h e G e n o m e . . . . . . . . . . . . . . . . . . . . . . . . . C. Polypeptides and Enzymatic Activities . . . . . . . . . . . . . . 111. Transcription. . . . . . . . . . . . . . . . . . . . . . . . . . . A. Properties of Virion-Associated Transcriptase Activity. . . . . . . B. The Transcripts. . . . . . . . . . . . . . . . . . . . . . . IV. Translation. . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Expression of Viral Polypeptides in Cultured Vector Cells . . . . . B. Expression of Viral Polypeptides in Cell-Free Systems. . . . . . . C. Regulation of Viral Gene Expression in Cultured Vector Cells . . . V. Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . A. Transmission by Leafhopper Vector . . . . . . . . . . . . . . . B. Loss of Transmissibility . . . . . . . . , . . . . . . . . . . C. Characterization of Exvectorial Isolates . . . . . . . . . . . . . D. Characterization of Remnant RNAs Associated with Exvectorial Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . E. Resemblance of Exvectorial Isolates to Defective Interfering Particles VI. Infection of the Plant Host . . . . . . . . . . . . . . . . . . . . . A. Tumor Induction . . . . . . . . . . . . . . . . . . . . . B. Tissue Culture Studies. . . . . . . . . . . . . . . . . . . . . VII. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . . . . . . . . . . .

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57 58 58 59 61 63 63 66 67 67 69 71 72 72 73 74 78 81

85 85 87 89 90

I. INTRODUCTION Plant virus members of the family Reoviridae possess a segmented, double-stranded1 (ds) RNA genome, exhibit a propensity to induce tumors in the plant host, and have evolved a transmission mechanism dependent Abbreviations: BHK, baby hampster kidney; BTV, blue tongue virus; CPV, cytoplasmic polyhedrosis virus; DI, defective interfering; ds, double-stranded; pi, postinfection; RB, standard reference preparation of WTV (Reddy and Black, 1972); SAM, S-adenosyl-L-methionine; ScV, Saccharomycescerevisiae virus; SDS, sodium dodecyl sulfate; WTV, wound tumor virus; m7G(5’)ppp(5’)Nm,7-methylguanosine 5 -trophosphoryl-5 -triphosphoryl-5 -2 -0methylnucleoside. 57

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039829-X

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DONALD L. NUSS

upon replication in the insect vector. Of the viruses comprising this group, wound tumor virus (WTV) has been the most extensively characterized, due primarily t o the efforts of its discoverer, Lindsay M. Black. Both the discovery and subsequent study of WTV are distinctive among the plant viruses. WTV was first described following an attempt to find new strains of known plant viruses by collecting the insect vectors (Black, 1944). Since its isolation in 1941WTV has not been identified in nature, and its natural plant hosts are unknown. Nevertheless, this virus has played an important role in several significant scientific developments. The technique of density gradient centrifugation was developed by Myron Brakke during his attempts to purify WTV (Brakke, 1951; Black, 1981). Studies with WTV were crucial in confirming the hypothesis that some insect-borne plant viruses actually replicate in the vector (Black and Brakke, 1952; Black,1953). Attempts to understand the fine details of WTV transmission led to the establishment and utilization of a continuous cell culture system derived from the leafhopper vector (Chiu etal., 1966; Chiu and Black, 1967). This tissue culture system remains aunique and powerful tool for the study and manipulation of WTV outside the plant host. The intent of this article is to view WTV primarily from the perspective of its suitability as a model system for studying the molecular details of virus transmission by insect vectors and its potential as a probe for studying the molecular biology of its plant hosts. The value of a model system or probe is dependent in part on the knowledge of its properties. Consequently a portion of this article will deal with the properties of the virion, its genome, and its transcription and translation products. Where deemed useful, WTV will be compared with other Reoviridae that replicate exclusively in animals, in insects, or in both. Several excellent accounts by L. M. Black (1944,1953,1959,1979,1982)provide details of his discovery of WTV and subsequent studies, including the classic studies which established that WTV multiplies in the insect vector.

11. THEVIRION A. Physical Properties and Morphology Larger than other viruses that replicate in plants, WTV is approxi1954;Bils and Hall, 1962;Streissle mately 70 nm in diameter (Brakke etal., and Granados, 1968; Lewandowski and Traynor, 1972; Reddy and MacLeod, 1976), with a sedimentation coefficient (sZ,J of 510 S (Black etal., 1962; Kalmakoff etal., 1969). Approximately 22% RNA by weight and

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS

59

78% protein (Black and Markham, 1963; Kalmakoff etal., 1969), it is icosahedral (Bils and Hall, 1962) and contains a dense RNA-protein core surrounded by two protein shells (Bils and Hall, 1962; Streissle and Granados, 1968; Reddy and MacLeod, 1976). Intact, purified particles exhibit a diffuse appearance under the electron microscope (Streissle and Granados, 1968). However, when WTV particles are treated with chymotrypsin to remove the outer protein layer, they take on a well-defined, ordered appearance with clearly discernible capsomeres (Streissle and Granados, 1968; Reddy and MacLeod, 1976). The number of capsomeres is apparently 92 (Bils and Hall, 1962; Streissle and Granados, 1968), although a count of 32 has also been cited (I. Kimura, personal communication, in Reddy and MacLeod, 1976). It is important for later discussion to note that the polypeptides of the outer protein layers are easily removed by mechanical disruption during purification (Reddy and MacLeod, 1976),or by exposure to CsCl (Reddy and MacLeod, 1976). The probable arrangement of structural polypeptides within the particle will be discussed in Section I1,C.

B. The Genome The dsRNA nature of the WTV genome has been demonstrated by base composition analysis (Black and Markham, 1963; Gomatos and Tamm, 1963), thermal denaturation measurements (Gomatos and Tamm, 1963), electron microscopy (Bils and Hall, 1962; Kleinschmidt etal., 1964), and X-ray diffraction analysis (Tomita and Rich, 1964). The genome, which possesses a guanosine cytidine content of 38% (Black and Markham, 1963; Gomatos and Tamm, 1963) and a melting out temperature of 90°C (Gomatos and Tamm, 1963), can be electrophoretically resolved into 12 individual segments (Fig. 1)with a combined molecular weight of approximately 16 X lo6 (Black and Markham, 1963; Kalmakoff etal., 1969; Wood and Streissle, 1970; Reddy and Black, 1973b). Analysis of the 3’-termini of the WTV genome segments has revealed equal amounts of uridine and cytidine (Lewandowski and Leppla, 1972), suggesting that one strand of each dsRNA segment terminates in 3’-terminal cytidine, while the complementary strand contains a 3’-terminal uridine. Analysis of the 5’-terminus of the genome segments has not yet been performed. For other members of the Reoviridae, e.g., cytoplasmic polyhedrosis virus (CPV) (Furuichi, 1974) and human reovirus (Shatkin, 1974), viral transcripts and messenger-sense (plus) strands of genome segments both contain the 5‘-terminal structures m7GpppNm(where N is adenosine for CPV and guanosine for human reovirus). Since transcripts synthesized by WTV particles contain the 5’-terminal structure

+

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DONALD L. NUSS

FIG.1. Relative electrophoretic mobility of WTV genome segments in a 7.5%polyacrylamide gel. Genome segments are designated Sl-SI2 (Reddy and Black, 1973b) based on relative mobilities: S1 is the slowest-migrating segment. The estimated molecular weights (Reddy and Black, 1977) of the fastest and slowest migrating segment are indicated to the right.

m7GpppAm(Rhodes et al., 1977), it will be interesting to determine whether the WTV genome segments also contain this structure. The genome segments of CPV (Furuichi and Miura, 1973, 1975; Shimotohno and Miura, 1974)and human reovirus (Muthukrishnan and Shatkin, 1975; Li etal., 1980; McCrae, 1981) are perfect duplex molecules in which the penultimate 5’-terminal nucleotide of the plus strand is complimentary to the 3’-terminal nucleotide of the negative strand. Since the penultimate

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61

5'-terminal nucleotide of the WTV transcripts is adenosine, it can be predicted by analogy that it is the negative strand of the WTV genome segment that contains the 3'-terminal uridine. Evidence that every WTV particle contains one copy of each genome segment is the finding that the genomic RNA prepared from purified virus particles is composed of equimolar amounts of each segment (Kalmakoff et al., 1969; Reddy and Black, 1973b) and that, as judged by infectivitydilution curves (Chiu and Black, 1969; Kimura and Black, 1972),an infection can be initiated by a single WTV particle. The mechanism responsible for the packaging of one and only one copy of each independent genome segment in each virus particle remains a complete mystery. Studies directly examining the replication of the WTV genome have not yet been performed.

C. Polypeptides and Enzymatic Activities The polypeptide composition of purified WTV particles has been investigated by two laboratories. Lewandowski and Traynor (1972) identified four structural polypeptides by analyzing the gel electrophoretic patterns of 1251-iodinatedvirus particles. Taking advantage of an improved purification procedure (Reddy and Black, 1973a) that yielded standard virus particles of high specific infectivity, Reddy and MacLeod (1976) identified seven virus-associated polypeptides with estimated molecular weights of 160, 131, 118,96,58,36,and 35 X lo3. Significantly, virus particles purified from infected plants, infected leafhopper vectors, and infected vector cell monolayers exhibited the same set of structural polypeptides. The probable locations of the structural polypeptides within the WTV virion were determined by examining the influence of various physical and enzymatic treatments on virus integrity and infectivity (Reddy and MacLeod, 1976). Digestion of purified virus particles with chymotrypsin resulted in the selective loss of two polypeptides with estimated molecular weights of 131and 96 X lo3. Subsequent analysis of theprotease-digested particles by electron microscopy revealed a concomitant removal of the outer protein layer. Chymotrypsin digestion of WTV particles caused no loss of infectivity on vector cell monolayers. Centrifugation of virus particles through CsCl gradients generated particles of densities ranging from 1.30 to 1.44 g/cm3, depending on the pH. Particles banding at a density of 1.43 g/cm3 contained polypeptides of 160, 118, and 58 X lo3 and the entire WTV genome but were devoid of capsomeres. All particles recovered from CsCl gradients were reduced in specific infectivity by greater than 1000-fold. From these combined observations Reddy and MacLeod (1976) con-

TABLE I ESTIMATED AND APPARENT MOLECULAR WEIGHTS OF WTV GENOME SEGMENTS AND GENEPRODUCTS MW of gene products Segment 1

2

6 7 8 9 10 11 12

MW of genome segment (X 106)o

Estimated"

In vivab

In vitrob

2.9 2.40-2.48 2.20-2.25

161,000 133,000- 137,000 122,000- 124,000

155,000 130,000 108,000

1.78-1.80

99,000- 100,000

1.10-1.20 1.05- 1.15 0.83-0.90 0.57-0.63 0.55-0.61 0.54-0.60 0.32-0.35

62,000-67,000 58,000-64,000 46,000- 50,000 32,000- 35,000 31,000- 34,000 30,000-33,000 18,000-19,000

155,000 130,000 108,000 76,000 74,000 57,000 52,000 42,000 41,500 39,000 35,000 19,000

[ ;;:% 57,000 52,000 42,000 41,500 39,000 35,000 19,000

Location'

Nomenclatured

Core Outer-protein coat Core Outer-protein coat

P1

Core Capsid Capsid

P2 P3 P5 Pns4 P6 Pns7 P8 P9 PnslO Pnsll Pnsl2

Estimated molecular weights for genome segments and gene products are from Reddy and Black (1977). The assignment of genome segments to corresponding gene products has not yet been established. The apparent molecular weights of in vivo and in vitro synthesized WTV gene products are from Nuss and Peterson (1980). Other estimates for the molecular weights (X 103) of WTV structural polypeptides include those of Lewandowski and Traynor (1972),listed first in each pair, and Reddy and MacLeod (1976):P1,156,160; P2, -, 131; P3, 122, 118; P5,-, 96; P6, 63, 58; P8, 44, 36; P9, 44, 35. The probable location of each structural protein within the WTV particle is from Reddy and MacLeod (1976). The nomenclature is that proposed by Nuss and Peterson (1980) with slight modifications according to Nuss (1983a). P indicates structural polypeptide, Pns indicates nonstructural polypeptide. The numbers, with the exception of 5 in P5 and 4 in Pns4 (Nuss, 1983a), indicate relative electrophoretic migration positions in polyacrylamide gels with 12 being the fastest migrating species.

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cluded that the polypeptides of 160,118, and 58 X lo3are intimately associated with the dsRNA genome in a structure termed the core. The structural polypeptides of 35 and 36 X lo3 were assigned to the capsomers surrounding the core. The 131 and 96 X lo3polypeptides were designated as the components of the amorphous outer protein layer. The molecular weights for WTV gene products recently reported by Nuss and Peterson (1980)differ from those reported by both Lewandowski and Traynor (1972) and Reddy and MacLeod (1976). All three sets of data and the probable locations of the virion-associated polypeptides are summarized in Table I. Enzymatic activities associated with purified WTV particles include a RNA transcriptase, first described by D. R. Black and Knight (1970), a mRNA guanyltransferase, a guanine-7-methyltransferase, and a mRNA-2’-O-methyltransferase (Rhodes et al., 1977; Nuss and Peterson, 1981b). The guanyl- and rnethyltransferases are responsible for modifying the 5’-termini of WTV transcripts to the structure m7G(5’)ppp(5’)Am (Rhodes et al., 1977). Interestingly, the mRNAs of insects and their viruses generally contain 2’-O-rnethylated penultimate 5’-terminal nucleotides, while the mRNAs of plants and their viruses do not (see Banerjee, 1980, for a current list of 5’-terminal cap structures of viral and cellular mRNA). The methylation pattern of the WTV transcripts may bear on the evolutionary origin of this virus. The presence of a 2’-O-methylated adenosine residue in the 5’-terminal cap structure of WTV transcripts suggests the possibility that WTV evolved as an insect virus that later adapted to replicate in plants. Since the enzyme activities involved in mRNA synthesis and modification are lost upon solubilization of purified virus particles, no individual activities have been assigned to structural polypeptides for the Reoviridae. Indeed, it has not been rigorously demonstrated that any of the enzyme activities associated with viral particles are encoded by the virus genome. However, the demonstration that WTV particles retain mRNA-2’-O-methyltransferase activity after 30 years of passage in a plant host (Nuss and Peterson, 1981b) strongly suggests that the WTV-associated 2’-O-methyltransferase is virus encoded. 111. TRANSCRIPTION A. Properties of Virion-Associated Transcriptme Activity Transcription by all members of the Reoviridae is dependent on the presence of divalent cations (Mg2+,Mn2+),is conservative and asymmet-

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DONALD L. NUSS

ric, and yields single-stranded transcripts that exhibit mRNA activity (Joklik, 1981). However, when the transcriptase activities of various Reoviridae are compared, several notable differences become apparent, some of which may be related to the quite different environments in which these viruses replicate (insects, plants, and animals). Most Reoviridae that replicate in vertebrates must be activated before they exhibit transcriptase activity in uitro. Human reovirus, blue tongue virus (BTV), and rotaviruses can be activated by digestion with chymotrypsin (Shatkin and Sipe, 1968; Skehel and Joklik, 1969; Martin and Zweerink, 1972;Verwoerd and Huismans, 1972),by heat shock (Borsa and Graham, 1968; Cohen etal., 1979), or by removal of Ca2+by EGTA (Cohen etal., 1979);all of these treatments disrupt the outer capsid polypeptides. However, no prior treatment is required to activate transcription by WTV (Black and Knight, 1970; Reddy etal., 1977) or CPV (Lewandowski etal., 1969). Moreover, transcriptase activity of WTV (Reddy and Black, 1977) and CPV (Smith and Furuichi, 1980) is not diminished when the outer capsid polypeptides are removed by chymotrypsin treatment, indicating that these polypeptides are not involved in the transcription process. The temperature optimum for in uitro transcription by human reovirus (Kapuler, 1970) and by rotaviruses (Cohen, 1977)is a surprising 47-52"C, while the optimum for WTV, CPV, and BTV, which replicate alternatively or exclusively in poikilothermic organisms, is 28-31 "C. A differential influence of the methyl donor S-adenosyl-L-methionine (SAM) on transcription by individual members of the Reoviridae has also been reported. SAM stimulates BTV transcription by 2-fold (Van Dijk and Huismans, 1980)and CPV transcription by more than 70-fold (Furuichi, 1974)but has no influence on transcription by human reovirus (Furuichi etal., 1975) or WTV (Rhodes etal., 1977). A detailed characterization of the virion-associated transcriptases of the Reoviridae has been hampered by the inability to solubilize the enzyme from the virus particle in an active form.

FIG.2. Resolution and genome assignment of WTV transcripts. (a) 32P-labeledtranscripts of WTV (lane 1)and reovirus (lane 2) were resolved by electrophoresis through a2.5 to 5% polyacrylamide gel containing sodium dodecyl sulfate (SDS) and 7 M urea. The letters a-k were picked arbitrarily to identify the individual WTV transcripts. The migration position of the large (l), medium (m), and small (s) classes of the reovirus transcripts are shown on the right. (b) Isolated, 32P-labeledtranscripts a-k were hybridized individually to a mixture of unlabeled WTV genome RNAs. The resulting hybrids were analyzed by electrophoresis through a 7.5%polyacrylamide gel. The letters indicate whether a lane contains a hybrid formed by individual transcripts (a-k) or total transcripts (T). Lane a' is a longer exposure of lane a. The migration positions of WTV genome segments are indicated at the right. (From Nuss and Peterson, 1981a.)

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DONALD L. NUSS

B. The Transcripts The products of the WTV in vitro transcription reaction remain sensitive t o ribonuclease digestion after incubation under conditions that would permit annealing of complimentary strands (Reddy et.al., 1977). This finding indicates that the transcripts are single-stranded and of the same polarity. Experiments in which the transcription products were annealed to genome RNA revealed that all 12 genome segments are transcribed into apparently full-length copies (Black and Knight, 1970; Reddy et.al., 1977). Although the results of sucrose gradient analysis of the WTV transcripts are consistent with this interpretation, an absolute determination as to whether the WTV transcripts are full-length copies of the genome segments or truncated species, as is the case for influenza mRNA (Hay etaL, 1977), will require direct sequence analysis of the transcripts and genome segments. Conditions of polyacrylamide gel electrophoresis have been defined for the resolution of the WTV transcripts into discrete species and has allowed the isolation of individual transcripts (Nuss and Peterson, 1981a) (Fig. 2). When the isolated, uniformly 32P-labeledtranscripts were independently

S6

FIG.3. Results of assignment analysis and proposed nomenclature for WTV transcripts. The lines connect the genome segments (Sl-S12) with the transcripts (T) which they encode. (From Nuss and Peterson, 1981a.)

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS

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hybridized to a mixture of the WTV genome segments, each transcript hybridized selectively to one genome segment (Fig. 2). In this manner the coding assignment of 10 of the 12 WTV transcripts was established. The assignment of the remaining transcripts, which hybridized to the unresolved mixture of genome segments 4 and 5, was determined by analyzing the transcripts synthesized by an isolate of WTV that contains no detectable genome segment 5. On the basis of this assignment of the WTV transcripts to their corresponding genome segments, Nuss and Peterson (1981a) proposed a nomenclature in which each transcript is denoted by the letter T and a number indicating the genome segment from which it is derived (Fig. 3). Thus T1 is derived from the genome segment S1. IV. TRANSLATION

A. Expression of Viral Polypeptides in Cultured Vector Cells A prerequisite to understanding the molecular biology of a virus is the identification of its primary gene products. Results obtained from in uitro studies initiated to gain such information are generally difficult to intertranslation products can be compared directly with pret unless the in vitro the viral polypeptides expressed in the infected host. Cell culture systems that support viral replication have provided animal virologists with a convenient method for making such comparisons. Although plant cell protoplasts provide plant virologists with certain advantages over cell-walled plant cells or tissue for such studies (Cocking, 1966; Takebe, etal., 1968), the protoplasts are limited in comparison to animal cell systems, primarily because of the difficulty of identifying viral gene products against the background of cellular polypeptides (Sakai and Takebe, 1974; Zaitlin and Beachy, 1974,1975; Takebe, 1975a,b, 1977; Sarkar, 1977. Cultured vector cells offer a powerful alternative to plant protoplast for studying gene expression by plant viruses, such as WTV, which also replicate in their insect vector. If a virus is to be transmitted efficiently by an insect vector, it generally must not affect the vector adversely. For viruses that are transmitted in a propagative manner, mechanisms have evolved to allow efficient multiplication in the vector cells with no deleterious effects on cell functions. WTV establishes a noncytopathic infection of cultured vector cells (line AC20; see Black, 1979, for history of line AC20) which persists during repeated subculturing (Black, 1969, 1979). Persistently infected and uninfected vector cell cultures have a similar appearance and growth rate

68

DONALD L. NUSS

- PI

- P2 - P3 HP1 4% PNS4

- PG .PNS 7

-PNS 11

‘PN5 12

FIG.4. Autoradiograph of a polyacrylamide gel analyzing [36S]methionine-labeledextracts of WTV-infected AC20 cells. The number above each lane indicates the time (in hours postinfection) at which pulse-labeling was performed. Lane M contains the lysate of mockinfected cells labeled at 8 hours postinfection. Lane PI contains labeled extracts of subcul-

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS

69

(Chiu and Black, 1967), even though more than 90% of the cells in the infected culture exhibit evidence of massive infection, as determined by fluorescent antibody staining (Black, 1979;A. J. Peterson and D. L. NUS, unpublished observation). Initial attempts to identify WTV-specific polypeptides in [36S]methionine-labeled lysates prepared from pulse-labeled, persistently infected cells were unsuccessful (D. L. Nuss and A. J. Peterson, unpublished observations. By using the immunofluorescence assay to monitor the progress and extent of infection, it was determined that a concentrated lysate, freshly prepared from persistently infected AC20 cells, produced a highmultiplicity infection of the majority of cultured AC20 cells in a fairly synchronous manner (Nuss and Peterson, 1980). Gel analyses of lysates prepared from cells infected under these conditions revealed 12 new polypeptides (Fig. 4),which increased in intensity against the background of host proteins with time after infection. Five of these polypeptides were identified as viral structural components on the basis of coelectrophoresis with polypeptides of purified virus preparations. Unfortunately, virus preparations obtained during this study did not contain appreciable amounts of the outer protein coat polypeptides, which presumably were lost during purification. Consequently, the assignment of the remaining two structural polypeptides was tentatively made on the basis of comparison to the results of Reddy and MacLeod (1976). The tentative assignments were later confirmed by coelectrophoresis studies with less extensively purified virus preparations and the sensitive silver-staining technique (Nuss, 1983a). The remaining five polypeptides observed in the infected cell lysates were identified as virus encoded nonstructural gene translation studies (Nuss and Peterson, 1980). Alproducts by invitro though pulse-chase experiments with infected cells revealed no evidence for gross posttranslational modifications of the newly synthesized polypeptides, additional studies will be required to rule out subtle modifications.

B. Expression of Viral Polypeptides inCell-Free Systems Both etal. (1975) set three criteria to define the primary gene products of human reovirus: (1)they are present in infected cells after pulse-labeling, tured (49 passages) infected cells. The migration positions of WTV structural (P) and nonstmctural (Pns) polypeptides are indicated. The nomenclature is that proposed by Nuss and Peterson (1980) with recent modifications (Nuss, 1983a),as illustrated in Table I. Also indicated are the positions of three host proteins (HP1-3) that increase in intensity after infection. (From Hsu etal., 1983.)

70

A

DONALD L. NUSS

1

2

3

4

B

1

2

FIG.5. Autoradiogram of polyacrylamide gels analyzing 3SS-labeledtranslation products synthesized in WTV-infected vector cells and in a wheat embryo cell-free system programmed by in vitro synthesized WTV mRNA or purified WTV particles. The arrows to the left of each gel indicate the positions of molecular-weight marker proteins. The arrows on the far right of (A) indicate the positions of WTV structural polypeptides. The numbers to the immediate right of the gels correspond to the apparent molecular weights of the WTV gene products. (A) W-labeled extracts from mock-infected and WTV-infected AC20 cells

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS

71

(2) they coelectrophorese with cell-free translation products synthesized in response to purified viral mRNA, and (3) they have molecular weights consistent with the coding capacity of the genome segments. To confirm that the polypeptides newly expressed in WTV-infected cells were virusencoded primary gene products rather than virus-induced host proteins, WTV mRNA synthesized in vitro was subjected to cell-free translation. Products synthesized in a wheat germ translation system programmed with WTV mRNA coelectrophoresed with all of the presumptive viral polypeptides except the 72 X lo3 polypeptide (Fig. 5 ) .However, a major in vitro product of 74 X lo3 presumably related to the 72 X lo3 in vivo polypeptide was observed. A final determination of the relatedness of these two polypeptides will require additional studies. The apparent molecular weights most recently determined for the WTV polypeptides synthesized in uivo and in uitro are listed in Table I, along with the expected molecular weights based on the size of the genome segments and a proposed general nomenclature for the WTV-specified polypeptides (Nuss and Peterson, 1980). The assignment of genome segments to corresponding gene products has not yet been established. To avoid the difficulty inherent in manipulating plant tissue when studying viral gene expression in infected plants, a system was established in which transcription by WTV particles was coupled to translation in a wheat germ cell-free extract (Nuss and Peterson, 1980). The polypeptides made in response to WTV mRNA synthesized directly in the cell-free system exhibited the same gel pattern as the translation products made in response to exogenous WTV mRNA (Fig. 5 ) . The availability of a homologous plant cell-free system which supports the simultaneous transcription and translation of WTV mRNA should be useful for examining certain details of the regulation of WTV gene expression in the plant host.

C. Regulation of Viral Gene Expression i n Cultured Vector Cells From kinetic measurements of WTV polypeptide synthesis after infection and subsequent subculturing of infected cells, the followingpicture of viral gene expression has emerged (Nuss and Peterson, 1980; A. J. Peterwere analyzed in lanes 1 and 2, respectively. Lane 3 contains the wheat embryo cell-free translation products programmed by WTV mRNA. Lane 4 contains translation products synthesized in the coupled transcription- translation system. In this case the wheat embryo translation lysate was supplemented with nucleoside triphosphates, SAM, and purified WTV particles. Under these conditions WTV transcripts were synthesized and translated in the product corresponding to the same reaction mixture. (B) Lane 1 shows that the in uitro fastest migrating WTV structural component is composed of two polypeptides. Lane 2 demonstrates the in uitro synthesis of the 155 X lo3structural polypeptide after addition of a ribonuclease inhibitor to the wheat embryo lysate. (From Nuss and Peterson, 1980.)

72

DONALD L. NUSS

son and D. L. NUSS,unpublished observation). Viral protein synthesis increases in a linear fashion from 4 hours postinfection (pi) and reaches a plateau between 24 and 36 hours pi (Fig. 4). Viral polypeptide synthesis then declines relative to the synthesis of host proteins during subsequent passage of infected cells. In persistently infected cells viral polypeptide synthesis is less than 5 % of that observed during the acute phase of infection (Lane PI, Fig. 4). However, as judged by immunofluorescence staining and Western gel analysis, a large pool of viral polypeptides exists in the individual persistently infected cells (A. J. Peterson and D. L. NUSS,unpublished observations). Recent studies also indicate that intact viral mRNA is present in the persistently infectedcells at approximately 50%of the level found at the peak of viral protein synthesis (A. J. Peterson and D. L. NUSS,unpublished observations). These results strongly suggest that a form of posttranscriptional regulation of viral gene expression operates in vector cells persistently infected with WTV. The term regulated infection, as defined by Walker (1964),best describes the persistent infection established in vector cells by WTV. While recent results suggest that one point of regulation in this type of infection is at the level of translation, continued studies with this system are expected to uncover additional regulatory mechanisms involving other viral and possibly cellular components and functions. The WTV-vector cell system is particularly well suited for studying the molecular details of regulated persistent infections because the virus - cell interaction observed in the laboratory closely reflects that operating in nature during biologic transmission of viruses to plant and animal hosts.

V. TRANSMISSION A. Transmission by Leafhopper Vector The complex process of biologic transmission of WTV involves acquisition of the virus by the leafhopper during feeding on an infected plant, sequential infection of the vector’s filter chamber and ventriculus, hemolymph, and salivary glands, and passage of the virus to uninfected plants via the salivary fluid during subsequent feeding (Shikata etal., 1964; Sinha, 1965; Shikata and Maramorosch, 1965; Granados etal., 1967). The time required for the virus to migrate t o the salivary gland and to multiply sufficiently for successful transmission with the salivary fluid (the extrinsic incubation period) varies from 13 to 30 days, depending on the environmental conditions (Maramorosch, 1950). Infection of the leafhopper by WTV results in no obvious pathology (Black, 1957; Hirumi etaL, 1967);

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and once infected, the insect retains the ability to transmit the virus for life (Maramorosch, 1950; Black, 1953). Efficient transmission is limited, however, to only two species: Agallia constricta van Duzee and Agalliopsis novella (say) (Black, 1944). Mechanical transmission of WTV to test plants has been reported but not conclusively demonstrated (Brakke et al., 1954). The establishment in the 1960s of continuous cultures of leafhopper cells that support the replication of WTV (Chiu et al., 1966; Chiu and Black, 1967) greatly influenced the direction and pace of research into WTV molecular biology in general and especially into transmission. The development of a fluorescent cell-counting technique for measuring WTV infectivity on cultured vector cell monolayers (Chiu and Black, 1969) reduced the time required to titrate WTV preparations from 12 weeks to 2 days. Consequently it became possible to measure conveniently the growth rate of WTV in the vector after virus acquisition and to derive a good estimate of the absolute concentration of the virus in the infected insect (Reddy and Black, 1972). It also became possible to measure the absolute specific infectivity of various virus preparations by comparing the number of virus particles estimated by electron microscopic examination with infectivity data derived from the immunofluorescent assay on vector cell monolayers (Reddy and Black, 1973a). This capability was particularly useful in studies examining the specific transmissibility of various virus preparations (Black, 1969; Reddy and Black, 1974).

B. Loss of Transmissibility In 1958 Black et al. described a strain of WTV that had lost the ability to be transmitted by the leafhopper vector. This strain was isolated from an infected sweet clover plant that had been maintained by vegetative propagation for several years after inoculation. Subsequent studies revealed that individual virus populations maintained in vegetatively propagated plants for varying numbers of years exhibited differing levels of transmissibility (Black, 1969; Liu et al., 1973). Reductions in transmissibility were accompanied by reductions in specific infectivity on cultured vector cells (Reddy and Black, 1969, 1974; Liu et al., 1973). In discussing loss of transmissibility, it is helpful to define several terms introduced by Black and co-workers during their discovery and examination of this phenomenon. Although individual virus preparations may have consisted of mixed populations of transmissible and nontransmissible virus particles, the individual populations were termed “isolates” because they were physically isolated from each other within aplant that was maintained by vegetative propagation (Reddy and Black, 1974). That is,

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each virus population evolved in the isolated environment of the infected plant without the introduction of exogenous virus or the opportunity to replicate in the insect vector. Since the isolates differed in the degree to which transmissibility had been lost, each isolate was designated by a prefix indicating whether it was fully transmissible (vectorial, VI), partially transmissible (subvectorial, SV), or nontransmissible (exvectorial, EV) and a number indicating the year in which the virus was inoculated into sweet clover (Black, 1969). For example, EV49 is an exvectorial isolate obtained from a vegetatively propagated plant originally inoculated in 1949. C. Characterization of Exvectorial Isolates An extensive electrophoretic analysis (Reddy and Black, 1974) of the genome segments of the subvectorial and exvectorial isolates revealed each isolate to have a distinctive electrophoretic pattern. The genome profiles of many isolates contained remnants of genome segments that had arisen as a result of deletion mutation events. By observing reductions in the molar proportion of a particular genome segment and the concomitant appearance of the remnant RNA, it was possible to make a tentative determination of the genome segment from which a remnant was derived. While mutation events were restricted to only 4 of the 12 genome segments (segments 1,2,5, and 7), they occurred much more frequently in segments 5 and 1than in 2 and 7. In several cases remnants of identical electrophoretic mobility were generated from the same genome segments in different isolates (Reddy and Black, 1974). Improved techniques allowed a determination of the genome pattern of virus particles derived from single sweet clover plants (Reddy and Black, 1977). Starting with plants infected with subvectorial or exvectorial isolates, successive cuttings of infected plants were selected which exhibited a progressive loss of virus genome segments 2 or 5 and their remnants. In this way viral isolates which appeared to be completely free of these segments and their remnant RNAs were eventually obtained -S2(70), - S5(60), and - S5(64) (nomenclature from Reddy and Black, 1977; the number in parentheses indicates the year in which the vectorial parent population was introduced into sweet clover). Other isolates were selected that contained 10% of the normal complement of segment l,lO%S1(49) and lO%S1(60),or a full complement of a remnant of segment 7 in place of the intact segment, MS7(57), where M signifies mutant. After the 1977 report by Reddy and Black the islolates were maintained by vegetative propagation without further selection or analysis. These isolates were recently examined by labeling the 3' ends of each genome

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segment with [32P]pCp, followed by electrophoretic analysis (Nuss, 1983a). As shown in Fig. 6, many of the remnant RNAs -and in one case an intact segment [segment 7 of isolate MS7(57)] thought to be absent in the 1977 study-were observed in the recent gel profiles. Reddy and Black (1977) estimated that they could detect a segment or remnant RNA when it was present in as few as 0.1%of the virus particles in the population. When they reversed their selection procedures to favor the presence of standard virus, starting with subvectorial isolates (which only partially lacked certain segments), they could eventually obtain virus populations that exhibited the standard genome pattern and could infect cultured vector cells. The apparent increase of remnant RNAs from less than one copy per 1000 virions in the isolates examined in 1977 to the concentrations shown in Fig. 6 suggests that in the absence of negative selection remnant RNAs may be preferentially replicated and/or packaged in the systematically infected plants. The isolate reported to lack genome segment 2 was clearly devoid of that segment (Fig. 6) and failed to synthesize the transcript encoded by that segment, T2 (Fig. 7). Analysis of the transcripts synthesized in uitro by purified isolates (Fig. 7) revealed that the transcripts encoded by genome segment 5, T5 (Nuss and Peterson, 1981a), and presumably segment 5 itself were undetectable in the two isolates reported to lack segment 5 [-S5(60) and -S5(64)]. The presence of segment 5 could be determined only by analysis of the transcripts synthesized by the individual isolates, because segments 4 and 5 comigrate under most conditions of gel electrophoresis, whereas the transcripts of these two segments are readily resolved (Fig. 2 4 Nuss and Peterson, 1981a). Detectable levels of transcript 5 were synthesized by only two isolates, MS7(57) and - S2(70), and even in these isolates it was synthesized in low amounts. In this regard, the exvectorial virus populations from which the selected isolates were derived lacked segment 5 partially or completely. The exception was the parent of MS7(57), EV57 (Reddy and Black, 1974). Transcripts corresponding in size to remnants of genome segments are also evident in Fig. 7. This result clearly indicates that the remnants must contain nucleotide sequences required for recognition and initiation by the virion transcriptase. Previous studies (Nuss and Peterson, 1981b) have shown that the selected isolates all synthesize transcripts that are correctly capped and methylated a t the 5'-ends. The combined results exclude the possibility that the loss of transmissibility involves a lesion at the level of transcription or mRNA modification. The products of genome segments 2 and 5 are clearly not involved in these functions. The report that the mutation events associated with loss of transmissi-

FIG.6. Autoradiogram of a polyacrylamidegel analyzing 3'-3ZP-labeledgenome segments of selected exvectorial WTV isolates. Genome segments extracted from purified standard and selected exvectorialpopulations were labeled at the 3' end with [5'-3*P]pCpand analyzed by electrophoresis through a 7.5% polyacrylamide gel. The genome segments are numbered (left) as described in Fig. 1. The dots to the right of individual lanes indicate the positions of remnant RNAs observed among the segments of individual virus populations. Lanes: 1, standard WTV; 2,MS7(57); 3, -S5(64); 4, -S5(60); 5, -S2(70); 6, 10%S1(60); 7, lO%S1(49). The nomenclature for the exvectorial variants is taken from Reddy and Black (1977), as discussed in the text. (From Nuss, 1983a.) 76

T1

T2

-

-

T3 T5 -

T4r

T6 T7

-

-

T8 -

T9

-

FIG.7. Autoradiogram of a SDS -poiyacrylamide gel analyzing transcripts of standard and exvectorial WTV populations. Uniformly 32P-labeledtranscripts synthesized in uitro by purified virus particles were resolved by electrophoresis through a 2.5 to 5% polyacrylamide gel containing SDS and 7 M urea. The dots to the right of individual lanes indicate the positions of transcripts thought to correspond to remnants of genome segments in Fig. 6. Lanes: 1, standard WTV; 2, MS7(57); 3, -S5(64); 4, -S5(60); 5, -S2(70); 6, 1O%S1(60); 7, 10%S1(49). The transcripts are numbered (left) as described in Fig. 3. (From Nuss, 1983a.) 17

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bility occur most frequently in genome segment 5 (Reddy andBlack, 1974), coupled with the observation that the selected isolates all contain little or no detectable segment 5, suggests that the product of this segment is important for successful transmission of WTV. To identify the polypeptide encoded by segment 5, the cell-free translation products specified by standard virus and by an exvectorial isolate that contained no detectable genome segment 5 ,-S5(60), were analyzed. The WTV-specified polypeptide of 76 X lo3 (Table I) was not synthesized in response to - S5(60) transcripts and was consequently assigned as the product of genome segment 5 (Nuss, 1983a). The molecular weight of this polypeptide is in agreement with the coding capacity of genome segment 5 (Table I). Coelectrophoresis of [3SS]methionine-labeledinfected cell lysates with purified virus particles indicated that the 76 X lo3 polypeptide is structural (Nuss, 1983a). This polypeptide corresponds to the smaller of the two polypeptides as identified by Reddy and MacLeod (1976) comprising the outer protein coat. Identification of the product of genome segment 5 as the smaller of the two outer-protein coat polypeptides, coupled with the report that the isolate lacking segment 2 lacks the larger component of this coat (Reddy and Black, 1977), suggests that both of these polypeptides play an important role in the transmission of WTV. This theory is clouded, however, by the report that removal of the outer-protein coat polypeptides by protease treatment causes no loss of infectivity on vector cells (Reddy and MacLeod, 1976). This inconsistency raises the interesting possibility that loss of infectivity is a consequence of the absence of these genome segments, rather than of the structural components themselves. If so, the products of these genome segments must perform multiple functions in the replication cycle of WTV.

D. Characterization of Remnant R N A s Associated with Exvectorial Isolates Remnant RNAs associated with WTV exvectorial isolates are transcribed in vitro (Nuss, 1983a; Fig. 7) and presumably in uiuo and are replicated and packaged into virus particles in systematically infected plants (Reddy and Black, 1974,1977; NUSS,1983a). Clearly these RNAs retain nucleotide sequences that are important for the efficient transcription, replication, and packaging of the WTV genome. They are therefore potentially useful tools with which to examine the role of WTV genome structures in these events. Remnant RNAs have been observed in preparations of other segmented

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dsRNA viruses: human reovirus (Ahmed and Fields, 1981; Brown et al., 1983) and Saccharomyces cerevisiae virus (ScV) (Vodkin etal., 1974; Tzen et al., 1974). While 3‘-end sequence analysis has established that ScV remnant RNAs are the product of internal deletion events (Bruenn and Brennan, 1980), the remnant RNAs of human reovirus have not been characterized. A recent analysis of two remnant RNAs associated with exvectorial isolates of WTV indicates that these RNAs are also derived from the parental genome segments via internal deletion events (Nuss, 1983b). 3’-end- labeled remnant RNAs with molecular weights of 2.05 X lo6and 0.26 X lo6 (Reddy and Black, 1974) were isolated from other end-labeled WTV genomic RNAs by preparative polyacrylamide gel electrophoresis (Fig. 8). The genome segment from which each remnant RNA was derived was identified by hybridizing the end-labeled remnant RNA against isolated unlabeled genome segments fixed to nitrocellulose (Thomas, 1980). The remnant RNA of 2.05 X lo6 molecular weight associated with exvectorial isolate -S2(70) (lane 2, Fig. 8) hybridized to segment 2 and was designated 2d2.05 after the nomenclature of Reddy and Black (1974), with the modification that d stands for deletion rather than defective. Thus the designation 2d2.05 indicates a molecule of approximately 2.05 X lo6 molecular weight that was derived by a deletion event from genome segment 2. The remnant RNA with a molecular weight of 0.26 X lo6associated with the exvectorial isolate lO%S1(49) (lane 5, Fig. 8) hybridized exclusively to the unresolved mixture of genome segments 4 and 5. The origin of this remnant RNA was established as segment 5 by its reduced level of hybridization to the genome of an exvectorial isolate which contained nearly undetectable levels of segment 5 (Nuss, 1983a,b). It was designated 5d.26. To determine whether the genesis of these remnant RNAs involved a terminal or internal deletion, the 3’-end-labeled parental and remnant RNAs were denatured and partially or completely digested with ribonuclease T,. Since this nuclease cleaves specifically a t the 3’-side of guanosine residues, partial digestion of the denatured dsRNAs yielded a series of 3’-end-labeled oligonucleotides for each RNA strand. By analyzing these TI digests on 20% polyacrylamide sequencing gels (Maxam and Gilbert, 1977) against an alkali-generated oligonucleotide ladder, the guanosine positions relative to the 3‘ -terminal end were compared simultaneously for each strand of a parental genome segment and remnant RNA. The partial and complete ribonuclease T, digestion patterns for segment 2 and remnant RNA 2d2.05 were indistinguishable for a t least 40 nucleotides from the 3’ -end of each RNA strand (Fig. 9). Similar results were obtained for genome segment 5 and remnant RNA 5d.26 (Fig. 10). A separate analysis of the 12 individual WTV genome segments by this method

FIG.8. Polyacrylamide gel electrophoretic analysis of 32P-pCp-end-labeledtotal and isolated genomic RNAs from standard and exvectorial isolates of WTV. The nomenclature for exvectorial isolates and remnant RNAs is from Reddy and Black (1974, 1977), as describedin the text. Lane 1, genome RNA of exvectorial isolate - S2(70) containing remnant RNA 2d2.05. Lane 2, 2d2.05 isolated from preparative gel containing genome RNA of exvectorial isolate - S2(70). Lane 3, genome segments 4 5, also isolated from the -S2(70) preparative gel to show the separation of 2d2.05 from segments 4 5. Lane 4, genome segment 2 from standard virus. Lane 5, remnant RNA 5d.26 isolated from a preparative gel containing genome RNA from exvectorial isolate lO%S1(49). Lane 6, genome RNA of exvectorial isolate 10%51(49)containing remnant RNA 5d.26. Genome segments S1-S12 are designated as in Fig. 1.

+

+

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has revealed a distinctive pattern for each segment (D. L. NUSS,unpublished observation). The remnant RNA 5d.26 is associated with an exvectorial isolate that evolved during vegetative propagation of a sweet clover plant that was inoculated in 1949. The standard virus population from which genome segment 5 was prepared for comparison with 5d.26 has a quite different history. The standard virus inoculum (RB) prepared in 1972 (Reddy and Black, 1972) was used directly from the freezer to infect AC20 cell monolayers in 1983. WTV genomic RNA was purified from the infected cells after 20 cell passages. Given the different histories of 5d.26 and genome segment 5, it is surprising that no divergence in the guanosine positions near the 3’ -termini was observed for these molecules. Generation of 2d2.05 from genome segment 2 involves the deletion of 15% of segment 2, while 85% of genome segment 5 is deleted to form 5d.26. The partial ribonuclease T, digestion analysis of both remnant RNAs indicated that the deletion boundary is located more than 40 base pairs from either end of the molecules. The finding that both ends of the genome segment are retained when viable remnant RNAs are formed suggests a critical role for the terminal nucleotide sequences in genome replication and packaging. Molecular cloning of cDNA copies of the WTV genome segments and remnant RNAs has been initiated (T. Asamizu and D. L. NUSS,unpublished). By using recombinant DNA technology it should be possible to engineer the WTV genome at the DNA level so as to investigate in detail the role of specific nucleotide sequences in transcription, replication, and packaging of genome segments. The feasibility of such studies is enhanced by the availability of cultured vector cells for the possible selection and amplification of engineered viruses.

E. Resemblance of Exvectorial Isolates toDefective Interfering Particles In several respects WTV exvectorial resemble the defective interferring (DI) particles described for human reovirus. Both the WTV exvectorial isolates and the reovirus DI particles lack certain genome segments (Reddy and Black, 1974, 1977; Nonoyama and Graham, 1970; Nonoyama etal., 1970; Schurech etal., 1974; Spandidos and Graham, 1976; Ahmed and Graham, 1977) and often contain remnant RNAs generated by deletion mutation events (Reddy and Black, 1974; Ahmed and Graham, 1977; Ahmed and Fields, 1981). Furthermore, WTV exvectorial isolates have been reported to interfere with standard virus infection of vector cells (unpublished observations, cited in Reddy and Black, 1977). Both DI particles and WTV exvectorial isolates are transcriptionally active invitro and in vivo (Spandidos etal., 1976; Nuss and Peterson, 1981b; Nuss,

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FIG.9. Polyacrylamide gel electrophoretic analysis of partial and complete ribonuclease T1 digests of [3'-32P]pCp-end-labeled genome segment 2 and remnant RNA 2d2.05. Lane 1, oligonucleotide ladder generated by partial alkali digestion of end-labeled genome segments

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1983a). In the case of WTV the remnants are active as templates for transcription (Nuss, 1983a). The WTV exvectorial isolates also differ from the reovirus DI particles in several important respects. Unlike the DI particles the WTV exvectorial isolates can replicate and cause disease symptoms in the apparent absence of helper virus. That is, isolates lacking detectable genome segment 2 or 5 replicate in the plant host in the apparent absence of virus containing all 12 genome segments (Reddy and Black, 1977; NUSS,1983a). Yet the exvectorial isolates do not replicate in vector cells. Whether the exvectorial isolates would replicate in vector cells upon mixed infection with standard virus remains an important point to be determined. It is conceivable that the exvectorial isolates act as DI particles in the vector cells but not in the plant host. Precedents for host control of DI replication and/or interference exist for other viruses that replicate in both an insect vector and a noninsect host. Sindbis virus DI particles isolated from baby hamster kidney (BHK) cells failed to interfere or replicate in Aedesalbopictus mosquito cells (Stollar etal., 1975). Vesticular stomatitis virus DI particles from A. albopictus cells failed to interfere with standard virus in BHK cells, whereas the DI particles from BHK cells interfered with standard virus infection in both BHK and A. albopictus cells (Gillies and Stollar, 1980). An additional difference between reovirus DI particles and WTV exvectorial isolates lies in the kinetics of appearance of virus populations containing remnant RNAs or lacking specific genome segments. Deletion mutants of reovirus containing remnants of genome segments appear after less than a dozen serial undiluted passages in mouse L cells (Ahmed and Fields, 1981). Subvectorial populations of WTV containing deletion mutants appear within 1to 2 years after infection of the plant host (Reddy and Black, 1974). Recent studies have revealed that remnants are also generated in WTV-infected vector cells but only after extensive subculturing of persistently infected cells, e.g., 150 passages over approximately 1.5 years (A. J. Peterson, M. P. Fox, and D. L. NUSS,unpublished observations). It will be interesting to determine whether the remnant RNAs in the infected vector cells are derived from the same genome segments as the remnant RNAs found in systematically infected plant hosts.

+

4 5. Lane 2, partial digest of genome segment 2. Lane 3, partial digest of 2d2.05. Lane 4, complete digest of genome segment 2. Lane 5, complete digest of 2L2.05. The migration positions of bromphenol blue (BPB) and xylene cyanol (XC) markers are indicated. Gel conditions followed the method of Maxam and Gilbert (1977). (From Nuss, 1983b.)

FIG.10. Polyacrylamide gel electrophoretic analysis of partial and complete ribonuclease T1 digests of [3’-S*P]pCp-end-labeled genome segment 5 and remnant RNA 5d.26. Genome segment 5 was separated from segment 4 by hybrid selection using 5d.26 fixed to nitrocellulose. Lane 1, oligonucleotide ladder generated by partial alkali digestion of end-labeled genome 4 5. Lane 2, partial digest of genome segment 5. Lane 3, partial digest of 5d.26. Lane 4,complete digest of genome segment 5. Lane 5, complete digest of 5d.26. The migration positions of bromphenol blue (BPB) and xylene cyanol (XC) markers are indicated. Gel conditions followed the method of Maxam and Gilbert (1977). The additional band that migrates near the BPB marker in lane 4 represents a 3’4erminal T1 fragment of contaminating genome segment 4.

+

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Recent studies by Fields and co-workers (Ahmed and Fields, 1981; Fields and Green, 1982; Brown et al., 1983) indicate that the biologic properties of reovirus DI particles may result from mutations in segments other than those that are deleted. In this respect Reddy and Black (1974) reported that several exvectorial isolates of WTV had genome patterns indistinguishable from that of a vectorial isolate. It is also interesting that Ahmed and Fields (1981) found interference by DI particles to be associated with a mutated genome segment, S4, which codes for a component (03) of the outer-protein coat of the virus. Clearly more information is required before an understanding of the molecular basis for DI particle generation and mechanisms of action can be achieved. A clear understanding of the loss of transmissibility of WTV may be linked to progress in this area. OF THE PLANT HOST VI. INFECTION

A. Tumor Induction WTV infects at least 55 species of plants (Black, 1965). The morphologic effects of WTV infection and the severity of disease symptoms vary depending on the plant host. Symptoms range from leaf-curling to dwarfing and suppression of flowering (Black, 1965). The most notable symptom, as suggested in the name given this virus, is tumor formation (Fig. 11). The morphologic, histologic, and cytologic symptoms, the origin and development of tumor formation, and the relationship between plant and animal neoplasia have been discussed in detail in numerous excellent reviews, several published recently (Black, 1965,1972,1979,1982; Reddy, 1981). Only information immediately relevant to current attempts to understand the molecular basis of WTV-induced tumorigenesis will be presented here. Most root tumors resulting from WTV infection occur at the emergence points of lateral roots. In considering this phenomenon, Black (1946) pointed out that the formation of a lateral root involves growth through overlying tissue, resulting in the wounding of that tissue as the root emerges. Tumors occur less frequently on stems of infectedplants but are readily induced by mechanical wounding, e.g., pinprick (Black, 1946; Kelly and Black, 1949). The close relationship between wounding and tumor induction led Black (1965) to propose the involvement of a “wound-inducible’’ hormone in tumorigenesis. Clearly, wounding causes many changes in plant metabolism, including changes in the concentration of plant growth regulators (Kahl, 1978; Conrad and Kohn, 1975; Rappaport and Sachs, 1967; Koda,

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FIG.11. Tumors induced in sweet clover by wound tumor virus. (A) Portion of the root system of a WTV-infected plant. The root tumors (RT) are easily distinguishable from nitrogen-fixing root nodules (N). (B) Root and stem portions of a WTV-infected plant, showing stem tumors (ST).

1982). In this regard Black and Lee (1957) demonstrated that application of plant growth regulators on infected plants stimulated tumor induction. Consistent with the wound-hormone hypothesis is the observation that the tumorigenic response of different clones of sweet clover is genetically determined (Black, 1951). That is, optimal tumor response in the WTV-

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infectedplant requires an appropriate genetic background, directing either the inducibility of enzymes involved in hormone synthesis or the response of the plant cells to growth regulators. Little information exists concerning the molecular basis of WTV-induced tumorigenesis. Nevertheless WTV offers considerable potential as a system for investigating the regulation of plant cell growth and differentiation in general and the role of growth regulators in the wound response in particular. Studies of tumorigenesis in the intact plant host suffer from technical difficulties in monitoring the synthesis of viral polypeptides, transcripts, and replication products, as well as in the general manipulation of responding tissue. The availability of sensitive new techniques which employ specific probes, e.g., cloned cDNA copies of viral genetic information, should allow the use of cultured tumor tissue to examine these parameters at a level approximating that already attained with cultured vector cells. Cultured plant tissue also offers the potential for development of an in uitro transformation assay to identify the genome segments responsible for tumor induction and to study the molecular details of the plant cell response to WTV infection.

B. Tissue Culture Studies The bulk of the studies on cultured tissue of WTV-induced tumors was conducted between 1949 and 1955 (Burkholder and Nickell, 1949; Nickell and Burkholder, 1950;Nickell et al., 1950;Nickell, 1950a,b,c, 1951;Brakke and Nickell, 1951,1952, 1955; Gentile and Naylor, 1955) on tissue derived from root tumors of a WTV-infected sorrel plant initiated by L. M. Black in 1944 (Black, 1957). Since nontumor tissue failed to grow under the conditions used to initiate and grow tumor callus (Black, 1957), these studies all suffered from the lack of normal control tissue. Nevertheless, it is clear that the cultured tumor tissue differed from most cultured plant tissue in several ways. A relatively high concentration of phosphate was required for optimal growth of the sorrel tumor tissue (Nickell, 1954). Subsequent studies revealed that tumor tissue derived from sweet clover also required high concentrations of phosphorus for optimal growth, while nontumor sweet clover tissue did not (Nickell, 1955). A most interesting property reported for the sorrel tumor tissue was the ability to utilize soluble starch effectively as a source of carbohydrate (Nickell and Burkholder, 1950). Utilization of starch was shown to be due to the synthesis and secretion of a-amylase (Nickell and Burkholder, 1950; Brakke and Nickell, 1955). Tumor tissue was therefore able to grow well on solid agar in which starch

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was the only source of carbohydrate (Nickell, 1955). While the balance of the information suggests that synthesis and secretion of large quantities of a-amylase is a property unique to the tumor tissue, a firm conclusion on this point must await further studies with appropriate control tissue derived from uninfected plants. WTV tumor callus was initiated and maintained in continuous culture in the absence of added growth hormones (Black, 1957; Streissle, 1971). Growth independent of exogenous growth hormone is a property that WTV tumor tissue shares with tissue derived from plant tumors initiated by Agrobacterium tumefaciens (Braun, 1956). Analysis by transmission studies, immunofluorescence, and electron microscopy indicated that the amount of WTV in tumor tissue decreased with time of culturing to undetectable levels (Black, 1957, 1972; Streissle et al., 1969; Streissle, 1971). Nevertheless, tumor tissue which appeared to be free of virus by these criteria retained the capacity for continuous growth in the absence of added growth hormones. With new tools now available, a reinvestigation into the properties of cultured WTV tumor tissue is warranted. The persistence of WTV genetic information in cultured tumor tissue, in particular, must be examined with the sensitive detection techniques made available by recent advances in molecular biology. Considerable progress has been recently achieved in elucidating the molecular basis of tumor induction by Agrobacterium tumefaciens in crown-gall disease of plants. The genetic information responsible for crown-gall tumors resides on large plasmids (Ti-plasmids) carried by the bacterium (Zaenen et al., 1974; Van Larebeke et al., 1974; Watson et al., 1975). Part of the DNA, T-DNA, is integrated into the plant host genome (Zambryski et al., 1980;Yadav et al., 1980;Thomashow et al., 1980;Chilton et a1.,1980; Willmitzer et al., 1980). Recent studies indicate that the T-DNA codes for products involved in growth hormone metabolism (Leemans et al., 1982; Liu et al., 1982). Furthermore, Ti-plasmid DNA can be used to transform plant protoplasts in uitro to the tumorigenic phenotype, conclusively showing this DNA to be the tumor-inducing principle (Krens et al., 1982). A similar strategy may be useful for identifying the genome segment(s) of WTV responsible for tumor induction. The distinguishing properties reported for WTV tumor tissue, hormone autonomy and growth on starch plates, form the basis for a selection system which can serve as an in uitro assay for WTV-mediated tumorigenesis. The ability to transform cultured plant protoplasts with intact virus or cloned cDNA copies of WTV genome segments would also permit an examination, a t the molecular level, of the response of the plant host to WTV infection.

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CONCLUDING REMARKS

In the decades following the discovery of WTV an impressive body of information accumulated concerning the biology of WTV transmission and tumor induction at the organism and tissue level. During the last decade the emphasis of research on this virus shifted to the molecular level with some degree of success. As a result WTV provides a particularly suitable system for studying the molecular details of virus transmission by insect vectors, as well as a potentially valuable reagent for probing the molecular biology of plant hosts. Several lines of evidence implicate the products of genome segments 2 and 5 as playing a critical role in transmission. The precise nature of that role may be determined by examining the details of the WTV -vector cell interactions that lead to virus internalization and uncoating. Alternative approaches to understanding the molecular details of transmission include experimentation designed to test the ability of standard virus to rescue exvectorial isolates from vector cells upon mixed infection, as well as the ability of exvectorial isolates to interfere with standard virus infection. Such studies may also provide important clues for the eventual understanding of the phenomenon of restricted vector range. WTV multiplies to a high titer in leafhopper cells without adversely affecting host functions, in contrast to the cytopathology that develops when insect or mammalian cells are infected by other Reoviridae. By determining the control points involved in the generation and maintenance of the “regulated” infection of WTV in vector cells, it may be possible to gain insights into the basis of virus-mediated cytopathology. While tumor induction is a general property of WTV infection in most plant hosts, our understanding of the molecular details of WTV-mediated tumorigenesis is meager. Tumors could arise as a result of a viral gene product acting directly at a control point involved in plant cell growth and differentiation. Alternatively, viral gene products could interact with the host genome to reprogram host gene expression, possibly by integrating WTV genetic information, after conversion to DNA, into the host genome. Any working hypothesis must include the role of growth hormones in tumor formation. Several of the possible mechanisms of tumorigenesis are readily amenable to testing, e.g., by probing DNA from tumor tissue for WTV specific sequences. Attempts to examine possible changes in host gene expression after WTV infection require more elaborate experimental designs. In particular, elucidation of the role of growth hormones in WTV tumorigenesis will be hampered by the paucity of available information on growth hormone metabolism and mechanisms of action. Paradoxically WTV may

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provide a useful tool with which to study the regulation of growth hormone function. Cultured vector cells provide the means for efficiently manipulating and studying WTV outside the plant host. This capability, coupled with recombinant DNA technology, provides an exciting range of future experimental approaches for the study of WTV molecular biology. Progress in this area, in turn, will increase the potential of WTV as a tool with which to investigate the molecular biology of its insect vector and plant host.

ACKNOWLEDGMENTS I thank Dr. Lindsay M. Black for assistance in the early stages of establishing the WTV system in our laboratory and for his continued interest in and encouragement of our work. Without his pioneering work and generous advice, this article would not have been written. The contributions of my co-workers, Andrew Peterson, M. Patricia Fox, and Dennis Summers, to our common research effort are gratefully acknowledged. I also thank Nancy Miller for her expert assistance during the preparation of this manuscript. Work described in this review from the author’s laboratory was supported in part by Public Health Service Grant 1 RO1-A117613 from the National Institute of Allergy and Infectious Diseases, PHS/ DHHS.

REFERENCES Ahmed, R., and Fields, B. N. (1981). Virology 111, 351-363. Ahmed, R., and Graham, A. F. (1977). J. Virol. 2 3 , 250-262. Banerjee, A. K. (1980). Microbiol. Reu.44, 175-205. Bils, R. F., and Hall, C. E. (1962). Virology 17,123-130. Black, D. R., andKnight, C. A. (1970). J. Virol. 6,194-198. Black, L. M. (1944). Proc. Am. Philos, Soc. 88,132-144. Black, L. M. (1946). Nature (London) 158,56-57. Black, L. M. (1951). Am. J.Bot. 38,256-267. Black, L. M. (1953). Ann. N . Y.Acad. Sci. 56,398-413. Black, L. M. (1957). J. Natl. CancerInst. (U.S.) 1 9 , 663-685. Black, L. M. (1959). In “The Viruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. 2, pp. 157-185. Academic Press, New York. Black, L. M. (1965). In “Handbuch der Pflanzenphysiologic” (W. Ruhland and A. Lang, eds.), Vol. 15, Part 2, pp. 236-266. Springer-Verlag, Berlin and New York. Black, L. M. (1969). Annu.Rev.Phytopathol. 7,73-100. Black, L. M. (1972). Prog. Exp.Tumor Res.1 5 , 110-137. Black, L. M. (1979). Adu. Virus Res.25,191-271. Black, L. M. (1981). Annu. Reu.Phytopathol. 1 9 , 1-19. Black, L. M. (1982). In “Molecular Biology of,Plant Tumors” (G. Kahl and J. Schell, eds.), pp. 69-105. Academic Press, New York. Black, L. M., and Brakke, M. K. (1952). PhytopathoZogy 42,269-273. Black, L. M., and Lee, C. L. (1957). Virology 3 , 146-159. Black, L. M., and Markham, R. (1963). Neth. J.Plant Pathol. 69,215.

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS

91

Black, L. M., Hills, G. J., and Markham, R. (1962). J . Gen. Microbiol. 28, 1. Black, L. M., Wolcyrz, S., and Whitcomb, R. F. (1958). Int. Cong. Microbiol., Symp., 7th 1958 p. 255. Borsa, J., and Graham, A. F. (1968). Biochem. Biophys. Res. Commun. 33, 896-901. Both, G. W., Lavi, S., and Shatkin, A. J. (1975). Cell 4,173-180. Brakke, M. K. (1951). J. Am. Chem. SOC.73, 1847. Brakke, M. K., and Nickell, L. G. (1951). Arch. Biochem. Biophys. 32, 28-41. Brakke, M. K., and Nickell, L. G. (1952). Bot. Gaz. 113, 482-484. Brakke, M. K., and Nickell, L. G. (1955). AnneeBid. 59 [Ser. 111, 311, 215-226. Brakke, M. K., Vatter, A. E., and Black, L. M. (1954). Brookhaven Symp. Biol. 6,137- 156. Braun, A. C. (1956). Cancer Res. 16, 53-56. Brown, E. G., Nihert, M. L., and Fields, B. N. (1983). I n “Double Stranded RNA Viruses” (D. H. L. Bishop and R. W. Compans, eds.), pp. 275-287. Elsevier, Amsterdam. Bruenn, J. A., and Brennan, V. E. (1980). Cell 19,923-933. Burkholder, P. R., and Nickell, L. G. (1949). Bot. Gaz. (Chicago) 110, 426-437. Chilton, M. D., Saiki, R. K., Yadav, N., Gordon, M. P., and QuCtier, F. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 4060-4064. Chiu, R. J., and Black, L. M. (1967). Nature (London)215, 1076-1078. Chiu, R. J., and Black, L. M. (1969). Virology 37, 667-677. Chiu, R. J., Reddy, D. V. R., and Black, L. M. (1966). Virology. 30,562-566. Cocking, E. C. (1966). Planta 68, 206-214. Cohen, J. (1977). J. Gen. Virol. 36, 395-402. Cohen, J., Laporte, J., Charpilienne, A., and Schemer, R. (1979). Arch. Virol. 60,177- 186. Conrad, K., and Kohn, B. (1975). Phytochemistry 14,325-328. Fields, B. N., and Green, M. I. (1982). Nature (London)300, 19-23. Furuichi, Y. (1974). Nucleic Acids Res. 1, 809-822. Furuichi, Y., and Miura, K.-I. (1973). Virology 55, 418-425. Furuichi, Y., and Miura, K.-I. (1975). Nature (London) 253, 374-375. Furuichi, Y., Morgan, M., Muthukrishnan, S., and Shatkin, A. J. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 362-366. Gentile, A. C., and Naylor, A. W. (1955). Arch. Biochem. Biophys. 58,270-275. Gillies, S., and Stollar, V. (1980). Virology 107, 497-508. Gomatos, P. J., and Tamm, I. (1963). Proc. Natl. Acad. Sci. U.S.A. 50, 878-885. Granados, R. R., Hirumi, H., and Maramorosch, K. (1967). J. Inoertebr. Puthol. 9,147- 159. Hay, A. J., Lomniczi, B., Bellamy, A. R., and Skehel, J. J. (1977). Virology 83, 337-355. Hirumi, H., Granados, R. R., and Maramorosch, K. (1967). J. Virol. 1,430-444. Hsu, H. T., Nuss, D. L., and Adam, G. (1983). I n “Current Topics in Pathogen-Vector-HostResearch” (K. F. Harris, ed.), pp. 189-214. Praeger, New York. Joklik, W. K. (1981). Microbiol.Rev. 45, 483-501. Kahl, G. (1978). “Biochemistry of Wounded Plant Tissue.” de Gruyter, Berlin. Kalmakoff, J., Lewandowski, L. J., and Black, D. R. (1969). J. Virol. 4, 851-856. Kapuler, A. M. (1970). Biochemistry 9, 4453-4457. Kelly, S. M., and Black, L. M. (1949). Am. J. Bot. 36,65-77. Kimura, I., and Black, L. M. (1972). Virology 49, 549-561. Kleinschmidt, A. K., Dunnebacke, T. H., Spendlove, R. S., Schaffer, F. L., and Whitcomb, R. F. (1964). J. Mol. Biol. 10, 282-288. Koda, Y. (1982). Plant Cell Physiol. 23, 851-857. Krens, F. A., Molendijk, L., Wullems, G. J., and Schilperoort, R. A. (1982). Nature (London) 296,72-74. Leernans, L., Deblaere, R., Willmitzer, L., DeGrere, H., Hernalsteens, S. P., Van Montagu, M., and Schell, J. (1982). EMBO J. 1, 147-152.

92

DONALD L. NUSS

Lewandowski, L. J., and Leppla, S. H. (1972). J. Virol. 10,965-968. Lewandowski, L. J., and Traynor, B. L. (1972). J. Virol. 10,1053-1070. Lewandowski, L. J., Kalmakoff, J., and Tanada, Y. (1969). J. Virol. 4,857-865. Li, J . K.-K., Keene, J. D., Scheible, P. O., and Joklik, W. K. (1980). Virology 1 0 5 , 41-51. Liu, H. Y., Kimura, I., and Black, L. M. (1973). Virology 5 1 , 320-326. Liu, S.-T., Perry, K. L., Schardl, C. L., andKado, C. I. (1982). Proc. Natl. Acad. Sci. U.S.A. 79,2812-2816.

McCrae, M. A. (1981). J. Gen. Virol. 55,393-403. Maramorosch, K. (1950). Phytopathology 40, 1071-1093. Martin, S. A., and Zweerink, H. J. (1972). Virology 50,495-506. Maxam, A. M., and Gilbert, W. (1977). Proc. Natl. Acad. Sci. U.S.A. 74,560-564. Muthukrishnan, S., and Shatkin, A. J. (1975). Virology 64,96-105. Nickell, L. G. (1950a). Bot. Gaz. (Chicago) 112,225 - 228. Nickell, L. G. (1950b). Nature (London)166,351-352. Nickell, L. G. (1950~).Am. J. Bot. 37,829-835. Nickell, L. G. (1951). Bot. Gaz. (Chicago)1 1 2 , 290-293. Nickell, L. G. (1954). Brookhauen Symp. Biol. 6,174-186. Nickell, L. G. (1955). Annee Biol. 59 [ser. 111, 311, 107-121. Nickell, L. G., and Burkholder, P. R. (1950). Am. J. Bot. 3 7 , 538-547. Nickell, L. G., Greenfield, P., andBurkholder, P. R. (1950). Bot. Gaz. (Chicago)112,42-52. Nonoyama, M., and Graham. A. F. (1970). J.Virol. 6,693-694. Nonoyama, M., Watanabe, Y., and Graham, A. F. (1970). J. ViroE. 6,226-236. Nuss, D. L. (1983a). In “Double Stranded RNA Viruses” (D. H. L. Bishop and R. W. Compans, eds.), pp. 415 -423. Elsevier, Amsterdam. Nuss, D. L. (198313). In “Plant Viruses.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Nuss, D. L., and Peterson, A. J. (1980). J. Virol. 34,532-541. Nuss, D. L., and Peterson, A. J. (1981a). Virology 1 1 4 , 399-404. Nuss, D. L., and Peterson, A. J. (1981b). J. Virol. 39,954-957. Rappaport, L., and Sachs, M. (1967). Nature (London)214,1149-1150. Reddy, D. V. R. (1981). In “Neoplasma-Comparative Pathology of Growth in Animals, Plants, and Man” (H. E. Kaiser, ed.), pp. 423-429. Williams & Wilkins, Baltimore, Maryland. Reddy, D. V. R., and Black, L. M. (1969). Annu. Reu. Phytopathol. 7,87-92. Reddy, D. V. R., and Black, L. M. (1972). Virology SO, 412-421. Reddy, D. V. R., and Black, L. M. (1973a). Virology 54,150- 159. Reddy, D. V. R., and Black, L. M. (1973b). Virology 64,557-562. Reddy, D. V. R., and Black, L. M. (1974). Virology 61,458-473. Reddy, D. V. R., and Black, L. M. (1977). Virology 80,336-346. Reddy, D. V. R., and MacLeod, R. (1976). Virology 70,274-282. Reddy, D. V. R., Rhodes, D. P., Lesnaw, J. A., MacLeod, R., Banerjee, A. K., and Black, L. M. (1977). Virology 80, 356-361. Rhodes, D. P., Reddy, D. V. R., MacLeod, R., Black, L. M., and Banerjee, A. K. (1977). Virology 7 6 , 554-559. Sakai, F., and Takebe, I. (1974). Virology 62, 426-433. Sarkar, S. (1977). Methods Virol. 6, 435-456. Scherch, A. R., Matsuhisa, T., and Joklik, W. K. (1974). Interuirology 3, 36-46. Shatkin, A. J. (1974). Proc. Natl. Acad. Sci. U.S.A. 7 1 , 3204-3207. Shatkin, A. J., and Sipe, J. D. (1968). Proc. Natl. Acad. Sci. U.S.A. 6 1 , 4162-4169. Shikata, E., and Maramorosch, K. (1965). Virology 27,461-475.

MOLECULAR BIOLOGY OF WOUND TUMOR VIRUS

93

Shikata, E., Orenski, S. W., Hirumi, H., Mitsuhashi, J., and Maramorosch, K. (1964). Virology 23, 441-444. Shimotohno, K., and Miura, K. (1974). J. Mol. Biol. 86, 21-30. Sinha, R. C. (1965). Virology 26, 673-686. Skehel, J. J., and Joklik, W. K. (1969). Virology 39, 822-831. Smith, R. E., and Furuichi, Y. (1980). Virology 103,279-290. Spandidos, D. A., and Graham, A. F. (1976). J. Virol. 20, 234-247. Spandidos, D. A., Krystal, G., and Graham, A. F. (1976). J. Virol. 18, 7-19. Stollar, V., Shenk, T. E., Koos, R., Igarashi, A., and Schlesinger, R. W. (1975). Ann N.Y. ACMJ!. S C ~266,214-231. . Streissle, G. (1971). Colloq. Int. C.N.R.S. 193 (32), 383-411. Streissle, G., and Granados, R. R. (1968). Arch. Gesamte Virusforsch. 25, 369-372. Streissle, G., Rizza, J. J., and Granados, R. R. (1969). Proc. 20thAnnu. Meet. Tissue Culture Assoc. Abstract, p. 58. Takebe, I. (1975a). Annu. Reu. Phytopathol 13, 105-127. Takebe, I. (1975b). Reu. Plant Prot. Res. 8 , 136-153. Takebe, I. (1977). Comp. Virol. 11,237-283. Takebe, I., Otsuki, Y., and Aoki, S. (1968). Plant Cell Physiol. 9, 115-124. Thomas, P. S. (1980). Proc. Nutl. Acud Sci. U.S.A. 77, 5201-5205. Thomashow, M. F., Nutter, R., Postle, K., Chilton, M.-D., Blattner, F. R., Powell, A., Gordon, M. P., and Nester, E. W. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,6448-6452. Tomita, K., and Rich, A. (1964). Nature (London)201, 1160-1163. Tzen, J. C., Somers, J. M., and Mitchell, D. J. (1974). Heredity 33, 132. Van Dijk, A. A., and Huismans, H. (1980). Virology 104, 347-356. Van Larebeke, N., Engler, G., Holsters, M., Van den Elsacker, S., Zaenen, I., Schilperoort, R. A., and Schell, J. (1974). Nature (London)252, 169-170. Verwoerd, D. W., and Huismans, H. (1972). OnderstepoortJ. Vet. Res.39,185-192. Vodkin, M., Katterman, F., and Fink, G. R. (1974). J. Bacteriot. 117,681-686. Walker, D. L. (1964). Prog. Med. Virol. 6, 111-148. Watson, B., Currier, T. C., Gordon, M. P., Chilton M.-D., and Nester, E. W. (1975). J. Bacteriol. 123, 255-264. Willmitzer, L., De Beuckeleer, M., Lemmers, M., Van Montagu, M., and Schell, J . (1980). Nature (London)287,359-361. Wood, H. A,, and Streissle, G. (1970). Virology 40, 329-334. Yadav, N. S., Postle, K., Saiki, R. K., Thomashow, M. F.,andChilton, M.-D. (1980). Nature (London)287,458-461. Zaenen, I., Van Larebeke, N., Tuechy, H., Van Montagu, M., and Schell, J. (1974). J . Mol. Biol. 86, 109-127. Zaitlin, M., and Beachy, R. N. (1974). Ado. Virus. Res.19, 1-35. E. Street, Zaitlin, M., and Beachy, R. N. (1975). In “Tissue Culture and Plant Science” (H. ed.), pp. 265-285. Academic Press, New York. Zambryski, P., Holsters, M., Kruger, K., Depicker, A., Schell, J., Van Montagu, M., and Goodman, H. M. (1980). Science 209, 1385-1391.

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ADVANCES IN VIRUS RESEARCH, VOL. 29

THE APPLICATION OF MONOCLONAL ANTIBODIES I N THE STUDY OF VIRUSES Michael J. Carter’ and Volker ter Meulen lnstitut fur Virologie der Universitat Wurzburg Wurzburg, Federal Republic of Germany

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Consideration of Immunological Techniques Applied to Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Virus Identification . . . . . . . . . . . . . . . . . . . . . . . . . A. Diagnostic Virology . . . . . . . . . . . . . . . . . . . . . . . B. Taxonomy and Epidemiology . . . . . . . . . . . . . . . . . . . C. Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Further Definition of Virus-specific Protein Structure, Function and Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Protein Structure and Function . . . . . . . . . . . . . . . . . . B. Monoclonal Antibodies as Probes for Virus Protein Expression . . . . C. Protein Purification and Quantitation . . . . . . . . . . . . . . . IV. Investigation of Virus Pathogenesis and Protection from Virus Infection . . A. Isolation of Virus Variants and their Pathogenicity. . . . . . . . . . B. Alteration in the Course of Infection . . . . . . . . . . . . . . . . C. Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95 96 99 100 102 104 105 105 112 118 119 119 120 121 122 124

I. INTRODUCTION Since their initial description (Kohler and Milstein, 1975) monoclonal antibodies have made a profound impression on all areas of biological research and biotechnology. The basic property of a monoclonal antibody, that of combining specifically with one epitope (or family of related epitopes), can be used to supply a wealth of data. A single monoclonal antibody can provide information on protein “relatedness,” structure, function, synthesis, processing, cellular or tissue distribution and on the association between molecules. Any attempt to separate the ways in which these data are gathered must be to some extent artificial since one study frequently yields results relevant to other areas. However, it is Present address: Department of Virology, Royal Victoria Infirmary, Newcastle upon Tyne, England. 95

Copyright 0 1984 by Academic Press, Inc. All rights of‘reproductionin any form reserved. ISBK W12-039823-X

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appropriate to differentiate between the broad areas of virological investigation. The contribution of monoclonal antibodies within each area can then be assessed. Thus, diagnostic virology, taxonomy, and epidemiology (Section 11)can be considered separately from the biochemical and molecular biological investigations described in Section 111; Section IV deals with the interaction of virus and host a t the whole organism level. Consequently, the selection of virus variants is dealt with in Section I11 where information concerning protein structure or function has been obtained, and also in Section IV where pathogenesis was investigated. It is not intended in this review to provide an exhaustive list of all publications in which monoclonal antibodies have been used, but rather to illustrate how monoclonal antibodies can be applied in virology. Other reviews of different aspects have recently been published (Antczak, 1982; Blann, 1981; Kennett, 1981;Nowinski et al., 1983; Sinkovics and Dreesman, 1983;Yewdell and Gerhard, 1981). It is important to emphasize that monoclonal antibody techniques are rarely applied in isolation, but are normally backed up with investigations using the more classical biochemical approaches. For instance the recognition of separate active sites for hemagglutination and neuraminidase activity on the Sendai virus HN protein (Portner, 1981) involved experiments using temperature-sensitive mutations and chemical inhibitors as well as competitive antibody binding assays and selection of virus variants, and the proof of relationship between turkey (H1N1) influenza, and swine viruses involved RNA/RNA hybridization and replication studies as well as the observation of monoclonal antibody cross reactions (Hinshaw et al., 1983). Furthermore, there are limitations to the information that antibody studies can provide; structural studies for instance ultimately depend on the orientation of an antibody binding site on the various structural levels of the protein, amino acid sequence, three-dimensional structure, and even intermolecular associations (Section 111).

General Consideration of Immunological Techniques Applied to Monoclonal Antibodies The principle of monoclonal antibody production, that of fusing a short lived antibody-producing cell derived from an immunized animal with a permanent myeloma cell line, is well known. This has been recently reviewed (Gerhard et al., 1980). It is, however, necessary to consider the production of the immunogen, since it may be necessary to enrich for, or preferentially expose, a particular antigen. Thus, in the production of measles virus-specific antibodies (Bohn et al., 1982),heating of the antigen in the presence of 1%sodium dodecyl sulfate (SDS) led to the derivation of

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antibodies specific for the matrix (M) protein. Similar treatment in the presence of reducing agents favoured the production of antibodies directed to the phosphoprotein (P) and nucleocapsid protein (N). If the protein against which monoclonal antibodies are desired is sufficiently immunogenic, it is not necessary to use pure preparations. Whole cell antigen was used in the production of antibodies directed against respiratory syncitial virus (RSV) (CGte etal., 1981) and against polyoma virus T antigen (Dilworth and Griffin, 1982). Also, if cross reacting, e.g., group specific antibodies are required, it is possible to perform the primary inoculation with one antigen, and the boosting inoculation with the cross reacting antigen (Gerhard etal., 1978). It is not appropriate to consider here the immunological techniques used in detail, but the interested reader is referred to a review of techniques used in conjunction with polyclonal sera (van Regenmortel, 1981). However, it is relevant to consider the limitations of these processes when applied to monoclonal antibodies. First, the majority of monoclonal antibodies dc not cause immunoprecipitation of the antigen with which they combine. This may be related to the fact that only one epitope is recognized per molecule. Consequently, extensive cross linking and stabilization of immune complexes is not possible. Therefore, immunoprecipitation by monoclonal antibodies requires the addition of a cross-linking agent such as staphylococcus protein A or antiimmunoglobulin. The antigen is only retained by one binding site and only the most strongly combining monoclonal antibodies will succeed. This problem may be diminished in the case of a multimeric protein. Second, most immune precipitation procedures involve the use of detergent in the precipitation buffer. This is necessary to prevent nonspecific contamination of the precipitate. However, this detergent will have some destabilizing effect on protein structure. Monoclonal antibodies are often sensitive to minor conformational change (Section II1,B) and consequently may be rendered nonreactive in this test. It may be necessary to experiment with detergent type and concentration in order to achieve satisfactory precipitation. The specificity of monoclonal antibodies, compared with hyperimmune serum, is illustrated in Fig. 1. The adverse effect of alterations in protein conformation may be aggravated in western blot procedures where antigens are often totally denatured for separation on sodium dodecyl sulfate (SDS)polyacrylamide gels before testing with antibody (Towbin etal., 1979). However, the method does have applications (Braun etal., 1983;Roseto et al., 1983). Immune precipitation is not suitable for quantitation of antigens. This type of information is extracted from radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). These processes are exten-

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sively applied during the initial screening for successful production of monoclonal antibodies, in diagnostic virology (Section I1,A) or in a modified form in competitive binding assays (Section 111). These procedures score only the combination of antibody and antigen and give no information on the nature of that antigen. Detergent conditions are frequently milder than those used in immunoprecipitation, but the attachment of antigen to the solid support necessary in many cases for these tests can itself result in conformational alteration (Bruck et al., 1982a). Information on the histological and intracellular location of antigen is obtained from techniques such as immune fluorescence (IF) and immune electron microscopy (IEM) using electron-dense or enzymatic markers (reviewed by van Regenmortel, 1981). These techniques have been widely used in diagnostic virology (Section II,A) as well as in studies of viral protein synthesis and virus maturation (Section 111,B). In addition to the traditional techniques of serology, neutralization (NT),hemagglutination inhibition (HI), complement fixation (CF), and hemolysin inhibition (HLI) have been extensively employed. In the following it is shown how application of these techniques in experiments utilizing monoclonal antibodies can be made in the study of all areas of virological research.

11. VIRUSIDENTIFICATION The reaction of a single antibody with an epitope is characterized by the equilibrium constant of the combination; this is reflected in the binding assays described above (Section I). Although any one antibody is elicited by the exposure of an epitope to the immune system, it will react with many other epitopes to a greater or lesser extent, depending on the degree of fit between the would-be target site on the antigen and the combining site on the antibody itself. In practical terms the epitope must possess some form of similarity to the parent epitope before any combination at all is detectFIG.1. Specificity of monoclonal antibodies illustrated by radioimmunoprecipitation. Vero cells were infected with measles virus Edmonston and labeled with [35S]methionine. Lysates were prepared and used in immunoprecipitation experiments. Proteins immunoprecipitated by track 1, rabbit hyperimmune antimeasles serum; track 2, rabbit preimmune control serum; tracks 3-5, monoclonal antibodies specific for the H protein; tracks 6-8, monoclonal antibodies specific for the Nprotein; tracks 9- 16, monoclonal antibodies specific for the matrix protein; track 17, control monoclonal antibody, raised against coronavirus antigen; track 18, total infected cell lysate without immunoprecipitation. Note that the antibodies used in tracks 6,7, and 8 are strongly mutually competitive in radioimmunoassay competitive binding experiments, but only one is also active in the immunoprecipitation reaction. Reproduced with permission from Carter etal. Nature(London) 1983.

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able in the binding assays. This specificity of reaction provides the basis for the detection of subtle differences between the gene products of related viruses and thus the differentiation of one virus from another. An alteration in the efficiency with which any one antibody combines with its target constitutes only a small part of the overall reaction of polyclonal antiserum with antigen and is consequently not detectable. The use of antibodies with uniform specificity thus permits very fine analysis of epitope/antibody interactions and therefore of structural alteration in the target molecule. Indeed this approach greatly exceeds conventional serology in specificity, and has led to the recognition of differences between viruses previously thought identical. Monoclonal antibodies are capable of detecting differences of one amino acid, formerly only demonstrated by recourse to peptide fingerprinting or oligonucleotide mapping of the viral genome. This approach has therefore revolutionized the techniques of virus identification, providing in many cases a simplification in the test procedure and yielding a more rapid result. Used in this way, monoclonal antibodies find application in two main areas, first, in the field of rapid diagnosis of virus disease in man, animals, and plants, and second, in the extension of virus taxonomy. These investigations could lead to more effective vaccination programs or disease treatment, and to a greater understanding of the relationships between viruses, their evolution, and epidemiology.

A. Diagnostic Virology Rapid and accurate diagnosis of viral infection is of obvious importance to the clinician and to public health. Wands etal. (1981)have summarized the criteria for selection of a monoclonal antibody for use as a diagnostic reagent as follows: 1. High affinity for antigen, both to permit efficient combination with low concentrations of antigen and to more efficiently displace host antibodies which may already coat primary biopsy samples. 2. It may be directed toward an area of the molecule not normally recognized efficiently by the host’s own antibodies, and not therefore masked. 3. The antibody should be directed against an often repeated antigenic determinant that is readily accessible, e.g., a virus coat protein. 4. Multivalence: this may increase sensitivity, and IgM was found more effective than IgG.

If the antibody is not carefully chosen, it may give less satisfactory results than usual serological procedures (Phillips etal., 19821, and cross reaction

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with host cell polypeptides should be carefully excluded (Fujinami etal., 1983). The application of monoclonal antibodies in diagnosis can be illustrated by reference to the following viruses; herpes, hepatitis B, rabies, flaviviruses, picornaviruses, and influenza; this list is not exhaustive. Cytomegalovirus is a human herpesvirus (human beta herpesvirus 5) which has gained prominence in recent years following latent virus reactivation and disease in immunosuppressed, organ transplant recipients or patients with some other immune deficiency syndrome (Peterson etal., 1980; Thomas etal., 1975a,b). Recently monoclonal antibodies have been developed which are directed against virus structural polypeptides (Goldstein etal., 1982; Pereira etal., 1982~).Some of these have proven able to detect CMV infection in biopsy specimens by the IF test (Goldstein etal., 1982). Antibodies specific for herpes simplex viruses types 1and 2 (Balachandran etal., 1981, 1982b; Pereira etal., 1982b; Showalter etal., 1981) have also been used to type these viruses directly in cells from herpetic lesions (Balachandran etal., 1982a; Peterson etal., 1983). These tests yielded identical results to the more time consuming process of virus isolation and typing by restriction endonuclease mapping, and results were superior to those obtained by standard serology. A similar procedure has also proved successful in the identification of varicella zoster (human herpesvirus 3) in cells obtained from vesicular skin lesions (Forghani etal., 1982)’and monoclonal antibodies are now available which could be useful for typing Epstein -Barr virus (human gamma herpesvirus 4) (MuellerLantzsch etal., 1981). Herpes viruses can also be distinguished by a rapid enzyme immune filtration test (Richman etal., 1982). Marek’s disease (MD) virus is a herpes virus of chickens normally diagnosed by reference antisera directed against the Marek’s associated tumor surface antigen (MATSA). Monoclonal antibodies have been obtained (Lee etal., 1983b) specific for MATSA, andused with success in the identification of Marek’s disease in chickens (Lee etal., 1983a). Use of this test should help prevent confusion with lymphoid leukosis, since both diseases produce similar symptoms and are of economic importance. Monoclonal antibodies produced against hepatitis B virus (HBV) (Wands etal., 1981) have been used to obtain a similar improvement in sensitivity over conventional diagnosis. The application of monoclonal antibodies in RIA was found to detect HBV surface antigen even in patients who were negative by conventional testing (Wands etat., 1982). This was subsequently confirmed by the demonstration that the new test detected antigen - antibody complexes invisible by the former procedure. These complexes were shown to be virus related by DNA hybridization techniques (Shafritz etal., 1982). All four serotypes of Dengue virus can be distinguished by monoclonal

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antibodies (Gentry etal., 1982; Henchal etal., 1982) and have been applied with success to the identification of fresh low passage, virus isolates in an IF test (Henchal etal., 1983). This was found just as reliable, but faster, than the usual plaque reduction tests. Influenza A and B viruses could be readily distinguished and, in some cases, typed satisfactorily even when there was insufficient virus present to permit hemagglutination inhibition testing (Schmidt etal., 1982). Finally monoclonal antibodies may find widespread future use in the diagnosis of plant virus disease. Recent work with antibodies specific for tobacco mosaic virus was able to distinguish two orchid strains which were previously thought identical with the tabomavirus type strain (Briand et al., 1982). The further use of monoclonal antibodies in the wider aspects of diagnosis has been reviewed (Nowinski et al., 1983).

B. Taxonomy and Epidemiology Further application of monoclonal antibodies to virus identification has been made in the fields of virus epidemiology and taxonomy. In this manner the antibodies permit a fine analysis of intervirus relationships at both structural and biological levels. Monoclonal antibodies have been used to compare vaccine strains of yellow fever virus (YFV) derived in different laboratories. This confirmed the required similarity between vaccines in use (Monath etal., 1983) and these strains could also be differentiated from virulent field isolates (Schlesinger etal., 1983). A similar comparison of wild type polio virus andvaccine strains, Saukett and Sabin, has also been conducted (Crainic et al., 1981; Ferguson et al., 1982; Humphrey etal., 1982;Minor et al., 1982;Osterhaus etal., 1981a). The use of monoclonal antibodies in the study of rabies infection has provided an important insight into the mechanism of virus pathogenicity (Section IV) and into the variation between virus strains. Many workers have compared field isolates using monoclonal antibodies directed against the virus glycoprotein and nucleocapsid protein. In this way differences have been found between viruses previously considered identical (Blancou etal., 1982; Charlton et al., 1982; Digoutte, 1982; Schneider, 1982; Witkor and Koprowski, 1980;Wiktor etal., 1980). This may have an important bearing on instances in which vaccine failure has been observed. Variation in the nucleocapsid protein is probably not so important in this respect (Charlton et al., 1982). Such differences also exist between morbilliviruses (Giraudon and Wild, 1981a,b; Server etal., 1982; ter Meulen etaL, 1981). In addition, monoclonal antibodies have permitted analysis of the similarities and differences between hapadnaviruses (C8te etal., 1982),

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parvoviruses (Burtonboy etal., 1982; Parrish etal., 1982), rotaviruses (Appleton and Letchworth, 1983; Gary etal., Greenberg etal., 1983),bunyaviruses (Gonzalez-Scarano etal., 1982) and to identify Zinga virus as Rift Valley fever (Meegan etal., 1983). At the present time the virus best characterized with monoclonal antibodies is that of influenza. The three-dimensional structure has been determined for both the hemagglutinin (HA) (Wilson etal., 1981) and neuraminidase (NA) (Varghese etal., 1983; Section III,A,3). The maintenance of influenza within the human population is related to two mechanisms. First, exchange of the hemagglutinin gene with one derived from an animal virus accomplishes a complete antigenic change (antigenic shift) in the virus and permits widespread human infection. Preexisting immunity is thus overcome and a worldwide pandemic may result. In the periods between antigenic shift, a more gradual alteration occurs in the virus proteins (antigenic drift) which in some cases may permit reinfection of the host population. Monoclonal antibodies have naturally been applied to these most interesting phenomena in an attempt to demonstrate the relationships between human, animal, and avian virus species, and also to demonstrate antigenic drift both in the field and the laboratory. Monoclonal antibodies provide the sensitivity required to analyze subtle structural alterations and are therefore well suited to the task. The seal influenza virus A/seal/Massachusetts/l/80 (H7N7) has been compared with viruses of avian origin (Kida etal., 1982). A close relationship was noted between hemagglutinin molecules from those sources suggesting avian viruses might have played a role in the evolution of the seal virus strain. In other studies differences have been noted between 25 avian (H4) influenza strains which may be related to the original host (Fukushi etal., 1982) and differences have been observed between neuraminidase (NA) molecules (Holmes etal., 1982). Antigenic drift has been demonstrated among field isolates of influenza A (Underwood, 1982) and B (Webster and Berton, 1981). In addition nonneutralized HA or NA variants are readily selected by using monoclonal antibodies to apply selection pressure (Natali etal., 1981; Webster etal., 1982). This demonstrates how such a process might occur under immunological pressure applied by the host. At least some of these laboratory selected variants might have been able to spread in the population since they were not neutralized by many samples of sera from potential hosts which did recognize the parent virus (Natali etal., 1981). In contrast to the ready demonstration of variability among the influenza viruses, tick-borne encephalitis virus (TBE) isolates have remained very similar over the last 26 years (Heinz etab, 1982). Ready differentiation between viruses permits analysis of virus spread

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within the population. At least two variants of influenza B could coexist in the population (Webster etal., 1981). The respective epidemiologies of herpes simplex virus 1and 2 have also been studied and one patient may suffer from both viruses simultaneously (Peterson etal., 1983). Monoclonal antibodies have permitted the analysis of mixed infections with Thus, interference and exclusion were demclosely related viruses in uitro. onstrated between Dengue virus types 2 and 3, but a small proportion of cells could be doubly infected (Dittmar etal., 1982). The use of these reagents permits the identification of the polypeptides carrying virus taxonomic markers (group, type, etc.) as demonstrated by Simian agent 12 (Benton etal., 1982), adenovirus 5 (Russell etal., 1981), and herpes virus (McLean etal., 1982;Showalter etal., 1981). This type of analysis could also indicate polypeptides which have been structurally conserved during the course of virus evolution.

C. Limitations The advantges of a technique which permits rapid and efficient virus identification are enormous. However, it is essential that the monoclonal antibody is chosen with care. Many will recognize carbohydrate determinants of glycosylated polypeptides, and this modification is influenced by the host cell in which the virus is grown (Klenk and Rott, 1980). At least one instance is known where the type-specific target present on HSV glycoproteins synthesized in Hep-2 cells was not reactive when the virus was grown in Vero cells (Pereira etal., 1981). Second, the ability to detect differences is influenced by the technique used (Kendal etal., 1981). Binding of monoclonal antibody to antigen may or may not disrupt the biological function of that protein; this could lead to errors if the tests involved monitor antibody binding indirectly by inhibition of target protein function. For this reason, procedures which simply demonstrate antibody binding are to be preferred in diagnostic virology. Immunofluorescence is the method of choice for this purpose since it is rapid and relatively simple. Furthermore, it is applicable directly to biopsy samples and provides additional information on the histological distribution of the antigen examined. However, binding alone is an insufficient guide for research purposes, since the functional significance of the same epitope may vary with the virus strain (ter Meulen etal., 1981) and mutations may render an antibody unable to inhibit protein function without abolishing antibody binding (Kendal etal., 1981). It is necessary therefore to perform a number of tests in order to extract the maximum possible information.

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111. FURTHER DEFINITION OF VIRUS-SPECIFIC PROTEIN STRUCTURE, FUNCTION, AND SYNTHESIS Monoclonal antibodies provide us with a tool which is able to examine the individual protein within a structure as complicated as the whole cell. It is possible to investigate, both in uitro and in viuo, the role of a protein. It is also possible to determine the active areas of the molecule and to monitor its synthesis and processing within the infected cell. The ability to distinguish related proteins can be used to provide a genetic marker in recombination experiments (Gonzalez-Scarano et al., 1982).

A. Protein Structure and Function The combination of antibody with antigen can be used to provide structural information on several levels. First, just as it is possible to identify proteins carrying a taxonomic marker, so it is possible to identify a protein as performing a particular function once an antibody is available which is capable of inhibiting that function. Second, antibodies binding to the same target protein differ in their capacity to produce function inhibition of that protein. A panel of monoclonal antibodies is frequently divided into groups according to their effects on the activity of a target protein, or family of proteins. In this way epitopes may be assigned a functional significance. If certain assumptions are made, it is then possible to conduct competitive binding analyses to arrange the antibody sites relative to each other, and a schematic map of the molecule’s surface can be drawn up. Frequently virus variants can be selected to which a given antibody can no longer bind. These may also be analyzed for loss of protein function or loss of capacity to bind other monoclonal antibodies. In this way the map can be refined, or indeed constructed entirely. Third, in order to explain function in terms of structure it is necessary to orientate the map thus obtained with the physical structure of the antigen. This can be done by determination of the amino acid alterations produced in the variants described above, either directly by partial proteolysis and examination of those molecule fragments bound by the antibody, or from genome sequence determination in the region of the alteration. Active amino acid residues can thus be distinguished and positioned in the primary structure of the protein. The location of the antibody binding site is then known if the three-dimensional structure of the protein has been determined, or clues may be obtained from immune electron microscopy (IEM). The whole process described above has met with varying success in application to a number of viruses. The most complete of such determina-

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tions has been derived in the study of the influenza virus hemagglutinin and neuraminidase. 1. Identification of the Functional Protein

The target protein of a monoclonal antibody is usually detected by radioimmune precipitation or western blot procedures (Section I). In this manner, those proteins to which neutralizing antibodies are directed have been determined, e.g., VP1 in poliovirus (Osterhaus et al., 1981b;Thorpe et al., 1982), VP2 in Bluetongue virus (Appleton and Letchworth, 1983; Letchworth and Appleton, 1983),and gp 56in Venezuelan equine encephalitis virus (Roehrig et at., 1982a). The glycoprotein GP-1 of the coronavirus MHV was proven responsible for cell attachment and virus spread through cell/cell fusion (Collins et al., 1982). A modified immunoprecipitation experiment has demonstrated that a subset of the SV40 T antigen present in the infected cell is able to bind to the virus origin of DNA replication. The antibody used in this work was apparently capable of distinguishing between T antigen with this activity and T antigen which could not perform this function (Scheller et al., 1982). A similar analysis has identified the cellular RNA polymerase which binds to the adenovirus 2 promotor in vitro (Dahmus and Kedinger, 1983), and microinjection techniques have demonstrated that antibody specific to the transformation related protein p53 in 3T3 cells is capable of preventing the seruminduced onset of DNA synthesis (Mercer et al., 1982). Although the last two experiments are concerned with the function of cellular proteins, this approach has an obvious application in the study of virus replication and transcription. Such an approach could also investigate the involvement of host -cell proteins in these processes. Further insight into the function of tumour virus proteins in transformed cell survival has come from work involving monoclonal antibodies specific for p15 E, a structural component of type C retroviruses. This protein was detected in cancer effusions and found to be highly efficient a t blocking monocyte response to chemoattractants, a function which could protect a cancer from attack (Cianciolo et al., 1981). 2. Differentiation of Active Areas on the Protein

This analysis seeks to define active areas of the protein in terms of epitopes recognized by a panel of monoclonal antibodies. In the main these experiments have been conducted on virus surface proteins which have easily monitored biological functions such as hemagglutination, neutralization, hemolysis, or neuraminidase activity. In principle, the same approach could be extended to any protein with a measurable activity. Combination of antibody with antigen can have a variety of effects, de-

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pending on the site of attachment. Some functions which might previously have been thought associated, have been found separable when highly specific monoclonal antibodies were used. A polyclonal monospecific serum which coats the protein surface with antibody will normally inhibit all protein functions, but a monoclonal antibody, which binds away from the active site need not have any detectable effect at all (Burstin etal., 1982; Massey and Schochetman, 1981; Pinter etal., 1982; Thorpe etal., 1982). By means of this function inhibition analysis, a panel of monoclonal antibodies specific for the measles virus hemagglutinin was divided into five groups (ter Meulen etal., 1981): group 1antibodies had no detectable activity, in HI or N T assays; group 2 exhibited HI activity but failed to neutralize virus; group 3 consisted of antibodies with similar activities in both tests. Antibodies of group 4 revealed a significantly higher activity in the HI than the N T tests whereas this was reversed in group 5. The antibodies used in that study were deliberately selected from those which had undetectable HLI activities. This report therefore documents the separation of hemagglutination (a model for virus attachment to its target cell) from hemolysis (a model for virus penetration by fusion reactions). This indicates that the requirements underlying these reactions must differ. Both of these tests in fact are artificial, since the virus does not normally act to cross link cells, or to lyse the cell it is about to attack, and these results indicate that it is possible to prevent stable cell cross linking in circumstances which still permit sufficient virus/cell association for action of the fusion protein. Similarly, antibodies may inhibit hemagglutination but still permit sufficient cell contact such that infection is not inhibited (Burstin etal., 1982; Gentry etal., 1982;Gonzalez-Scarano etal., 1982; Kimura-Kiroda and Yasui, 1983; ter Meulen etal., 1981. This type of analysis provides clues that the functional areas of a multifunctional protein are distinct. For instance the paramyxovirus HN protein has two activities, cell attachment and neuraminidase. These probably reside at different areas of the molecule since some monoclonal antibodies inhibit only neuraminidase (Orvell and Grandien, 1982), and virus variants have been obtained under monoclonal antibody selection pressure which are deficient only in neuraminidase (Portner, 1981). In the same way, antibodies are known which neutralize virus without blocking hemagglutination. Presumably these antibodies bind to sites important in virus penetration but not absorption. This phenomenon is exemplified by Sendai virus (Miura etal., 1982), influenza virus (Kida etal., 1982), Sindbis virus (Chanas etal., 1982a), and reovirus (Burstin etal., 1982). Other biological functions have been used to characterize epitopes defined by competition ELISA on the envelope glycoprotein of bovine

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leukemia virus (Bruck etal., 1982a). This analysis concentrated on BLV/ VSV pseudotype formation inhibition, polykaryotosis inhibition (cell/cell fusion following infection), and complement fixation (Bruck etal., 1982b). Monoclonal antibodies directed to virus surface proteins are generally more readily produced, but as more become available specific for virus internal proteins, this approach can be expected to yield further information on their function. A function inhibihon assay has been applied to delineate that area of the SV40 T antigen involved in ATPase activity (Clark etd., 1981)and to demonstrate that some areas of the influenza and VSV nucleocapsid proteins may be associated with the transcription process (De etal., 1982; van Wyke etd., 1981). 3.Correlation of Antibody BindingSites withProtein Structure

Once epitopes are identified it is necessary to determine their topological relationship to each other on the surface of the molecule. This is conveniently achieved by a competitive binding analysis although selection of variants has also been extensively used. Competitive binding is frequently examined in RIA or ELISA. One monoclonal antibody carries the appropriate marker and unlabeled antibodies are assessed for their capacity to inhibit the combination of the labeled antibody with antigen. If competition is observed, the antibodies are said to belong to the same binding group. However, several precautions must be taken in the interpretation of data before meaningful conclusions can be reached. It is assumed that two antibodies are mutually competitive if their binding sites overlap. The binding site of a second antibody is then wholly or partially masked by the binding of the first antibody and binding is truly competitive in nature. However, this is not the only explanation for such an observation. Binding sites may be distinct but close together, such that the bulk of the first antibody molecule sterically prevents access of a second to its binding site. These two possibilities cannot be readily distinguished but do at least both imply proximity of attachment sites. In fact, a monoclonal antibody bound to a single epitope on its target protein may have considerable “swivel” movement available to it rendering steric effects relatively unimportant. Stone and Nowinski (1980) reported that two IgG molecules (MW 150K)could bind noncompetitively to a retrovirus 15K protein. However, immunoglobulin class may well be important here since oligomeric IgM would obviously provide a greater steric hindrance than IgG. If steric effects are suspected they may be minimized by the use of monomeric antigen-binding submolecular antibody fragments. It is however quite possible that allosteric inhibitory effects might also occur which would be indistinguishable from true competitive binding.

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Indeed positive allosteric effects have been observed and antibody pairs identified which bind synergistically (Lefrancois and Lyles, 1982a,b; Lubeck and Gerhard, 1982). Similar effects resulting in competition may be widespread but are difficult to demonstrate. Combination of monoclonal antibody with influenza neuraminidase modifies the reaction kinetics (Mountford etal., 1982) and combination with HSV glycoprotein can alter the pattern obtained by partial proteolysis (Eisenberg et al., 1982a). These effects could also be mediated by antibody-induced conformational change. Finally, two antibodies directed a t the same site will compete in a manner related to their relative avidities or concentrations. Taking an extreme case, a weakly binding antibody would not show detectable competition with a labeled, strongly binding immunoglobulin, but the reverse is clearly not true. In practice a spectrum of relative interference efficiencies will probably be obtained (Lefrancois and Lyles, 1982a,b; Stone and Nowinski, 1980). The extent of interference obtained will be influenced by the antibody avidity as well as the proximity of the binding sites. For this reason binding sites should not be distinguished on the basis of the extent of competition, it is the observation of competition itself which is significant. Furthermore, while the observation of competition assigns two antibodies to the same binding group, it is not sufficient to separate them merely on the basis of failure to compete. Any such noncompeting antibodies should be examined in the reverse experiment (i.e., with the other partner carrying the marker) to exclude avidity effects as described above. If this procedure fails to reveal competition, then allocation to separate binding groups is justified. Alternatively, determination of antibody avidity may obviate the need for this analysis but one-way competition effects can still occur even in the case of antibodies of similar avidity and concentration (Carter etal., 1982). The reasons for this are unknown but might involve some form of allosteric effect. Determination of antibody avidity may be performed by a rapid method (Jackson etal., 1983) or relative antibody avidities may be estimated from RIA saturation curves (Carter etal., 1982; Massey and Schochetman, 1981; Stone and Nowinski, 1980). The competitive binding procedure has been applied with success to many viruses. Monoclonal antibodies specific for the measles virus hemagglutinin were grouped into five sets on the basis of their ability to influence the biological functions of that molecule (ter Meulen etal., 1981) and these were then sorted into three binding groups by competition radioimmunoassay (Carter etul., 1982). A similar approach has produced a detailed map of the VSV G protein including epitopes instrumental in virus neutralization in which determinants were identified as cross-reacting or

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specific to the strains Indiana or New Jersey (Lefrancois and Lyles, 1982a,b; Volk et al., 1982). Japanese encephalitis virus (Kimura-Kiroda and Yasui, 1983) and RNA tumor viruses (Bruck et al., 1982a,b; Massey and Schochetman, 1981; Niman and Elder, 1982; Pinter et al., 1982) have also been extensively analyzed. The binding group analysis forms a sensitive probe for alterations in protein surface structure (Carter et al., 1983). The manner in which the sample is prepared can also influence the conformation and/or exposure of the reacting epitopes and this should be considered in the design of such experiments (Bruck et al., 1982b). The second approach to the problem of arranging binding sites relative to each other is to select virus variants under monoclonal antibody selection pressure. In the case of neutralizing antibodies this is relatively straightforward. Nonneutralizing antibodies could also be applied to enveloped viruses if used in conjunction with complement. Virus variants may then be isolated to which the selecting antibodies can no longer bind, or in which antibody combination no longer results in neutralization. These viruses are then tested with a panel of monoclonal antibodies to determine which other antibodies also show altered binding characteristics. In this way, a map of epitopes can be constructed (Yewdell and Gerhard, 1981). The virus variants are commonly already present in the population at a frequency of (Emini et al., 1982; Portner et aL, 1980;Prabhakar et al., 1982;Webster et al., 1982)which presumably represents the error rate in virus transcription. This results in amino acid substitutions either within the antibody binding site or at a position which influences the structure of the binding site. This type of experiment provides a finer analysis of the relationships between binding sites because steric hindrance is eliminated. Variant selection and mapping has been applied in the main to the HA and NA proteins of influenza. The extremely detailed studies performed by Laver et al., (1979,1980a) and Gerhard et al., (1981) exemplify this approach. However, regardless of the method in which epitope topography is determined, the schematic map thus produced must be oriented on the physical structure of the protein and this necessitates a detailed knowledge of the molecule. Two approaches have been used to achieve this objective. First, antibody binding sites can be localized to a particular peptide fragment following partial proteolysis, and second, the amino acid substitutions occurring in the antibody-selected variant proteins can be determined. Niman and Elder (1982) have conducted a comprehensive analysis of the Rauscher virus gp 70. Products of partial proteolysis were identified by reactivity with antisera directed against synthetic polypeptides predicted from the gene sequence. The other polypeptides were assigned by peptide map-

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ping. In this way a linear proteolytic leavage map could be derived for 8 different enzymes. Monoclonal antibodies were then analyzed for their ability to immunoprecipitate various polypeptide fragments and their binding sites could then be assigned in the molecule’s primary structure. It was found that antibody binding sites were closely grouped in two areas which presumably reflected regions of the protein exposed for immunological attack. A similar analysis located eight epitopes, previously defined by their reaction with a panel of monoclonal antibodies, in the proteolytic cleavage map of gp 70 from murine leukemia virus (Pinter etal., 19821,and antibody sites have also been localized on gp 51 of bovine leukemia virus, a 15K molecular weight fragment contained areas involved in biological activity (Bruck etal., 1982b). Similarly, a herpes simplex virus glycoprotein type common determinant has been localized to a 12K fragment of a larger (38K) peptide which also contained two type-specific determinants (Eisenberg etal., 1982a). Other studies have located the active site of SV40 T antigen ATPase by means of well characterized deletion mutants. It was found that enzyme activity and antibody binding required the region of the A gene from 0.37 to 0.29 map units (Clark etal., 1981). Provided the primary sequence of the molecule is known, virus variants can supply highly specific information on the location of antibody binding sites. Unlike antibody binding to a peptide fragment, however, it is not possible to be absolutely certain that the amino acid affected actually constitutes part of the binding site itself. This method has been extensively applied in the study of influenza. Amino acid alterations can be detected by peptide mapping (Laver etal., 1979, 1980b) or genome sequencing (Caton etal., 1982). Since the three-dimensional structure of the influenza hemagglutinin is now available (Wilson etal., 1981) these binding sites could be easily located (Wiley etal., 1981). Analogy with this model has permitted the location of such sites on other strains of influenza (Caton etul., 1982; Jackson etal., 1982). These studies have revealed the presence of distinct areas on the HA molecule within which the amino acid substitutions predominantly occurred, These areas were also implicated in the production of antigenic variation associated with the process of antigenic drift (reviewed by Ward, 1981). The three-dimensional structure of the influenza virus NA molecule is now known (Varghese etal., 1983) permitting the location of monoclonal antibody binding sites to be identified (Colman etal., 1983). This also provided insight into those areas of the molecule involved in enzyme function and previously identified by monoclonal antibodies (Jackson and Webster, 1982) as well as locations in which antigenic drift could occur. This level of refinement in our knowledge of structure, function, and anti-

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genicity has so far been reached only in influenza but a similar approach has located the neutralization sites of poliovirus type 3. This was found to be a small (eight amino acid) sequence in protein VP1 (Evans et al., 1983; Minor et al., 1983);however no three-dimensional structure is yet available for this virus. A further clue to antibody binding site location may be obtained from immune electron microscopy in which the antibody is used in conjunction with an electron-dense material such as ferritin or hemocyanin (Gonda et al., 1981). Chanas et al. (1982a) demonstrated that hemagglutination inhibiting monoclonal antibodies attached near the tip of the Sindbis virus peplomer, while hemolysin inhibiting antibodies attached lower down, near the virus membrane. A similar situation has also been demonstrated in influenza (Webster et al., 1981) and may explain the separation of hemagglutination inhibition and neutralization exhibited by monoclonal antibodies specific for the HA protein of seal influenza (Kida et al., 1982).

B. Monoclonal Antibodies as Probes for Virus Protein Expression In just the same way as monoclonal antibodies provide a sensitive probe for virus detection in diagnosis (Section I1,A) so they can also be used to detect low amounts of individual virus proteins within the infectedcell. In this way they can answer questions of molecular and cell biology. They can thus provide information concerning temporal and spatial separation of protein formation and accumulation, and data on protein modification and processing in the infected cell. 1. Protein Synthesis in Persistently Infected and Transformed Cells

Many studies have been performed to demonstrate temporal control of protein synthesis within the lytic virus infection, e.g., Sendai virus (Orvell and Grandien, 1982; Kristensson and Orvell, 1983) and measles virus 1982). It is not the object of this review to list all these (Norrby et al., studies. Rather we shall consider persistent infections where normal protein synthesis is modified or controlled such that virus and cells may coexist. Interesting information has been gathered in this area which frequently involves low levels of infected cells or proteins within those cells and monoclonal antibodies are thus admirably suited for investigation of this phenomenon. Measles virus is associated with a persistent slowly progressing fatal infection of the human central nervous system (CNS) termed subacute sclerosing panencephalitis (SSPE) (reviewed by ter Meulen et al., 1983). This disease is associated with a failure to produce mature virus or to

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develop the typical measles virus cytopathology, cell fusion in brain tissue. An examination of the patients’ immune response during this illness demonstrated a lack of antibodies directed against the virus matrix (M) protein (Hall etal., 1979). A direct search by immunoprecipitation and western blot techniques has failed to demonstrate the production of this protein in infected brain or brain explants (Hall and Choppin, 1979). The clinical importance of this disease has prompted an examination of measles virus polypeptide expression in persistently infected cells in uitro. Studies using several monoclonal antibodies failed to detect M protein in persistently infected tissue culture cell lines (SSPE cell lines) derived from SSPE patient brain (Carter etal., 1984). Although this method did permit identification of M protein in a laboratory-established persistent infection (Carter and ter Meulen, 1983) which was previously thought not to express this polypeptide (Stephenson etal., 1981). Monoclonal antibodies thus provide excellent evidence that matrix protein is not synthesized in SSPE cell lines. The lack of this major structural polypeptide is thought to prevent virus particle maturation and account for the slowly progressing nature of the disease. Measles virus persistently infected cells show a low expression of membrane glycoproteins (Norrby etal., 1982)’although expression is normally sufficient to permit development of syncytia. The expression of virus polypeptides has also been investigated within mouse brain persistently infected with lymphocytic choriomeningitis (LCM) virus (Oldstone and Buchmeier, 1982; Rodriguez etal., 1983), Sendai virus (Kristensson etal., 1983), and on the surface of spleen cells from mice infected with Friend leukemia virus (Britt etal., 1981). In these cases the level of virus glycoproteins detected on the cell surface was also reduced. Perhaps this is one way in which persistently infected cells evade the immune system and thus permit the maintenance of infection. Monoclonal antibodies permit the selective detection of virus-specific transformation markers on the transformed cell surface, e.g., Epstein Barr virus (Kintner and Sugden, 1981; Slovin etal., 1982) and Marek’s disease virus (Lee etal., 1983b). Antibodies further permit the detection and characterization of transformation specific proteins as well as some investigations of their function. Monoclonal antibodies directed against the v-ras gene product of Harvey sarcoma virus have been produced. The products of the v-rczs gene family in viruses, transformed and normal cells were shown to share similarities, and the antibodies were able to confirm that the protein is located at the inner surface of the membrane and binds guanine nucleotides (Furth etal., 1982). In addition, a tumor antigen of adenovirus 5 has been shown to accumulate in the cell nucleus (Sarnow et al., 1982).

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2. Search for NovelVirus Proteins

The specificity of the monoclonal antibody confers the ability to react with a family of related proteins. This property is particularly relevant where precursor - product relationships exist (as in proteolysis), or where similar sequences of proteins might occur in different molecules, e.g., following a recombination or readthrough event in transcription. If the precursors never reach appreciable levels within the cell or the shared sequences are small, the cross reacting proteins are difficult to detect with polyclonal serum. Small virus-specific polypeptides which reacted with monoclonal antibodies specific for glycoproteins A and B were detected in HSV-infected 1981). These were not identified in virus-inVero cells (Pereira etal., fected Hep-2 cells and were later shown to be proteolytically related to the intact forms of the glycoproteins (Pereira etal., 1982a). Families of related proteins were also detected in this manner in CMV infected cells (Pereira etal., 1982~).A 15K cleavage product of the measles virus nucleocapsid protein has also been identified (Carter and Baczko, 1983). This type of analysis defines aprotein family but does not indicate whether the submolecular fragments serve any purpose. Furthermore, care should always be taken to ensure that the protein precipitation is not caused by a spurious cross reaction with a host cell polypeptide (Section V). Monoclonal antibodies have also permitted the identification of novel virus proteins. A new cell surface marker was discovered on B cell lines 1982), a new HSV glycoprotein has known to harbor EBV (Slovin etal., also been detected and characterized (Balachandran etaL,1981) and a new protein (gp 70) was detected in leukemic cells from mice infected with Friend leukemia virus. This protein was apparently derived from a virus produced by a recombination event during infection, and had lost cross reactivity with a monoclonal antibody specific for the ecotropic Friend murine leukemia virus (Britt etal., 1981). An unusual form of composite protein was demonstrated in measles virus infected BGM cells and was apparently not produced in other infected cells. The protein was detected because it cross-reacted with a monoclonal antibody specific for nucleocapsid protein, but the new molecule was 24K larger. Peptide mapping analysis showed that it apparently contained all the nucleocapsid peptides plus some new ones whose origin could not be determined (Wild and Giraudon, 1982). No precursor -product relationship could be demonstrated between this new protein and the nucleocapsid protein. The origin of this protein is still obscure but a readthrough mechanism was suggested. A similar finding has also been reported using monoclonal antibodies in the study of HSV 1 and 2 infections (Zweig etal., 1980). A

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polypeptide was observed which was larger than the nucleocapsid protein and contained additional peptides but apparently had no precursor product relationship to the smaller nucleocapsid protein. 3.Protein Processing and Maturation

Following its synthesis, a protein may mature through posttranslational processing. Processing in the sense used here includes covalent modification through glycosylation, posttranslational cleavage etc, as well as maturation by conformational changes often associated with the formation of intermolecular associations. This concept is particularly relevant in the context of virus assembly where stable associations must occur leading to the production of a viable particle. Conformational change is probably involved in this process and may perform a regulatory function as in tobacco mosaic virus assembly (Butler, 1971; Klug and Durham, 1971). a. Modification and Transport. The novel proteins formed by proteolysis in some HSV-infected cells have already been mentioned (Section III,B,2). These proteins are degraded only in some cell types (Pereira et al., 1981,1982a)which suggests this process is not a functionally important processing phenomenon. However, monoclonal antibodies have been used in pulse-chase experiments to demonstrate genuine modification of proteins through glycosylation (Balachandran etal., 1981) and also to demonstrate temporal regulation of their synthesis (Showalter et al., 1981). Misra etal., (1982) have studied the maturation of an HSV-1 early glycoprotein (GVP-11). These workers discovered that tunicamycin-sensitive glycosylation was necessary for the cell surface expression of this protein. However, monoclonal antibodies may not react with the unmodified form of a protein (only one antibody from a panel of 65 directed against measles virus H protein reacted with the unmodified product formed in vitro; M. J. Carter, unpublished observation) and consequently might not always provide an adequate probe for the expression of the polypeptide moiety. A polyclonal serum should therefore always be used. Opposite results have been obtained with measles virus H protein. Glycosylation was not required for insertion into the rough endoplasmic reticulum and intracellular transport of the protein to the plasma membrane. This detailed study also elucidated intermediates formed in the protein modification pathway leading to the mature molecule (Bellini et al., 1983). A comprehensive immunofluorescence study using monoclonal antibodies has examined the distribution of a number of Moloney murine leukemia proteins on the infected cell membrane (Satake et al., 1981; Satake and Luftig, 1983). These workers were able to demonstrate that the mem-

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brane sites a t which virus antigens accumulated were often distinct and differentially controlled by the cellular microtubule system. In addition, they were able to demonstrate by double dot immunofluorescence that two virus-specific antigens, P15 and P15 E, accumulated at the same sites. This association between a gagand an envgene product may be important for virus assembly. b. Conformational Changesand ComplexFormation.In the formation of a virus particle stable intermolecular associations must be formed. It is likely that protein combination to form virus structural components will involve conformational changes. Protein conformation could also be altered artifactually during extraction procedures. Although some antibodies seem to recognize a sequence of amino acids, most monoclonal antibodies possess a sensitivity to the conformation of the epitope that cannot be visualized using a polyclonal serum, and this permit investigations into this complex field of structural isomerism. Alternatively the development of intermolecular associations must result in the masking of some epitopes and therefore the inhibition of combination with antibody directed toward that area of the protein. Monoclonal antibodies could distinguish readily between native and unfolded forms of the Sindbis virus glycoprotein El (Roehrig etal., 1982b) and between native (N) and heated (H) forms of the poliovirus capsid (Brioen etal., 1982; Icenogle etal., 1981). In the latter case it was also shown that the irreversible conversion of N to H could be accomplished by some acidic isolation procedures (Rombaut etal., 1982). This explained an earlier finding that some capsids were in the H form inside the infected cell (Ferguson etal., 1981). More biologically relevant information concerning virus assembly has also been derived from studies of poliovirus. Two monoclonal antibodies were obtained which bound to poliovirion precursor structures. The sites recognized were different, one antibody bound to infectious virions, 80 S empty capsids in the N form and to 14 S precursor subunits, the other bound only to virions and empty capsids. Neither antibody bound to the heat-treated particles. Furthermore, the antibody binding sites were both shown to be located on VP1 by chemical cross linking, but neither antibody bound well to free VP1 in the native or denatured form. This indicated the sites recognized were formed only in the capsid structure. However, variants could readily be selected which did not express these sites, so it is likely that the areas recognized by these antibodies are not vital for structural integrity (Emini etal., 1982). Similar data have been reported for foot and mouth disease virus (FMDV) where monoclonal antibodies have been obtained which can distinguish between complete (146s) virions and their precursor (12 S) subunits

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(McCullogh and Butcher, 1982). These two studies demonstrate that unique antigenic sites are created during picornavirus maturation and explain earlier reports that neutralizing antibodies could only be elicited by using the mature polio virion as immunogen (Dernick, 1981). A similar effect has been demonstrated in the case of TMV where some monoclonal antibodies react only with the nucleocapsid, and others only with the protein monomer (Dietzgen and Sander, 1982). Although the former class must recognize a new site caused by conformational change or formed at molecular junctions, the latter class of antibodies could be directed a t a site which is either masked in the protein complex or destroyed through conformational change. The pathway of adenovirus hexon polymerization has also been investigated with conformation-specific monoclonal antibodies. The monomeric or denatured hexon protein lacks antigenic determinants in common with the trimeric capsomere, a situation reminiscent of the picornavirus capsid. Using these conformation-sensitive monoclonal antibodies, Cepko and Sharp (1982) have shown that trimerization of the hexon protein involves a nonstructural lOOK virus-specific polypeptide. Three hexon molecules bind to this protein in the monomeric configuration and are released as a trimeric capsomere. The capsomeric conformation is adopted concomitantly with release from the 100K “scaffolding” protein. A structural rearrangement may also occur during release of CMV since some antibodies are able to react with determinants and neutralize infectious virus but are unable to bind to the same proteins on the surface of infected cells (Pereira etal., 1982~).Conformational changes are believed important in the development of fusion protein activity by the influenza virus hemagglutinin at low pH (Skehel etal., 1982). Monoclonal antibodies have been used to demonstrate this structural change directly, and to locate areas involved in the molecule’s structure (Daniels etal., 1983). Virus architecture, that is the proximity of structural proteins, is also amenable to examination. For instance gp 56 and gp 50 have been shown closely associated on the Venezuelan equine encephalitis (VEE) virus envelope by means of competitive binding studies (Roehrig etal., 1982a). A similar examination showed that the influenza hemagglutinin was close to the cellular H-2K histocompatibility antigen on the surface of the infected cell. A complex is not apparently formed since these antigens did not cocap (Hackett and Askonas, 1982). On the mature influenza virus envelope however, the HA protein is often found in close association with the NA protein, since monoclonal antibodies specific for hemagglutinin may have a neuraminidase inhibiting activity (Webster etal., 1982). Frequently, virus-specific products associate with cellular proteins and this

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can be demonstrated by competitive binding assays or coprecipitation experiments (Carroll and Gurney, 1982; Ferracini etal., 1982; Sarnow et al., 1982). c.Mutations. Structural change can also result from the acquisition of mutations by the virus genome. During persistent infection a virus genome is able to accumulate mutations rapidly (Holland etal., 1979). The resulting structural alterations in virus proteins can be detected by monoclonal antibodies. Differences have been found in the glycoproteins synthesized by lytic transforming EBV and by the nontransforming virus shed from persistent infections (Qualtiere etal., 1982). Such changes have also been demonstrated in the measles virus hemagglutinin by immunofluorescence (ter Meulen etal., 1981). A similar process has been detected by alterations in competitive binding behavior using monoclonal antibodies specific for the measles virus matrix protein (Carter etal., 1983). In this study an apparent separation of binding groups was observed. A monoclonal antibody was found to have lost its capacity to compete with a second even though both still immunoprecipitated the M protein. This example illustrates that the detection of structural change is dependent on the method used. An examination based solely on radioimmune precipitation may be superior in detecting certain changes which “loosen” the protein and render it more susceptible to the action of detergents in the buffer used. However, changes in relative avidity, topography, or allostery would not be detected provided antibody avidity was still sufficiently high to permit precipitation (Section I). Similarly, detergent produced effects might not be detected in the competition RIA alone.

C. Protein Purification and Quantitation In any analysis of virus-specific protein structure and function, it is always desirable to obtain pure preparations of the protein in question to permit inuitro experimentation or vaccine production. One obvious example of the use to which monoclonal antibodies can be put is in the preparation of affinity columns for such purifications. It is not appropriate to list here all instances in which this has been performed, but any review of the application of monoclonal antibody technology would be incomplete without mention of this subject. The application of the technique is exemplified in the purification of glycoproteins from HSV 1and 2 (Eisenberg etal., 1982b) and the measles virus hemagglutinin (Bellini et al., 1981). When used in RIA or ELISA monoclonal antibodies provide a convenient and accurate method for the quantitation of viral proteins (Lutz et al., 1983).

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IV. INVESTIGATION OF VIRUSPATHOGENESIS AND PROTECTION FROM VIRUSINFECTION The study of a virus as a pathogen is more complicated than studies conducted in uitro. These examinations frequently cross the boundary between virology and immunology. Monoclonal antibodies may be used to mimic a host immune response both in uitro and in uivo and thus modify the course of infection. Animal experiments have demonstrated the protective effect of monoclonal antibodies in uiuo. With the development of human monoclonal antibodies such methods may be of medical importance.

A. Isolation of Virus Variants and Their Pathogenicity The isolation of nonneutralizable virus variants has already been discussed (Section II1,A). These variants can also be applied in studies of pathogenicity as well as protein structure. Spontaneous virus variants occur at a high frequency (lo-*) in Coxsackie B4 virus populations (Prabhaker et al., 1982) and this may explain the high variation in disease pattern produced by these viruses. Possibly alterations in the virus attachment protein might result in an altered tissue tropism. However, the most interesting data have so far emerged from studies of rabies and influenza virus. The selection of naturally arising antigenic variants in the rabies virus glycoprotein was first reported by Wiktor and Koprowski (1980). Coulon et al.(1982a,b) applied this method to select variants using three monoclonal antibodies and noted that while two antibodies led to the isolation of variants indistinguishable in virulence from the parent virus stock, one antibody selected avirulent viruses. These variants were very similar to the parental virus by all other criteria, but differed in their capacity to evoke a strong and rapid immune response which could lead to successful defense against the infection (Coulon et al., 1982~). More monoclonal antibodies were then used to locate the virulence-related epitope on an antigenic map (Coulon etal., 1983). Similar experiments led Dietzschold et al., (1983) to the identification of a single amino acid change in nonpathogenic variants of rabies virus. The arginine residue present in the virulent virus glycoprotein a t position 333 was substituted in all avirulent variants tested. Such differences have been identified (Section I1,B) at other positions in the virus proteins. Broadly similar results have also been obtained in studies of reovirus. Monoclonal antibodies which neutralized the virus (Burstin et al., 1982)were used in the selection of variants. These variants were found

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to exhibit reduced neurovirulence compared with the parent strain (Spriggs and Fields, 1982). The altered epitope was also demonstrated to be the major site of recognition for virus specific cytotoxic lymphocytes (Finberg etal., 1982). The use of monoclonal antibodies in the study of antigenic drift in influenza virus has already been discussed in the context of the structural information derived from such experiments (Section II1,A). Although antigenic drift has been studied mainly in the HA molecule (Natali etal., 1981;Underwood, 1982;Webster and Berton, 1981) it may also occur in the neuraminidase but its extent may be restricted in some species of avian virus (Webster etal., 1982). Knowledge of the basis for such drift is now approaching that for the HA molecule (Colman etaZ., 1983). The subject is mentioned again here since it has a direct bearing on the pathogenesis and survival of influenza viruses in the population. The role of the HA in this drift has recently been reviewed (Ward, 1981).

B. Alteration intheCourseof Infection Monoclonal antibodies can be applied in order to mimic the immune response and to gain insight into the effect of immunological pressure on the virus. Experiments using monoclonal antibodies to block cytotoxic lymphocyte action have demonstrated that the epitopes recognized by these cells may be identical to (Finberg etal., 1982) or distinct from (Lefrancois and Lyles, 1983b) those regions to which neutralizing antibodies are directed. The host immune response has been implicated in the maintenance of measles virus persistence leading to SSPE. As described in Section JII,B, the disease is associated with a defect in the production of virus M protein. Some evidence has been gathered that suggests the hosts’ immune response itself may actually modulate the synthesis of virus polypeptides and consequently help to bring this situation about (Fujinami and Oldstone, 1979). This has been modelled inuiuo where inoculation of a hyperimmune anti-measles virus antiserum was found to alter the characteristics of disease in intracerebrally inoculated BALB/c mice, a delayed disease was then produced. Inoculation of monoclonal antibody specific for the virus hemagglutinin protein was also able to induce this effect although monoclonal antibody directed against the nucleocapsid protein could not (Rammohan etal., 1981). A similar process has been demonstrated with a measles virus persistent infection in uitto. Antiserum treatment of C6 cells persistently infected with measles virus led to a drastic reduction in the expression of virus polypeptides (Barrett and Koschel, 1983). Once again monoclonal antibody to H, but not N could

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produce this effect (P. N. Barrett, personal communication). Antibody to surface glycoprotein can also prolong experimental Sindbis virus infection (Chanas et al., 1982a). Antibodies with neutralizing activity were active in this respect while those which lacked this property were not. This is also reflected in the measles virus system described above. Monoclonal antibodies which neutralized the virus seemed to be exclusively active in producing the alteration in virus polypeptide expression (P. N. Barrett, personal communication). However, it is also true that under some circumstances monoclonal antibodies can enhance virus infection. Flaviviruses, in combination with antibody, are able to infect cells bearing Fc receptors through a cross bridging mediated by the antibody (Chanas et al., 1982b;Peiris et al., 1982; Schlesinger et al., 1983). However, use of monoclonal antibodies has demonstrated that the virus epitope to which the antibody is bound is also important. Brandt et al. (1982) found only flavivirus type common determinants were effective, and second, that some cells were unable to support YFV replication even when antibody enhancement was attempted. Consequently the presence of the Fc receptor on the target cell surface is not alone sufficient for productive infection. C. Protection Use of monoclonal antibodies for therapeutic purposes is as yet in its infancy. It is likely that the antibodies would be used in three main areas. First, in emergency, injection of a neutralizing antibody can aid recovery from disease. Human monoclonal antibodies are now becoming available (Crawford et al., 1983; Sikora and Neville, 1982) and are likely to produce fewer problems with allergic reaction. Animal experiments in the mouse have indicated that monoclonal antibodies will be beneficial in this area. Neutralizing antibodies are able to localize HSV infection and prevent spread to the nervous system (Kapoor et al., 1982),promote recovery from ocular infection (Rector et al., 1982) or protect against a later footpad challenge (Dix et al., 1981),and antibodies to any herpesvirus glycoprotein had protective activity. Intravenous inoculation of monoclonal antibody specific for the virus hemagglutinin was found to protect mice from the invariably lethal consequences of intracerebral injection with influenza WSN. This action was however dependent upon the breakdown of the blood-brain barrier (Doherty and Gerhard, 1981). The ability of antibody to function in this way is not simply correlated with its anti-virus action measured by in uitro assays. Nonneutralizing antibodies protect mice from HSV (Balachandran et al., 1982c; Rector etal., 1982) or Sindbis virus (Schmaljohn et al., 1982), and Mathews and Roehrig (1982) found

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nonneutralizing antibodies could also protect mice from Japanese equine encephalitis infection. Antibodies of high avidity protected with greater efficiency than those of lower avidity, but antibodies directed against epitopes instrumental in neutralizing the virus were effective at lower doses. This work therefore implicates the tightness of attachment as well as site of combination with antigen in efficiency of protection. Some evidence suggests that complement is not involved in protection by monoclonal antibodies and suggests the process is mediated by an antibody-dependent cell-mediated cytolysis (Balachandran etal., 1982~). Second, monoclonal antibodies will be applied to the selection of avirulent viruses for vaccine purposes. Rabies virus has already been described in this context (Section IV,A). Heterogeneity among Coxsackie (Prabhakar etal., 1982) or bluetongue viruses (Letchworth and Appleton, 1983) may permit such an approach. The antibodies could also be used in the preparation of pure subunit vaccines (Osterhaus etal., 1981b). Finally, when combined with a cytotoxic agent, monoclonal antibodies provide a specific cell killing tool (Davies, 1981). Specific cytotoxicity has already been 1981; Krolick etal., demonstrated using this technique (Blythman etal., 1980; Youle and Neville, 1980).

V. CONCLUSIONS Monoclonal antibodies allow us to view the fate of a protein in the infected cell, from synthesis to virus assembly, as a continuous process and hint at the interaction between virus-specific gene products and the cellular machinery. The use of monoclonal antibodies provides valuable insight into the working of the protein both as an enzyme and as a target for the host immune response, evolving in reaction to that response. As three-dimensional structures of more molecules are determined, so this work should extend our understanding of virus protein structure and function. In the foregoing discussion it has been emphasized how the specificity of a monoclonal antibody can be applied to the study of all areas of virology. In each case it is the specificity of the antibody which has proved useful, and even permitted the identification of related but hitherto unknown proteins. However, this very specificity also provides the basis for spurious cross reactivity not normally present in polyclonal serum. In general, an appropriately absorbed monospecific antiserum will recognize many epitopes and thus coat a considerable area of a protein’s exposed surface. A single epitope repeated on another protein would cross react, but this

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would be masked by the vast majority of antibodies reacting with the target antigen. In the case of a monoclonal antibody this is not so. A target molecule may have only one site for the antibody and if this is shared with another protein a considerable cross reaction could be detected, even in the abscence of extensive structural homology. It has been calculated (Crawford etal., 1982) that a primary sequence of four amino acids (a not unusual size for an antibody binding site) might occur some 15times in the average cell’s complement of protein sequences. However, monoclonal antibody cross reactions would be expected much less frequently than this because (1)the amino acid sequence must be expressed on the surface of the molecule, (2) antibodies may recognize a sequence in a given conformation or state of modification, (3) protein sequence does not normally occur at random, and (4) antibody binding sites are often larger than this (Atassi, 1975). Furthermore, it would be very difficult for an animal to produce an antibody response to an epitope which was likely to cross react, merely on a random basis, with a “self” epitope. However, this could perhaps be tolerated as a small constituent of a total non-self-cross-reacting immune response. Consequently, it is likely that fortuitous cross reaction may be comparatively rare. Nevertheless, many cross reactions have been reported which are at present not understood (reviewed by Lane and Koprowski, 1982). Several of these (Crawfordet al., 1982;Lane and Hoeffler, 1980) involve cross rections between SV40 T antigen and host proteins or between SV40 T and t antigens (Harlow et al., 1981). In the former case there is good reason to believe that some of these antibodies recognize epitopes on functionally related host proteins, and which therefore may be constrained to some degree of overall structural similarity. Whether this represents an evolutionarily conserved structure, or one which has arisen by independent convergent evolutionary processes is unknown. However, cross reaction a t the monoclonal antibody level cannot be assumed indicative of any extensive structural or functional similarity without considerable supporting evidence. The application of monoclonal antibodies in conventional serological reactions, permitting the differentiation of closely related, serologically cross-reacting proteins has already been discussed in this review. In addition, the process described above provides an entirely new dimension to these procedures and might thus permit the identification of functionally related proteins which bear no evidence of cross reaction with polyclonal sera. Consequently application of monoclonal antibodies has not only improved information obtained from well established assay procedures, but may also lead to processes yielding an entirely new type of information.

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ACKNOWLEDGMENTS The authors thank the Deutsche Forschungsgemeinschaft for financial support. We also thank Professor E. Wecker for constructive criticism and Helga Kriesinger for typing the manuscript.

REFERENCES Antczak, D. F. (1982). J. Am. Vet. Med. Assoc. 1 8 1 , 1005-1010. Appleton, J. A., and Letchworth, G. J. (1983). Virology 124, 286-299. Atassi, M. Z. (1975). Zmmunochemistgv 21,423-438. Balachandran, N., Harnish, D., Killington, R. A., Bacchetti, S., and Rawls, W. E. (1981). J. Virol. 3 9 , 438-446. Balachandran, N., Frame, B., Chernesky, M., Kraiselburd, E., Kouri, Y., Garcia, D., Lavery C., and Rawls, W. E. (1982a). J. Clin. Microbiol. 1 6 , 205-208. Balachandran, N., Harnish, D., Rawls, W. E., and Bacchetti, S. (198213). J. Virol. 4 4 , 344- 355.

Balachandran, N., Bacchetti, S., and Rawls, W. E. (1982~).Infect. Immun. 37,1132-1137. Barrett, P. N., and Koschel, K. (1983). Virology 1 2 7 , 299-308. Bellini, W. J., McFarlin, D. E., Silver, G. D., Mingioli, G. S., and McFarland, H. F. (1981). Infect. Immun. 32 , 1051-1057. Bellini, W. J., Silver, G. D., and McFarlin, D. E. (1983). Arch. Virol. 76, 87-101. Benton, C. V., Gilden, R. V., and Shah, K. V. (1982). Virology 117,490-495. Blancou, J., Andral, L., and Mannen, K. (1982). Comp. Zmmunol. Microbiol. Infect. Dis. 5 , 95-99.

Blann, A. D. (1981). Med. Lab. Sci.3 8 , 389-393. Blythman, H. E., Cassellas, P., Gros, O.,Gros, P., Jansen, F. K., Paolucci, F., Pau, B., and Vidal, H. (1981). Nature (London) 2 9 0 , 145-146. Bohn, W., Rutter, G., andMannweiler, K. (1982). Virology 1 1 6 , 368-371. Brandt, W. E., McCown, J. M., Gentry, M. K.,andRussell, P. K. (1982). Infect. Zmmun. 3 6 , 1036- 1041.

Braun, D. K., Pereira, L., Norrild, B., and Roizman, B. (1983). J. Virol. 4 6 , 103-112. Briand, J. P., A1 Moudallal, Z., and van Regenmortel, M. H. (1982). J. Virol. Methods 5 , 293 - 300. Brioen, P., Sijens, R. J., Vrijsen, R., Rombaut, B., Thomas, A. A., Jackers, A., and Boeye, A. (1982). Arch. Virol. 7 4 , 325-330. Britt, W. J., Collins, J. K., and Chesebro, B. (1981). J . Exp.Med. 164,868-882. Bruck, C., Mathot, S., Portetelle, D., Berte, C., Franssen, J. D., Herion, P., and Burny, A. (1982a). Virology 1 2 2 , 342-352. Bruck, C., Portetelle, D., Burny, A., and Zavada, J. (1982b). Virology 1 2 2 , 353-362. Burstin, S. J., Spriggs, D. R., and Fields, B. N. (1982). Virology 1 1 7 , 146-155. Burtonboy, G., Bazin, H., and Delferriere, N. (1982). Arch. Virol. 71, 291-302. Butler, P. J. G. (1971). ColdSpring Harbor Symp. Quant.Biol. 36,461-468. Carroll, R. B., and Gurney, E. G. (1982). J. Virol. 44,565-573. Carter, M. J., and Baczko, (1983). 2.Nuturforschung 38c, 887-889. Carter, M. J., and ter Meulen, V. (1983). Prog. Bruin Res. 5 9 , 163-171. Carter, M. J., Willcocks, M. M., Loeffler, S., and ter Meulen, V. (1982). J. Gen. Virol. 6 3 , 113-120.

MONOCLONAL ANTIBODIES IN VIRUS STUDY

125

Carter, M. J., Willcocks, M. M., Loeffler, S., and ter Meulen V. (1983). J. Gen.Virol. 64, 1801- 1805.

Carter, M. J. Willcocks, M. M., and ter Meulen, V. (1983). Nature (London) 305,153-155. Caton, A. J., Brownlee, G. G., Yewdell, J. W., and Gerhard, W. (1982). Cell 31,417-427. Cepko, C. L., and Sharp, P. A. (1982). Cell31,407-415. Chanas, A. C., Ellis, D. S., Stamford, S., and Gould, E. A. (1982a). AntiuiralRes. 2,191-201. Chanas, A. C., Gould, E. A., Clegg, J. C., andVarma, M. G. (1982b). J. Gen. Virol.58,37-46. Charlton, K. M., Casey, G. A,, Boucher. D. W., and Wiktor, T. J. (1982). Comp. Immunol. Microbiol. Infect. Dis. 5 , 113-115. Cianciolo, G., Hunter, J., Silva, J., Haskill, J. S., and Snyderman, R. (1981). J. Clin. Inuest. 68,831-844.

Clark, R., Lane, D. P., and Tjian, R. (1981). J. Biol.Chem. 2 6 6 , 11854-11858. Collins, A. R., Knobler, R. L., Powell, H., and Buchmeier, M. J. (1982). Virology 119, 358- 371.

Colman, P. M., Varghese, J. N., and Laver, W. G. (1983). Nature (London)300,360-362. Cbte, P. J., Jr., Fernie, B. F., Ford, E. C., Shih, J. W., and Gbrin, J. L. (1981). J . Virol. Methods 3, 137-147. Cbte, P. J., Jr., Dapolito, G. M., Shih, J. W., and GBrin, J. L. (1982). J. Virol. 4 2 , 135-142. Coulon, P., Rollin, P., Aubert, M., and Flamand, A. (1982a). Arch Inst. Pasteur Tunis 6 9 , 105-113.

Coulon, P., Rollin, P., Aubert, M., and Flamand, A. (1982b). J. Gen.Virol. 6 1 , 97-100. Coulon, P., Rollin, P., Blancou, J., and Flamand, A. (1982~).Comp. Immunol. Microbiol. Infect. Dis. 5 , 117 - 122. Coulon, P., Rollin, P. E., and Flamand, A. (1983). J. Gen. Virol. 6 4 , 693-696. Crainic, R., Couillin, P., Cabau, N., Boue, A., and Horodniceanu, F. (1981). Deu.Bid. Stand. 60,229-234.

Crawford, D. H., Callard, R. E., Muggeridge, M. I., Mitchell, D. M., Zanders, E. D., and Beverley, P. C. (1983). J. Gen.Virol. 64, 697-700. Crawford, L., Leppard, K., Lane, D., and Harlow, E. (1982). J. Virol. 4 2 , 612-620. Dahmus, M. E., and Kedinger, C. (1983). J. Biol. Chem.258,2303-2307. Daniels, R. S., Douglas, A. R., Skehel, J. J., and Wiley, D. C. (1983). J. Gen.Virol. 6 4 , 1657- 1662.

Davies, T. (1981). Nature (London)2 8 9 , 12-13. De, B. K., Tahara, S. M., and Banerjee, A. K. (1982). Virology 1 2 2 , 510-514. Dernick, R. (1981). Deu.Biol.Stand. 4 7 , 319-333. Dietzgen, R. G.,and Sander, E. (1982). Arch. Virol. 74,197-204. Dietzschold, B., Wunner, W. H., Wiktor, T. J., Lopes, A. D., Lafon, M., Smith C. L., and Koprowski, H. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 70-74. Digoutte, J. P. (1982). Arch. Inst. Pasteur Tunis 59,99-103. Dilworth, S. M., and Griffin, B. E. (1982). Proc. Natl. Acad. Sci. U.S.A. 7 9 , 1059-1063. Dittmar, D., Castro, A., and Haines, H. (1982). J. Gen.Virol. 5 9 , 273-282. Dix, R. D., Pereira, L., and Baringer, J. R. (1981). Infect. Immun.34, 192-199. Doherty, P. C., and Gerhard, W. (1981). J. Neuroimnunol. 1,227-237. Eisenherg, R. J., Long, D., Pereira, L., Hampar, B., Zweig, M., and Cohen, G. H. (1982a). J. Virol. 4 1 , 478-488. Eisenberg, R. J., Ponce de Leon, M., Pereira, L., Long, D., and Cohen, G. H. (1982b). J. Virol. 41,1099-1104.

Emini, E. A., Jameson, B. A., Lewis, A. J., Larsen, G .R., and Wimmer, E. (1982). J.Virol. 43,997-1005.

126

MICHAEL J. CARTER AND VOLKER TER MEULEN

Evans, D. M. A., Minor, P. D., Schild, G. S., and Almond, J. W. (1983). Nature (London) 304,459-462.

Ferguson, M., Schild, G. C., Minor, P. D., Yates, P. J., andSpitz, M. (1981). J.Gen. Virol. 6 4 , 437-442.

Ferguson, M., Yi-hua, Q.,Minor, P. D., Magrath, D. I., Spitz, M., and Schild, G. C. (1982). Lancet 2 , 122-124. Ferracini, R., Prat, M., and Comoglio, P. M. (1982). Int. J. Cancer 2 9 , 477-481. Finberg, R., Spriggs, D. R., and Fields, B. N. (1982). J. Immunol. 129,2235-2238. Forghani, B., Schmidt, N. J., Myoraku, C. K., and Gallo, D. (1982). Arch, Virol. 7 3 , 311317.

Fujinami, R. S., and Oldstone, M. B. A. (1979). Nature (London)2 7 9 , 529-530. Fujinami, R. S. Oldstone, M. B., Wroblewska, Z., Frankel, M. E., and Koprowski, H. (1983). Proc. Natl. Acad. Sci. U.S.A. 8 0 , 2346-2350. Fukushi, H., Yanagawa, R., and Kida, H. (1982). Arch. Virol. 72,217-221. Furth, M. E., Davis, L. J., Fleurdelys, B., and Scolnick, E. M. (1982). J. Virol. 43,294-304. Gary, G. W., Jr., Black, D. R., and Palmer, E. (1982). Arch. Virol. 7 2 , 223-227. Gentry, M. K., Henchal, E. A., McCown, J. M., Brandt, W. E., and Dalrymple, J. M. (1982). Am. J. Trop. Med. Hyg. 31,548-555. Gerhard, W., Croce, C. M., Lopes, D., and Koprowski, H. (1978). Proc. Natl. Acud. Sci. U.S.A. 75, 1510-1514. Gerhard, W., Yewdell, J. W., Frankel, M. E., Lopes, D., and Standt, L. (1980). In “Monoclonal Antibodies” R. H., Kennett, T. J. McKearn, and K. B. Bechtol, eds., pp. (1-423. Plenum, New York. Gerhard, W., Yewdell, J. W., Frankel, M. E., and Webster, R. (1981). Nature (London)2 9 0 , 713-717.

Giraudon, P., and Wild, T. F. (1981a). J. Gen. Virol. 54,325-332. Giraudon, P., and Wild, T. F. (1981b). J.Gen. Virol. 5 7 , 179-183. Goldstein, L. C., McDougall, J., Hackman, R., Meyers, J. D., Thomas, E. D., and Nowinski, R. C. (1982). Infect. Immun. 3 8 , 273-281. Gonda, M. A., Benton, C. V., Massey, R. J., and Schultz, A. M. (1981). Scanning Electron Micros. 2 , 45-62. Gonzalez-Scarano, F., Shope, R. E., Calisher, C. E., and Nathanson, N. (1982). Virology 120,42-53.

Greenberg, H., McAuliffe,V., Valdesuso, J., Wyatt, R., Flores, J., Kalica, A., Hoshino, Y., and Singh, N. (1983). Infect. Immun. 39,91-99. Hackett, C. J., and Askonas, B. A. (1982). Immunology 46, 431-438. Hall, W. W., and Choppin, P. W. (1979). Virology,99,443-447. Hall, W. W., Lamb, R. A., and Choppin, P. W. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 2047- 2051.

Harlow, E., Crawford, L. V., Pim, D. C., and Williamson, N. M. (1981). J. Virol. 3 9 , 861 - 869.

Heinz, F. X., Berger, R., Majdic, O.,Knapp. W., and Kunz, C. (1982). Infect. Immun. 3 7 , 869-874.

Henchal, E. A., Gentry, M. K., McCown, J. M., and Brandt, W. E. (1982). Am.J. Trop. Med. Hyg. 31,830-836. Henchal, E. A., McCcwn, J. M., Seguin, M. C., Gentry, M. K., and Brandt, W. E. (1983). Am. J . Trop. Med. Hyg. 32, 164- 169. Hinshaw, V. S., Webster, R. G.,Bean, W. J., Downie, J., and Senne, D. A. (1983). Science 220,206-208.

Holland, J. J., Graham, E. A., Jones, C. L., and Semler, B. L. (1979). Cell16,495-504.

MONOCLONAL ANTIBODIES IN VIRUS STUDY

127

Holmes, K. T., Hampson, A. W., Raison, R. L., Webster, R. G.,O’Sullivan, W. J., and Mountford, C. E. (1982). Eur. J . Immunol. 12, 523-526. Humphrey, D. D., Kew, 0. M., and Feorino, P. M. (1982). Infect. Immun. 36,841-843. Icenogle, J., Gilbert, S. F., Grieves, J., Anderegg, J., and Rueckert, R. (1981). Virology 1 1 5 , 211-215. Jackson, D. C., and Webster, R. G. (1982). Virology 123,69-77. Jackson, D. C., Murray, J. M., White, D. O., and Gerhard, W. (1982). Infect. Immun. 37, 912-918. Jackson, D. C., Howlett, G. J., Nestorowicz, A,, and Webster, R. G. (1983). J. Immunol130, 1313-1316. Kapoor, A. K., Nash, A. A., Wildy, P., Phelan, J., McLean, C. S., andField, H. J. (1982). J . Gen. Virol. 60, 225-233. Kendal, A. P., Phillips, D. J., Webster, R. G., Galland, G. G., and Reimer, C. B. (1981). J. Gen. Virol. 5 4 , 253-261. Kennett, R. H. (1981). In Vitro 17,1036-1050. Kida, H., Brown, L. E., and Webster, R. G. (1982). Virology 122, 38-47. Kimura-Kuroda, J., and Yasui, K. (1983). J .Virol. 45, 124-132. Kintner, C., and Sugden, B. (1981). Nature (London) 294,458-460. Klenk, H. D., and Rott, R. (1980). Curr. Top. Microbiol. Immunol. 90, 19-48. Klug, A., andDurham, A. C. H. (1971). Cold Spring HurborSymp. Quant. Biol.36,449-460. Kohler, G., and Milstein, C. (1975). Nature (London) 256,495-497. Kristensson, K., and Orvell, C. (1983). J. Gen.Virol. 64, 1673-1678. Kristensson, K., Orvell, C., Leestma, J., andNorrby,E. (1983). J . Infect. Dis. 147,297-301. Krolick, K. A,, Villeriez, C., Isakson, P., Uhr, J. W., and Videtta, E. S. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,5419-5423. Lane, D. P., and Hoeffler, W. K. (1980). Nature (London) 288,167-170. Lane, D. P., and Koprowski, H. (1982). Nature (London) 296,200-202. Laver, W. G., Air, G. M., Webster, R. G., Bernhard, W., Ward, C. W., and Dopheide, T. A. A. (1979). Virology 98,226-237. Laver, W. G., Air, G. M., Wehster, R. G., Gerhard, W., Ward, C. W., and Dopheide, T . A. A. (1980a). Philos, Trans. R. Soc. London, Ser.B 288,313-326. Laver, W. G., Air, G. M., Webster, R. G., Gerhard, W., Ward, C. W., and Dopheide, T. A. A. (1980b). In “Structure and Variation in Influenza Virus’’ (W. G. Laver and G. M. Air, eds.), pp. 295 -306. Elsevier/North Holland, New York. Lee, L. F., Liu, X., and Witter, R. L. (1983a). J. Immunol. 130, 1003-1006. Lee, L.F., Liu,X., Sharma, J. M., Nazerian, K.,andBacon, L. D. (198313). J.Immunol. 130, 1007- 1011. Lefranqois, L., and Lyles, D. S. (1982a). Virology 1 2 1 , 157-167. Lefrancois, L., and Lyles, D. S. (1982b). Virology 121, 168- 174. Lefranqois, L., and Lyles, D. S. (1983a). J. Immunol. 1 3 0 , 394-398. Lefranqois, L., and Lyles, D. S. (1983b). J . Immunol. 130,1408-1412. Letchworth, G. J., and Appleton, J. A. (1983). Virology 124, 300-307. Lubeck, M., and Gerhard, W. (1982). Virology 118, 1-7. Lutz, H., Pedersen, N. C., Durhin, R., andTheilen, G. H. (1983). J. Immunol. Methods 56, 209 220. McCullough, K. C., and Butcher, R. (1982). Arch.Virol. 74, 1-9. McLean, C., Buckmaster, A,, Hancock, D., Buchan, A,, Fuller, A., and Minson, A. C. (1982). J . Gen. Virol. 63, 297-305. Massey, R. J., and Schochetman, G. (1981). Virology 1 1 5 , 20-32. Mathews, J. H., and Roehrig, J. T. (1982). J.Immunol.129, 2763-2767. ~

128

MICHAEL J. CARTER AND VOLKER TER MEULEN

Meegan, J. M., Digoutte, J. P., Peters, C. J., and Shope, R. E. (1983.) Lancet1 , 641. Mercer, W. E., Nelson, D., DeLeo, A. B., Old, L. J., and Baserga, R. (1982). Proc. Natl. Acad. Sci. U S A .7 9 , 6309-6312. Minor, P. D., Schild, G. C., Ferguson, M., MacKay, A., Magrath, D. I., John, A,, Yates, J. P., and Spitz, M. (1982). J. Gen.Virol. 6 1 , 167-176. Minor, P. D., Schild, G. C., Bootman, J., Evans, D. M., Ferguson, M., Reeve, P., Sptiz, M., Stanway, G., Cann, A. J., Hauptmann, R., Clarke, L. D., Mountford, R. C., and Almond, J. W. (1983). Nature(London) 301,674-679. Misra, V., Gilchrist, J. E., Weinmaster, G., Qualtiere, L., vanden Hurk, S., andBabiuk, L. A. (1982). J.Virol. 4 3 , 1046-1054. Miura, N., Uchida, T., and Okada, Y. (1982). Exp.Cell Res.141,409-420. Monath, T. P., Kinney, R. M., Schlesinger, J. J., Brandriss, M. W., and Bres, P. (1983). J. 6 4 , 627-637. Gen.Virol. Mountford, C. E., Grossman, G., Holmes, K. T., O’Sullivan, W. J., Hampson, A. W., Raison, R. L., and Webster, R. (1982). Mol.Immunol.19,811-816. Mueller-Lantzsch, N., Georg-Fries, B., Herbst, H., zur Hausen, H., and Braun, D. G. (1981). Int. J. Cancer28,321-327. Natali, A., Oxford, J. S., and Schild, G. C. (1981). J. Hyg.87,185-190. Niman, H. L., and Elder, J. H. (1982). Virology 1 2 3 , 187-205. Norrby, E., Chen, S. N., Togashi, T., Shesberadaran, H., and Johnson, K. P. (1982). Arch. Virol. 7 1 , 1-11. Nowinski, R. C., Tam, M. R., Goldstein, L. C., Stong, L., Kuo, C. C., Corey, L., Stamm, W. E., Handsfield, H. H., Knapp., J. S., and Holmes, K. K. (1983). Science 219,637-644. 300,360-362. Oldstone, M. B. A., and Buchmeier, M. J. (1982). Nature(London) Orvell, C., and Grandien, M. (1982). J.Immunol.129,2779-2787. Osterhaus, A. D., van Wezel, A. L., van Steenis, B., Drost, G. A., and Hazendonk, A. G. (1981a). Intervirology 16,218-224. Osterhaus, A. D., van Wezel, A. L., van Steenis, B., Hazendonk, A. G., and Drost, G. (1981b). Deu.Biol. Stand. 50, 221-228. Parrish, C. R., Carmichael, L. E., and Antczak, D. F. (1982). Arch.Virol. 7 2 , 267-278. Peiris, J. S., Porterfield, J. S., and Roehrig, J. T. (1982). J. Gen.Virol. 5 8 , 283-289. Pereira, L., Dondero, D. V., Norrild, B., and Roizman, B. (1981). Proc.Natl. Acad.Sci. U.S.A. 7 8 , 5202-5206. Periera, L., Dondero, D. V., and Roizman, B. (1982a). J. Virol. 44,88-97. Pereira, L., Dondero, D. V., Gallo, D., Devlin, V., and Woodie, J. D. (1982b). Infect. Immun. 3 5 , 363-367.

Pereira, L., Hoffman, M., Gallo, D., and Cremer, N. (1982~).Infect, Immun.36,924-932. Peterson, E., Schmidt, 0. W., Goldstein, L. C., Nowinski, R. C., andCorey, L. (1983). J.Clin. Microbiol. 17, 92-96. Peterson, P. K., Balfour, H. H., Marker, S. C., Fryd, D. S., Howard, R. J., and Simmons, R. L. (Baltimore) 5 9 , 283-300. (1980). Medicine Microbwl. 15, Phillips, D. J., Galland, G. G., Reimer, C. B., andKendal, A. P. (1982). J. Clin. 931 -937.

Pinter, A., Honnen, W. J., Tung, J. S., O’Donnell, P. V., and Haemmerling, U. (1982). Virology 1 1 6 , 499-516. Portner, A. (1981). Virology 115, 375-384. Portner, A., Webster, R. G., andBean, W. J. (1980). Virology 1 0 4 , 235-238. Prabhakar, B. S., Haspel, M. V., McClintock, P. R., and Notkins, A. L. (1982). Nature (London) 300,374-376. Qualtiere, L. F., Chase, R., Vroman, B., and Pearson, G. R. (1982). Proc. Natl. Acad.Sci. U.S.A. 79, 616-620.

MONOCLONAL ANTIBODIES IN VIRUS STUDY

129

Rammohan, K. W., McFarland, H. F., and McFarlin, D. E. (1981). Nature (London) 290, 588-589. Rector, J. T., Lausch, R. N., andOakes, J. E. (1982). Infect. Zmmun. 3 8 , 168-174. Richman, D. D., Cleveland, P. H., and Oxman, M. N. (1982). J . Med. Virol. 9,299-305. Rodriguez,M., Buchmeier, M. J., Oldstone,M.B., andLampert, P. W. (1983). Am. J. Pathol. 100,95-100. Roehrig, J. T., Day, J. W., and Kinney, R. M. (1982a). Virology 188,269-278. Roehrig, J. T., Gorski, D., and Schlesinger, M. J . (192813). J. Gen.Virol. 5 9 , 421-425. Rombaut, B., Vrijsen, R., Brioen, P., and Boeye, A. (1982). Virology 1 2 2 , 215-218. Roseto, A., Scherrer, R., Cohen, J., Guillemin, M. C., Charpilienne, A., Feynerol, C., and Peries, J. (1983). J. Gen. Virol. 6 4 , 237-240. Russell, W. C., Patel, G., Precious, B., Sharp, I., andGardner, P. S. (1981). J. Gen. Virol. 5 6 , 393 - 408. Sarnow, P., Sullivan, C. A., and Levine, A. J. (1982). Virology 120,510-517. Satake, M., and Luftig, R. B. (1983). Virology 124, 259-273. Satake, M., McMillan, P., and Luftig, R. B. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 6266-6276. Scheller, A,, Covey, L., Barnet, B., and Prives, C. (1982). Cell 2 9 , 375-383. Schlesinger, J. J., Brandriss, M. W., and Monath, T. P. (1983). Virology 125,8-17. Schmaljohn, A. L., Johnson, E. D., Dalrymple, J. M., and Cole, G. A. (1982). Nature (London) 297,70-72. Schmidt, N. J., Ota, M., Gallo, D., and Fox, V. L. (1982). J. Clin. Microbiol. 1 6 , 763-765. Schneider, L. G. (1982). Comp. Immunol. Microbiol. Infect. Dis. 5, 101-107. Server, A. C., Merz, D. C., Waxham, M. N., and Wolinsky, J. S. (1982). Infect. Zmmun. 35, 179- 186. Shafritz, D. A., Lieberman, H. M., Isselbacher, K. J., and Wands, J. R. (1982). Proc. Natl. Acad. Sci. U.S.A. 7 9 , 5675-5679. Showalter, S. D., Zweig, M., and Hampar, B. (1981). Infect. Immun. 34,684-692. Sikora, K., and Nelville, A. M. (1982). Nature (London) 3 0 0 , 316-317. Sinkovics, J. G., and Dreesman, G. R. (1983). Reu.Infect. Dis.6 , 9 - 3 4 . Skehel, J. J., Bayley, P. M., Brown, E. B., Martin, S. R., Waterfield, M. D., White, J. M., Wilson, I. A., and Wiley, D. C. (1982). Proc. Natl. Acad. Sci. U.S.A. 7 9 , 968-972. Slovin, S. F., Frisman, D. M., Tsoukas, C. D., Royston, I., Baird, S. M., Wormsely, S. B., Carson, D. A., and Vaughan, J. H. (1982). Proc. Natl. Acad. Sci. U.S.A. 79,2694-2653. Spriggs, D. R., and Fields, B. N. (1982). Nature (London) 2 9 7 , 6 8 . Stephenson, J. R., Siddell, S. G., and ter Meulen, V. (1981). J . Gen. Virol. 6 7 , 191-197. Stone, M. R., and Nowinski, R. C. (1980). Virology 100,370-381. ter Meulen, V., Loeffler, S., Carter, M. J., and Stephenson, J . R. (1981). J. Gen.Virol.5 7 , 357 - 364. ter Meulen, V., Stephenson, J. R., and Kreth, H. W. (1983). Comp. Virol. 1 8 , 105-159. Thomas, E. D., Storb, R., Clift, R. A., Fefer, A., Johnson, F. L., Neiman, P. E., Lerner, K. G., Glucksberg, H., and Buckner, C. D. (1975a). N. Engl. J. Med. 292,832-843. Thomas, E. D., Storb, R., Clift, R. A., Fefer, A., Johnson, F. L., Neiman, P. E., Lerner, K. G., Glucksberg, H., and Buckner, C. D. (1975b). N. Engl. J. Med. 292,895-902. Thorpe, R., Minor, P. D., Mackay, A., Schild, G. C., and Spitz, M. (1982). J. Gen. Virol. 6 3 , 487- 492. Towbin. H., Staehelin, T., and Gordon, J. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,43504354. Underwood, P. A. (1982). J. Gen. Virol. 6 2 , 153-169. van Regenmortel, M. H. (1981). Comp. Virol. 17, 183-244. van Wyke, K. L., Bean, W. J., Jr., and Webster, R. G. (1981). J. Virol. 3 9 , 313-317.

130

MICHAEL J. CARTER AND VOLKER TER MEULEN

Varghese, J. N., Laver, W. G., and Colman, P. M. (1983). Nature (London) 3 0 3 , 41-44. Volk, W. A., Snyder, R. M., Benjamin, D. C., and Wagner, R. R. (1982). J. Virol. 42, 220-227.

Wands, J. R., Carlson, R. I., Shoemaker, H., Isselbacher, K. J., and Zurawski, U. R. Proc. Natl. Acad. Sci. U.S.A. (1981). 78, 1214-1218. Wands, J. R., Marciniak, R. A., Isselbacher, K. J., Varghese, M., Don, G., Halliday, J. W., and Powell, L. W. (1982). Lancet 1, 977-980. Ward, C. W. (1981). Curr. Top. Microbiol. Immunol. 9 7 , 1-74. Webster, R. G., and Berton, M. T. (1981). J. Gen. Virol. 54, 243-251. Webster, R. G . , Hinshaw, V. S.,Berton, M. T.,Laver, W. G., and Air, G. M. (1981). I n “ICN UCLA Meeting on Antigenic Variation in Influenza Viruses” (D. P. Nayak, ed.) Vol. 21, pp. 309-322. Academic Press, New York. Webster, R. G.,Hinshaw, V. S., and Laver, W. G. (1982). Virology 117,93-104. Wiktor, T. J., and Koprowski, H. (1980). J . Exp.Med. 152,99-112. Wiktor, T. J., Flamand, A., and Koprowski, H. (1980). J. Med. Virol. 1 , 33-46. Wild, T. F., and Giraudon, P. (1982). J. Gen. Virol. 6 3 , 247-250. Wiley, D. C., Wilson, I. A., and Skehel, J. J. (1981). Nature (London) 289,373-378. Wilson, I. A., Skehel, J. J., and Wiley, D. C. (1981). Nature (London) 2 8 9 , 366-373. Yewdell, J. W., and Gerhard, W. (1981). Annu. Reu.Microbiol. 3 5 , 185-206. Youle, R. J., and Neville, D. M. (1980). Proc. Natl. Acud. Sci. U.S.A. 77,5483-5486. Zweig, M., Heilman, C. J., Rabin, H., and Hampar, B. (1980). J. Virol. 35,644-652.

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Evamarie Sander and Ralf G Dietzgen lnstitut Biologie II Universitat Tubingen Tubingen. Federal Republic of Germany

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Production of Monoclonal Antibodies against Plant Viruses . . . . . . . . A . Immunization of Mice . . . . . . . . . . . . . . . . . . . . . . B . Myeloma Line and Cell Culture Conditions . . . . . . . . . . . . . C. Isolation of Spleen Cells . . . . . . . . . . . . . . . . . . . . . D . CellFusion . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Hybridomas and Cloning . . . . . . . . . . . . . . . . . . . . . F. ScreeningforVirus-SpecificHybridoma Antibodies . . . . . . . . . G . Large Scale Production of Monoclonal Antibodies (MCA). . . . . . . H . Purification of Monoclonal Antibodies . . . . . . . . . . . . . . . I. Conservation of Hybridoma and Myeloma Lines . . . . . . . . . . . 111. Characterization of Monoclonal Antibodies . . . . . . . . . . . . . . . A. Homogeneity of Hybridoma Lines . . . . . . . . . . . . . . . . . B. Determination of Antibody Specificity . . . . . . . . . . . . . . . C. Determination of Immunoglobulin (Ig) Isotype . . . . . . . . . . . D . Nomenclature of Monoclonal Antibodies . . . . . . . . . . . . . . IV . Monoclonal Antibody-Determined Antigenic Properties . . . . . . . . . A. Alfalfa Mosaic Virus . . . . . . . . . . . . . . . . . . . . . . . B. IlarViruses . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Luteo Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . D . Nepo Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . E. Poty Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . F. Sobemo Viruses . . . . . . . . . . . . . . . . . . . . . . . . . G . Tobamo Viruses . . . . . . . . . . . . . . . . . . . . . . . . . V . Application of Monoclonal Antibodies for Virus Diagnosis . . . . . . . . A. Prunus Necrotic Ringspot and Apple Mosaic Virus . . . . . . . . . . B. Soybean Mosaic Virus . . . . . . . . . . . . . . . . . . . . . . C. Potato Virus Y . . . . . . . . . . . . . . . . . . . . . . . . . VI . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131 133 133 137 137 138 142 144 145 146 146 146 146 147 148 149 150 150 150 151 152 153 154 154 161 161 161 162 163 165

I . INTRODUCTION Ever since antigenic properties of plant viruses were discovered (Dvorak. 1927; Purdy. 1928. 1929) antisera have been raised and used for plant virus diagnosis and for the analysis of virus structure as well (Tsugita 131

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EVAMARIE SANDER AND RALF G. DIETZGEN

etal., 1960; Anderer, 1963; van Regenmortel etal., 1983). From the early qualitative diagnosis method of precipitating the virus in clarified sap of an infected plant (Beale, 1931) and the first quantitative application of the precipitin test (Schramm and Friedrich-Freksa, 1941) vast progress has been made with regard to the development of highly sensitive and highly quantitative methods for virus detection. Of equal importance was the improvement of methods for separating virus from host cell components since the specificity of antisera raised against a virus could be increased by using an antigen for immunization highly concentrated and largely freed from contaminating host substances. The introduction of the enzymelinked immunosorbent assay (ELISA) into plant virology (Clark and Adams, 1977) allows detection of virus in nanogram quantities. Still, the conventionally raised antisera, no matter how pure an antigen was used for immunization, are polyclonal. They contain products of thousands of different antibody-secreting plasma cell clones which can be directed against all antigenic determinants (epitopes) of the virus, but also against antigens of the host plant that may not have been entirely separated from the immunizing virus during the purification procedure. Even after cross adsorption of polyclonal antisera some residual heterogeneity can be expected to remain. Within these boundaries the information gained with polyclonal antisera on virus structure and on virus diagnosis has to be interpreted. Separate homogenous antibodies of defined specificity can now be achieved by the method of somatic cell hybridization (Kohler and Milstein, 1975). Single immunocompetent B-lymphocytes can be rendered ‘‘immortal” by fusion with a myeloma cell and thus become separated from the multitude of different antibody-secreting clones in a warm-blooded animal. The antibodies secreted by a cell hybrid (hybridoma) clone are by definition monoclonal and therefore directed against one defined epitope on for instance the immunizing virus. It is with monoclonal antibodies that many of the results obtained with polyclonal antibodies have been reevaluated, often verified and often improved with regard to fine structure of virus coat protein and the corresponding biological function, location of epitopes, detection and discrimination of cryptotopes and neotopes, differentiation of virus strains and taxonomic groups, and also with regard to more sensitive and specific virus diagnosis. A host of information in this respect is already available in medical virology (Carter and ter Meulen, this volume). But after the first report on monoclonal antibodies directed against a plant virus (Dietzgen and Sander, 1981) rapid progress has been achieved by workers in plant virology as well, summarized in this review (Table I).

MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES

133

11. PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES For the production of antibody-secreting cell clones spleen cells of mice immunized with antigen are fused with myeloma cells of murine origin. The resulting hybrid cells (hybridomas) that secrete antigen-specific antibodies are then cloned so that cell lines may be obtained which originated from a single hybridoma. Each cell line or clone produces antibodies of only one type and antigen specificity, the monoclonal antibodies. A. Immunization of Mice

Most workers immunize mice of the BALB/c type (A1 Moudallal et al., 1982; Briand et al., 1982; Gugerli and Fries, 1983); others (Dietzgen and Sander, 1982) prefer the STU strain (Schafer, 1979) renowned for its high immune response. High antibody titers depict a large amount of activated B lymphocytes desirable to increase the chance to obtain in high number hybridomas which produce antibodies specific for the immunizing antigen. Usually 2- to 4-month-old mice are immunized with antigen quantities ranging from 60pg (Gugerli and Fries, 1983)to 4-6 mg (Dietzgen and Sander, 1982). With regard to the amount of immunizations and the loci of injection the immunization patterns vary widely. But all patterns have in common that the antigen for the first immunization is emulgated in Freund's Adjuvant (v/v), that the interval between two immunizations is not less than 2 weeks, and that the last immunization, usually intravenous (iv) (Dietzgen and Sander, 1982; Gugerli and Fries, 1983) or intraperitoneal (ip) (A1 Moudallal et al., 1982; Briand etal., 1982) and without adjuvant is 3 to 4 days previous to the fusion of spleen and myeloma cells (cell fusion). Differences in immune response between male or female BALB/c mice are not yet mentioned in the literature. With STU mice, female animals appear to withstand higher antigen concentratiow and show higher immune response than males. In order to raise antibodies, plant viruses can be injected iv, subAbbreviations: AMV, alfalfa mosaic virus; ApMV, apple mosaic virus; ArMV, arabis mosaic virus; BYDV, barley yellow dwarf virus; CERV, carnation etched ring virus; CLRV, citrus leaf rugose virus; CVV, citrus variegation virus; LMV, lettuce mosaic virus; MDMV, maize dwarf mosaic virus; PDV, prune dwarf virus; PLRV, potato leafroll virus; PNRV, prunus necrotic ringspot virus; PVA, potato virus A; PVY, potato virus Y; SBMV, southern bean mosaic virus; SCRLV, subterranean clover red leaf virus; SMV, soybean mosaic virus; SoMV, sowbane mosaic virus; TBV, tulip breaking virus; TMV, tobacco mosaic virus; TomRV, tomato ringspot virus; TobRV, tobacco ringspot virus; TRosV, turnip rosette virus; TSV, tobacco streak virus.

TABLE I MONOCLONAL ANTIBODIESAGAINSTPLANT VIRUSES Virus

+ w

+

Abbreviation

Virus group

Immunizing strain

Reference

Alfalfa mosaic

AMV

Alfalfa mosaic

ATCC PV 92

Apple mosaic

ApMV

Ilar

F (apple isolate)

Arabis mosaic Barley Yellow dwarf

ArMV BYDV

Nepo Luteo

ATCC PV 192 MAV, RPV, PAV

Carnation etched ring Citrus leaf rugose Citrus variegation Lettuce mosaic

CERV

Caulimo

Halk etal. (1982b,c); Halk etal. (1984); Hsu etal.(1983b); Halk etal. (1982a,b,c); Halk and Franke (1983); Halk etal. (1984); Hsu etal. (1983b) Dietzgen (1983) Diaco etal. (1983); Hewish etal. (1983) Hsu etal. (1983a,b) Hsu etal. (1983a,b,c)

CLRV

Ilar

Hsu etal. (1983b)

cvv LMV

Ilar Poty

ATCC PV 63

MDMV

Poty

Ap; B(ATCC PV 53)

Maize dwarf mosaic

Hsu etal. (198313) Hill eta1 (1983); Hill etal. (1984) Hill etal. (1983); Hill etal. (1984)

Potato leafroll

PLRV

Luteo

Suisse isolate

Potato A Potato Y Prunus necrotic ringspot

PVA PVY PNRV

Poty Poty Ilar

N605 G (ATCC PV 22)

Southern bean mosaic Soybean mosaic

SBMV

Sobemo

B, C

SMV

Poty

Ia 75-16-1

Subterranean clover red leaf Tobacco mosaic

SCRLV

Luteo

TMV

Tobamo

vulgare

Tobacco streak

TSV

Ilar

WC (ATCC PV 276)

Tomato ringspot Tulip breaking

TomRV TBV

Nepo Poty

Apple isolate

Dietzgen (1983) Gugerli (1983a,b); Martin and Stace-Smith (1983) Gugerli (1983a,b) Gugerli and Fries (1983) Halk etal. (1982a,b,c); Halk etal. (1984); Hsu etal. (1983b) Tremaine and Ronald (1983) J. H. Tremaine (personal communication) Hill etal. (1983); Hill eta2. (1984) Hewish etal. (1983) A1 Moudallal etal. (1982); Briand etal. (1982); Dietzgen and Sander (1981, 1982, 1983); Dietzgen (1983) Halk etal. (1982b,c); Halk etal. (1984); Hsu etal. (1983b) Powell and Marquez (1983) Hsu etal. (1983b,c)

136

EVAMARIE SANDER AND RALF G . DIETZGEN

cutaneously (sc), ip, and intramuscularly (im). With two 1OOpg ip injections of 3 poty viruses (SMV, LMV, MDMV)' into separate animals Hill et al. (1984) obtained an antibody titer of 1 :256 as determined by microprecipitin or interfacial ring test. Other authors (Dietzgen, 1983) used a pattern of 2 - 4 sc injections followed by an iv injection in separate mice comprising to 4 - 6 mg TMV, 300 pg ArMV, and 400 - 500 pg PLRV giving rise to an antibody titer of 1 :lo4- lo5 (TMV), 1 :lo4 (ArMV), and 1 : lo3lo4 (PLRV) detected by an indirect ELISA. Halk etal. (1984) deviated from these patterns by injecting a mixture of up to four viruses into one animal instead of only one virus. With 2 - 3 ip or iv injections, the last of which was always iv, a total of 120 - 180,ug of PNRV, the same quantity of ApMV, and 600- 750 ,ug each of TSV and AMV were applied. The resulting antibody titer for each virus was determined by an indirect ELISA and amounted to 1: 1.5 X lo5 (PNRV, ApMV), 1 : 4 X lo6 (TSV), and 1 :1 x 105 (AMV). In order to increase the amount of hybridomas that secrete antibodies against the immunizing virus it is recommended to booster the animal 3-4 days before explantation of the spleen since the activated B-lymphocytes specific for the immunizing virus will have assembled there within this span of time (Claflin and Williams, 1978; Sprent, personal communication). This may be indicated by an antibody titer lower at the date of spleen explantation than at the day of the last virus injection. All authors employed at least sucrose gradient purified virus (Halk etal., 1984;Hill etal., 1984; Gugerli and Fries, 1983) if not virus subjected to CsC1 density gradient centrifugation (Dietzgen and Sander, 1982; Hill etal., 1984). A comparison of the amounts of virus injected and the corresponding antibody titers seems to indicate that the amount of antigen injected may not have as much influence on antibody production as the antigenicity of the virus, the immune response of the recipient animal, and, with regard to variety in antibody specificity, the locus of injection (Section IV,G). It is as advisable for the production of monoclonal antibodies (MCA) as it is for the conventional production of antisera to immunize with highly purified antigen in order to obtain antigen-specific antibodies with the least amount of antibodies specific for any host cell components which may have contaminated the viral antigen. However, there are several viruses that cannot a t all or only partially be purified as yet or not in sufficient quantity (Halk etal., 1984). Partially purified virus preparations are highly contaminated with host material and thus give rise to an antibody population of many other specificities besides that directed against the virus. The great advantage of antibody production of hybridoma clones is that even though crude antigen is injected, by way of screening, clones that

MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES

137

secrete antibodies specific for viral antigens can be separated from those that secrete host specific antibodies. The use of highly purified virus for hyperimmunization of mice can be expected to lead to a high proportion of virus-specific antibody-secreting hybridomas within the population of growing hybridomas from the corresponding cell fusion (Dietzgen and Sander, 1982).

3.MyelomaLineand CellCulture Conditions The myeloma lines most commonly used in the production of monoclonal antibodies against plant viruses are either of the non-antibody-producing or of the non-antibody-secreting type, as described by Hammerling and Hammerling (1981). The lines X63-Ag8.653, SP2/0-Ag 14, and FO used by Halk etal.(1984), Martin and Stace-Smith (1983), Hill etal. (1984), and Gugerli and Fries (1983), respectively, did not produce any antibodies of their own, whereas the line P3-NSl/l-Ag4-1 (NS1) employed by A1 Moudallal etal. (1982), Briand etal. (1982), Dietzgen and Sander (1982),and Halk etal. (1984)produces kappa light chains intracellularly, which may appear in the virus-specific antibodies secreted by some hybridomas (Goding, 1980; Galfr6 and Milstein, 1981). Therefore, it is recommended that nonproducer myeloma lines are used instead, although they were found to be sometimes less efficient for the generation of specific antibody-secreting hybridomas (Fazekas de St. Groth and Scheidegger, 1980; McCullough etaL, 1983). For the cultivation of the myeloma and hybridoma cells the most commonly used media are RPMI 1640 (Roswell Park Memorial Institute, Moore etal., 1967) and DMEM (Dulbecco's modified Eagle's medium, Dulbecco and Freeman, 1959; Galfri! and Milstein, 1981). They are buffered with Hepes or sodium bicarbonate and supplemented with 10- 20% fetal calf serum (FCS) and various other cell growth-promoting substances according the modifications each author has developed to suit his experiments (complete medium). Hardly any modification is known of the conditions for cell growth which are 37"C, in a humidified atmosphere containing 5 - 10% CO,/air.

C. Isolation of Spleen Cells The explanted spleen of a mouse hyperimmunized with plant virus or 2 spleens combined (A1 Moudallal etal., 1982) are passed through a stain-

138

EVAMARIE SANDER AND RALF G. DIETZGEN

less-steel sieve of 75 mesh pore size (Dietzgen, 1983) and the resulting cells suspended in Dulbecco's phosphate-buffered saline (DPBS; Dulbecco and Vogt, 1954; Dietzgen and Sander, 1982) or in serum-free culture medium (Hill etal., 1984). Also homogenization of spleen tissue with a glass or Teflon homogenizer can be employed to yield single spleen cells. Another possibility is reported by Halk etal.(1984), and Siraganian etal.(1983) who repeatedly injected serum-free medium into the organ by which the spleen cells are gently dispersed.

D. CellFusion The fusion of spleen cells with myeloma cells has been achieved by different methods. A1 Moudallal etal.(1982) and Briand etal.(1982) followed the method described by Galfr6 etal. (1977); Hill etal. (1984) combined this method with one described by van Deusen and Whetstone (1981). Halk etal. (1984) conducted the fusion according to Galfr6 etal. (1977) combined with the method of Nowinski etal.(1979) and Dietzgen and Sander (1982) followed largely the procedure of Hammerling (1977). Usually a total amount of 5 X lo7 to 3 X lo8 cells were involved in one fusion experiment and the proportion of spleen cells/myeloma cells varied from 1 :1 to 10 :1 (Table 11). Successful fusion depends to a large extent on the physiological conditions of the myeloma cells (Galfr6 and Milstein, 1981). Therefore, many authors optimize the amount of fusion-competent, exponentially growing cells by diluting the myeloma culture with new medium about 3 days before fusion to improve growth conditions (Dietzgen and Sander, 1982; Halk et al., 1984; Nowinski etal., 1979). Cell fusion is mediated by PEG (polyethylene glycol) the proportion of which is adjusted according to the amount of cells involved. Source and concentration of PEG are critical as reviewed by Fazekas de St. Groth and Scheidegger (1980) and Goding (1980). Halk etal.(1984) added 1.5 ml 40%PEG 4000 (w/v) to 1 X 108ce11s,whereas Dietzgen and Sander (1982) added 0.7 ml50% PEG 4000 (w/v) to the same amount of cells. Hill etal. (1984) employed 0.5 ml45% PEG 1540 with 1 X lo8 cells. Fusion between a single spleen cell and a single myeloma cell can also be mediated by an electrophysical procedure described by Zimmermann (1982). The fusion mixture is finally plated into 24- or 96-well tissue culture plates (1 ml or 0.1-0.2 ml cultures) the wells containing either HAT (Littlefield, 1964) selective medium (Dietzgen and Sander, 1982; Halk et

MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES

139

al., 1984) or medium to which HAT is added 1 day after cell plating (A1 1983). A cell Moudallal etal., 1982;Briand etal., 1982; McCullough etal., density of 1X lo6 (A1 Moudallal etal., 1982; Briand etal., 1982; Dietzgen 1984) or 1 X lo6cells/well (Halk etal., 1984) and Sander, 1982; Hill etal., has proved successful. Since only hybridomas can grow in this medium the dead myeloma cells and spleen cells should be eliminated. This can be achieved by the addition of peritoneal macrophages (Fazekas de St. Groth and Scheidegger, 1980) usually in a concentration of 5 X 104-1 X 106/ml (Dietzgen and Sander, 1982; Halk etal., 1984). Macrophages serve a double purpose: they not only remove cellular debris but they also excrete substances promoting the growth of hybridomas. They should not be extracted from infected or immunized animals and should not contain erythrocytes since in both cases the macrophages will become activated and will then include hybridomas into their phagocytosis. Instead of macrophages hybridoma growth can also be promoted by media conditioned by the growth of myeloma cells (Hill etal., 1984), spleen cells, or fibroblasts. In principle, 1982; hybridoma cells also grow in HAT medium alone (A1Moudallal etal., Briand etal., 1982),but according to our experience cell growth is enhanced by the mentioned promoters. With antigens other than plant viruses, Fox etal. (1981) and Siraganian etal. (1983) cultured spleen cells from immunized mice in uitro in the presence of the immunizing antigen in order to concentrate antigen-specific antibody-producing B-lymphocytes before the fusion with myeloma cells. This way the low content of such B-lymphocytes in the spleen and thereby the frequency of antigen-specific antibody-secreting (so called positive) hybridomas after cell fusion can eventually be improved. According to Hammerling and Hammerling (1981) and Stahli etal. (1983)the frequency of positive hybridomas is directly correlated to the amount of antigen-specific B-lymphocyte blast cells in the spleen of immunized animals used for cell fusion. With this method R. G. Dietzgen (unpublished results) attempted to enhance the frequency of hybridomas secreting TMV-specific antibodies. He incubated the pooled spleen cells of 5 TMV-hyperimmunized mice with the virus (3.4 X los cells; 10 pg TMV/ ml) 3 days and subjected the recovered 3 X lo' viable cells with an equal amount of myeloma cells to fusion conditions. Of the resulting 72 growing hybridoma cultures 47% secreted TMV and/or TMV protein-specific antibodies. When mice were immunized with a total of 4-6 mg TMV, an average of 54.5% of hybridoma cultures secreting antibodies of such specificity were obtained without the additional in uitro booster (Table 11). An in uitro booster would therefore be a means to improve the yield of positive

TABLE I1 FREQUENCY OF VIRUS-SPECIFIC ANTIBODY-SECRETING HYBRID OM AS^

r 4

_

Number of plated cultures Immunizing virus and number of cell fusion

Total number of cells/fusion

Spleen cells/ myeloma cells

Total

Containing growing hybridomas

Containing positive hybridomas

_

~

~ __ ~ _

Positive hybridoma cultures/growing hybridoma cultures (%)

~

TMV- Ib TMV- 2 TMV- 3 TMV- 4 TMV- 5 TMV- 6 TMV- 7 TMV- 8 TMV- 9 TMV-10 ArMV- l b

9.3 x 107 1.15 X 10' 1.0 x 108 8.3 X 10' 1.57 X lo8 2.6 X lo* 1.38 X lo8 2.3 X 108 6.4 x 107 1.7 X 108 2.0 x 108

1.3 :1 1.3:l 1.0: 1 5.4: 1 3.2: 1 10.0: 1 1.7: 1 1.9 : 1 1.0: 1 1.8: 1 3.4: 1

89 89 86 70 96 96 96 120 72 144 168

45 (50.5%) 89 (100.0%) 74 (86.0%) 66 (94.3%) 85 (88.5%) 56 (58.3%) 94 (97.9%) 120 (100.0%) 72 (100.0%) 85 (59.0%) 19 (11.3%)

28 (30.3%) 85 (95.5%) 59 (68.6%) 46 (65.7%) 53 (55.2%) 47 (49.0%) 13 (13.5%) 23 (19.2%) 34 (47.2%) 40 (27.8%) 1 (0.6%)

62.2 95.5 79.7 68.2 62.3 83.9 13.8 19.2 47.2 47.1 5.3

ArMV- 2 ArMV- 3 ArMV- 4 PLRV- 1* PLRV- 2 PLRV- 3 PLRV- 4 PLRV- 5 PVY - 1' PVY- 2 Virus mix-ld Virus mix-2 O1

-

F

rp

2.2 x 1.4 X 1.38 X 1.65 X 2.25 X 2.25 X 2.0 x 3.08 X 1.0 x 1.0 x 1.1

1.1

108 los lo8 lo8 lo8 lo8 108 lo8 108 108

x 108 x 108

3.4: 1 1.3:l 1.8: 1 3.1:l 1.25: 1 3.5:l 3.0: 1 10.0: 1 1.0: 1 1.0: 1 10.0: 1 10.0: 1

]

168 168 168 112 120 144 144 144

35 (20.8%) 47 (28.0%) 89 (53.0%) 112 (100.0%) 120 (100.0%) 128 (88.9%) 131 (90.9%) 140 (97.2%)

10 (6.0%) 1 (0.6%) 10 (6.0%) 22 (19.7%) 17 (14.2%) 8 (5.6%) 25 (17.4%) 44 (30.5%)

28.6 2.1 11.2 19.6 14.2 6.3 19.1 31.4

480

447 (93.1%)

99 (20.6%)

22.1

15 (1.7%) 21 (2.4%)

30.0 35.0

864 864

50 60

(5.8%) (7.0%)

Frequency of virus-specific antibody-secreting (i.e., positive) hybridomas, 3-4 weeks after cell fusion.

* TMV-1 to 6 (Dietzgen and Sander, 1982); TMV-7 to 10, ArMV-1 to 4, PLRV-1 to 5 (Dietzgen, 1983);pg of viral antigen injected per mouse: TMV, 4000-6000; ArMV, 300; PLRV, 400-500. Gugerli and Fries (1983); 60 pg PVY injected per mouse. Halk et al. (1984):fusion 1:120pg of each PNRV and ApMV, 600pgofeach TSV and AMV injectedper mouse; fusion 2: 18Opgofeach PNRV and ApMV, 750 pg of each TSV and AMV injected per mouse.

142

EVAMARIE SANDER AND RALF G. DIETZGEN

hybridomas only when limited amounts of antigen are available and/or poor immunogens are used.

E. Hybridomas and Cloning After the fusion procedure the cells are plated into HAT medium designed to promote growth of true somatic hybrids between spleen cells and myeloma cells, the hybridomas. Nonfused myeloma cells and nonfused spleen cells lose their viability in this medium within 21 days andcannot be detected any longer. Since the spleen cells can liberate antigen-specific antibodies during this interval (Fig. 1)a screening for positive hybridoma cultures should begin only thereafter. The yield of positive hybridomas per fusion varies greatly in relation to the fusion efficiency, i.e., the amount of growing hybridoma cultures, and the amount and antigenicity of virus used for immunization. This is the case with different viruses as well as with the same virus in different fusion experiments (Table 11, viz. TMV, ArMV, PLRV). The ratio of spleen : myeloma cells when within 1- 10 :1 appears to be without impact on the fusion efficiency (Table 11; Goding, 1980). Not all the hybridomas which secrete the antibody of desired specificity develop into stable cell lines. They tend to lose their ability to secrete antibodies or their viability. Halk et al. (1984) state that within 5 to 6 weeks 26.7-90.5% of the positive hybridoma cultures survived; other authors speak of 20-40% (Claflin and Williams, 1978) or 50-70% (van Deusen and Whetstone, 1981); Powell and Marquez (1983) observed only 69.3% survival after a single passage of the cell cultures. Our observations are in agreement with those of the other authors. Clones secreting viral antigen-specific monoclonal antibodies are obtained by all plant virus workers by 2 - 3 cycles of limiting dilution cloning. This can be effected (1)in complete medium (Halk etal.,3 984), (2) in medium conditioned by growth of myeloma cells (Hill et al., 1984), of nonimmunized spleen cells, or fibroblasts (Halk etal., 1984; Hsu etal., 1983a), and (3) in the presence of a layer of feeder cells (Goding, 1980; Hammerling and Hammerling, 1981) such as 2 -5 X lo4peritoneal macrophages/ml (Dietzgen and Sander, 1982;Gugerli and Fries, 1983) or 1X lo7 thymocytes/ml (Briand etal., 1982; Siraganian etal., 1983). Halk etal. (1984) compared the effect on clone growth of two conditioned media and the complete but nonconditioned medium and obtained comparable amounts of viable clones and, therefore, continued to use the nonconditioned medium. According to our experience the amount of viable clones is increased by the use of peritoneal macrophages as described by Galfrk and Milstein (1981).

143

MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES

0.10

0.08

0.05

0

z P

0.04 3

3 0.02

0.00

1

7

14

20

DAYS AFTER CULTURE IN H A T MEDIUM FIG.1. Titer of TMV antibodies and longevity of nonfused spleen cells of a TMV immunized mouse. Spleen cells of the immunized mouse were cultured in HAT medium under the same conditions as were the fused cells. The number of spleen cells (.)was determined, the cells then sedimented, and supernatants (0)and resuspended cells (A) assayed for TMV antibodies by the indirect ELISA. From Dietzgen and Sander (1982), with permission of Springer-Verlag, Wien.

The efficiency of cloning, i.e., positive hybridoma clones as proportion of all growing clones, is reported to be 10-50% with PVY (Gugerli and Fries, 1983) and 24-30% with a mixture of 3 Ilar viruses and AMV (Halk etal., 1984). With TMV- and ArMV-specific antibody-secreting hybridomas, Dietzgen (1983) observed an increase in cloning efficiency from 40%after

144

EVAMARIE SANDER AND RALF G. DIETZGEN

the first, to 60- 100%after the third subcloning (TMV) and from 20 to 75% (ArMV). This confirms the general observation that with each cycle of limiting dilution more genetically stable and viable antibody-secreting hybridoma clones are selected. Besides the limiting dilution cloning in culture medium (Hammerling and Hammerling, 1981) used by all plant virus workers, there are other methods such as cloning in soft agar (McCullough etal., 1983), in methylcellulose (Davis etal., 1982),or automatically with FACS (fluorescent-activated cell sorter; Dangl and Herzenberg, 1982).

F. Screening forVirus-Specific HybridomaAntibodies The detection of positive hybridomas after the fusion as well as after each cloning is effected by an indirect ELISA (Douillard and Hoffman, 1983) with the immunizing antigen as antibody-detecting agent, using either antibody-precoated microtiter plates, or direct coating with the purified viral antigen (Koenig, 1981; Dietzgen, 1983; Gugerli and Fries, 1983). It is recommended to block unoccupied binding sites on the solid phase with 1%bovine serum albumin in saline (Kearney etal., 1979). In order to avoid nonspecific reactions in the ELISA we found it important to incubate the culture supernatants during the assay at a 10% CO,/air atmosphere (Dietzgen and Sander, 1982). According to most recent results (A1 Moudallal etal., 1984) the efficiency of detecting positive clones has been improved by multilayered sandwich ELISA as compared to the indirect ELISA. This way clones with low output of antibodies can be detected as well as clones secreting antibodies with low affinity to the immunizing antigen. Other screening methods besides the ELISA are reviewed by Hammerling and Hammerling (1981). After the fusion the screening of the supernatant of growing cell cultures should begin only after 21 days since then it can be expected that antigenspecific antibodies are derived from hybridomas and not from remaining spleen cells (Fig. 1). A confluence of cells between 25 and 50% of the culture wells and 1 week of incubation without changing the medium is recommended to obtain an antibody concentration sufficient for detection in the ELISA (Halk etal., 1984; R. G. Dietzgen, unpublished results). In order to detect secreted hybridoma antibodies of the main imunoglobulin classes, antiglobulins raised against mouse-IgG, -IgM, and -1gA should be employed as enzyme-marked antibodies in the indirect ELISA. Dietzgen and Sander (1982) screened with both, the entire antigen used for immunization (TMV) and the viral capsid monomers. Thus they

MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES

145

found hybridomas that secreted antibodies reacting with the nucleocapsid but not with the capsid monomers and such reacting vice versa or such that reacted with both. This indicates that by screening with the entire immunizing agent only, hybridomas that secrete antibodies directed against epitopes hidden in the nucleocapsid (cryptotopes), but important in the study of structure and conformation of the viral coat protein, are easily discarded. Cases are known where monoclonal antibodies with heterospecific activity, i.e., “antibodies that are better or only recognized by another antigen than the one used for immunization” can be detected by screening with a mutant of the immunizing virus instead of the wild type (van Regenmortel et al., 1983).

G. Large Scale Production of Monoclonal Antibodies (MCA) There are two ways generally used to obtain large quantities of MCA by either culturing the respective hybridoma clone in suspension in uitro (Galfr6 and Milstein, 1981) or in uiuo by growing the hybridoma cells as tumors in Pristan-primed mice and yield the antibodies from the ascitic fluid (van Deusen and Whetstone, 1981; Hoogenraad et al., 1983). The in uitro culture can be effected in vessels kept either stationary, as roller culture or as spinner culture either in a water-jacketted vessel (Galfr6 and Milstein, 1981) or in a cytostate (Feder and Tolbert, 1983; Fazekas de St. Groth, 1983). The yield of MCA from i n uitro cultures can be improved by spleen cell- or fibroblast-conditioned medium or by a feeder layer, for instance peritoneal macrophages (Galfr6 and Milstein, 1981). In culture supernatants quantities of plant virus-specific antibodies between 10pg/ml (Gugerli and Fries, 1983) and 2-46pg/ml (Dietzgen, 1983) were reported. This amount is well within the order of magnitude (10- 100 pg/ml) obtained with antibodies of specificity other than against plant viruses (Galfr6 and Milstein, 1981). When 2 X 105-1 X 10’ hybridoma cells were injected ip into Pristanprimed mice, the titer of antibodies directed against Ilar, Luteo, Sobemo, and Tobamo viruses (Table I) is reported to range between 1: lo2 and 1: lo8, but is mostly found to be 1: lo”. Amounts from 5 to 20 mg MCA/ml ascitic fluid can be expected (Galfr6 and Milstein, 1981). It should be kept in mind that it cannot be excluded that the desired MCA, when produced in ascites, will be accompanied by antibodies of other specificities contributed by the mouse, whereas from in uitro cultures only the desired MCA will be derived provided synthetic serum-free medium is used. A medium containing FCS will contribute calf antibodies

146

EVAMARIE SANDER AND RALF G. DIETZGEN

which can, however, be removed by affinity chromatography (Underwood et al., 1983). H. Purification of Monoclonal Antibodies

Methods to purify monoclonal antibodies are reviewed by Goding (1980) and Schreier et al. (1980). Antibodies contained in the supernatant of hybridoma cultures were concentrated with 50% saturated ammonium sulfate by Gugerli and Fries (1983) and Dietzgen (1983). The latter reports that by this step 30%of the proteins were precipitated and contained the entire antibody activity. Further purification was effected by ion exchange chromatography (Gugerli and Fries, 1983) or by affinity chromatography on protein A-sepharose (Dietzgen, 1983). From ascitic fluid the antibodies were purified by affinity chromatography on protein A-sepharose directly (Hill et al., 1984);usually the concentration of antibodies is so high that the concentration step can be omitted. It was obviously for that reason that A1 Moudallal et al. (1982) employed the untreated ascitic fluid in the ELISA to characterize antibody specificity. I. Conservation of Hybridoma and Myeloma Lines

The principles of methods to conserve hybridoma and myeloma cells are reviewed by Fazekas de St. Groth and Scheidegger (1980) and by Galfr6 and Milstein (1981). Most workers in the plant virus field have stored the cells in liquid nitrogen after freezing them slowly to - 70°C in a density of 1 X lo6-1 X lo7 cells/ml medium in the presence of 10%DMSO (Halk et al., 1984; Hill et al., 1984). When freezing the cells slowly and thawing them fast, Dietzgen and Sander (1982) recoveredviable antibody-secreting hybridoma cells in the order of 40-50% of the originally frozen number; similar results were obtained with myeloma cells.

111. CHARACTERIZATION OF MONOCLONAL ANTIBODIES A. Homogeneity of Hybridoma Lines

In order to ascertain that an antibody-secreting cell line is truely derived from one hybridoma cell only, all investigators subjected hybridoma cultures to 2- 3 cycles of limiting dilution cloning closely observing a Poisson distribution of cells (Hammerling and Hammerling, 1981). The homoge-

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neity of a hybridoma line can be assessed by labeling the secreted MCA with radioactive amino acids and subsequently subjecting them to SDS polyacrylamide gel electrophoresis or isoelectric focusing (GalfrB and Milstein, 1981). Several limiting dilution cycles serve a second purpose in so far as simultaneously with the establishment of a homogenous clone clones can be selected that are stable in both the secretion of antibodies and the property to grow. For this purpose, even established cell lines should be cloned from time to time as well as frozen cells immediately after thawing.

B. Determination of Antibody Specificity For the application of MCA in virus diagnosis, for serotyping of viruses, for virus purification, and for detection of location, structure, and biological function of antigenic determinants (epitopes) on the coat protein it is highly desirable if not imperative to know the MCA specificity. This task is achieved by most investigators with an indirect ELISA. Much information is gained with this binding test: (1)the reactivity of the MCA, i.e., its ability to react with an antigen or not, and (2) the titer which reflects the concentration and the specific activity of the MCA, i.e., its affinity or binding force to the respective antigen. Compared with the indirect ELISA a recently designed multilayered sandwich ELISA (A1 Moudallal etal., 1984) shows higher detection efficiency. Still another variant, a competition ELISA (Diaco etal., 1983),is highly efficient for epitope mapping, i.e., the determination if MCA secreted by different hybridoma clones are specific for separate or the same viral epitope(s). The interpretation of this test is reviewed by Yewdell and Gerhard (1981). The specificity of a MCA for the immunizing virus strain is detected by reaction with the homologous virion and/or coat protein (Dietzgen and Sander, 1982;Dietzgen, 1983). This discerns not only epitopes exposedon the surface of both nucleocapsid and capsomers, but also neotopes, i.e., epitopes present only on the nucleocapsid, and cryptopes, i.e., epitopes exposed only on the capsomers (van Regenmortel, 1982). A neotope can be comprised of discontinuous amino acid sequences on adjacent capsomers or can originate by conformational changes in the capsomers after assembly of the nucleocapsid. Cryptotopes are amino acid sequences located in regions hidden in the assembled nucleocapsid, but exposed on the separate capsomeres. Besides in the nascent state, virion and coat protein were employed in several forms, swollen or denatured by SDS or formaldehyde in the determination of MCA specificity (J. H. Tremaine, personal communication). To discern whether a MCA is specific not only to one strain but to a

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group of related virus strains or isolates, i.e., if they have an epitope in common thus being of common epitype, the entire virion and/or the coat protein of several of these strains are assayed for binding the MCA (Briand et al., 1982; Gugerli and Fries, 1983;Dietzgen, 1983). Mutants of the wild type virus with known amino acid exchanges also served this purpose (A1 Moudallal etal., 1982). In order to detect if a MCA is specific for more than one virus of the taxonomic group to which the immunizing virus belongs, several members of this virus group and their strains are employed. The MCA specificity for viruses of this taxonomic group is ascertained when no cross-reaction with viruses of other taxonomic groups occurs. Virus specificity of a MCA is ascertained when cross-reaction with proteins from healthy plants can be excluded. This is particularly important when MCA are intended for virus diagnosis. With peptides derived from enzymatic digests of the virus coat protein and with comparable chemically synthesized peptides the specificity of a MCA for a defined epitope on the nucleocapsid and capsomers can be detected (Dietzgen and Sander, 1983). The faculty of a MCA to precipitate the antigen against which it was raised can be assayed in agar gel diffusion tests (Tremaine and Ronald, 1983;Halk etal., 1984). The usefulness of a MCA for electron microscopic virus diagnosis can be tested by immunosorbent electron microscopy (J.H. Tremaine, personal communication). Since of the different Ig classes only IgG can bind to protein A, MCA of this isotype can be characterized by the binding capacity of protein A for each individual MCA (Hill etal., 1984). The specificity of a MCA for an epitope involved in the biological activity of a virus can be detected only by the neutralization of infectivity assayed in a biotest (Dietzgen, 1983; Dietzgen and Sander, 1983).

C. Determination of Immunoglobulin (Ig) Isotype The description of the isotope of an antibody comprises its heavy chain class and subclass and its type of light chain (kappa or lambda). Methods for this determination are described by Hammerling and Hammerling (1981). It is important for the application of MCA to define the isotype since different isotypes differ in several ways, such as in ability and affinity to bind to protein A, frequently chosen for purification, in their ability to fix complement and to bind to other cell types by their Fc region. Two assays were employed in the determination of the isotype of plant virus specific MCA, the indirect ELISA, used by most authors, and the

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agar double diffusion test (Halk etal., 1984). When assaying antibodies from in uitro cultures it is recommended to concentrate them prior to the assay which is not necessary when MCA are raised in uiuo since their concentration in ascitic fluid is usually superior by several orders of magnitude. Of the classes IgG, IgM, and IgA most investigators report having obtained IgG more frequently than the others. Among all, the kappa light chain occurred predominantly. However, Nowinski et al. (1979) and Dietzgen (1983) obtained also MCA containing both kappa and lambda light chains in one clone even after several cycles of limiting dilution cloning. Williams (1978) attributes this phenomenon to a limited heterogeneity of the chains in the MCA.

D. Nomenclature of Monoclonal Antibodies There is no general agreement as yet on the nomenclature of hybridoma clones and their secreted products and different designations are used by different authors. For instance, Dietzgen and Sander (1983, A1 Moudalla1 etal. (1982), Briand etal. (1982), and Dietzgen (1983) attached a running number to each hybridoma clone and the same number to the corresponding MCA which this clone secreted and added the abbreviation of the virus used as immunizing antigen. For each virus the running numbers were started anew. The abbreviation of the virus, virus strain, or isolate used to generate the MCA-secreting cell line together with the number of the hybridoma clone or of the well on the culture plate were used for cell line and MCA by Hill et al. (1984), Diaco etal. (1983), J. H. Tremaine (personal communication), and Hsu etal. (1983a). Gugerli and Fries (1983) attached letters from A to J to their PVY -specific antibody-secreting cultures and attached running numbers to the positive hybridomas after cloning. Still another nomenclature was applied by Halk etal. (1984) and Halk (1983) who attached the abbreviated name of the virus for which the MCA shows specificity as well as the number of the culture plate and the number of the well in which the hybridoma line originated. Perhaps an agreement could be reached t o attach to the term hybridoma clone the abbreviated name of the virus used as immunizing antigen followed by the running number of the antigen positive clone. For the antibody secreted by this clone the corresponding nomenclature would apply, i.e., hybridoma clone TMV 95 and MCA TMV 95. Other specificities like reactivity, affinity, Ig class, subclass, and light chain type and other properties may follow this basic designation in the suggested order.

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IV. MONOCLONAL ANTIBODY-DETERMINED ANTIGENICPROPERTIES Antigenic structures of plant viruses have been investigated already by means of conventional polyclonal antisera. Since the advent of monoclonal antibodies these served as very sensitive tools to evaluate the former results, many of which could be verified with monoclonal antibodies and interesting new aspects could be revealed. Since the information obtained by the reactivity of a MCA in different types of assay can reveal different properties of a viral epitope, more than one method should be employed to characterize the structure of this epitope and eventually its biological function. From the information gained by MCA so far due to their discriminatory faculty superior to that of polyclonal antibodies, contributions can be expected with regard to the fine structure of the viral coat protein (epitope mapping), taxonomy (serotyping), and epidemiology (virus diagnosis).

A. Alfalfa Mosaic Virus MCA against AMV secreted by 2 different clones, obtained by Halk etal. (1984),showed specific reaction with the immunizing virus in the indirect ELISA. They did not form any precipitate with the purified virus in the agar double diffusion test. They did not cross react with Ilar viruses such as PNRV, ApMV, and TSV. This indicated 2 AMV-specific epitopes not present on the Ilar viruses. With these MCA no serological relationship of AMV could be found with members of the Ilar virus group which are described as similar to AMV in physical, chemical, and biological properties (van Vloten-Doting etal., 1981). The information on MCA against AMV is still rather scanty and the report of Halk etal. is the only one yet on the subject.

B. Ilar Viruses Halk etal. (1984) obtained 12 stable hybridoma clones secreting MCA directed against PNRV and ApMV, serologically related viruses (Barbara etal., 1978) and against TSV, all members of the Ilar virus group (Fulton, 1981). By the reactivity of 7 MCA in the indirect ELISA assayed with several strains of PNRV and ApMV, 4 epitopes on each PNRV and ApMV were discerned. One epitope is common to all PNRV strains tested, but is not present on ApMV. Another epitope is also common to these PNRV strains except one and except ApMV which belongs to the same serological Ilar virus subgroup. There are 2 ApMV-specific epitopes, one common to all ApMV

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strains tested and one common to all the strains but one. According to the cross reactivity of 2 MCA, 2 distinct epitopes could be revealed that are common to both PNRV and ApMV. One of these epitopes is present only on some of the strains of both viruses. With the other epitope the binding of the MCA, as measured by the indirect ELISA, was identical with PNRV strains and was less and of differing strength within the ApMV strains (Halk, 1983). This phenomenon may be attributed to slight sequential or conformational changes of the viral protein resulting in similar but not identical epitopes which by MCA can still be discerned. None of the MCA reacted with PDV, a member of the same serological subgroup than PNRV and ApMV, with TSV, another Ilar virus, or with AMV. Two of the ApMV-specific epitopes and one epitope common to both, ApMV and PNRV, are most probably cryptotopes. This is concluded from the result that the respective MCA did not react with the intact, but only with the disrupted viruses in thp DAS (double antibody sandwich) ELISA. This could not be detected in the indirect ELISA when coating the plates with the viruses because thereby, these labile viruses may become partially disrupted thus exposing inner surfaces of the coat protein (Halk, 1983). With MCA directed against TSV secreted by 5 different clones 4 different epitopes were discerned each common to 2,3, or 4 strains of TSV (Halk, 1983; Halk etal., 1984). Since, as far as is known all TSV isolates cross react with each other’s antiserum, antigenic differences are postulated to be rather slight (Fulton, 1981), but they can be recognized by the MCA obtained. Of the 14 MCA directed against Ilar viruses only 5 could precipitate virus in agar double diffusion tests. If such tests are intended for diagnostic purposes precipitating MCA may be a much better tool since conventional precipitating antisera against Ilar viruses are known to be difficult to raise (Halk etal., 1984).

C. LuteoViruses Several MCA directed against strains of BYDV (MAV, RPV, PAV) were obtained and revealed 5 different epitopes on BYDV (Hsu etal., 1983a; Diaco etal., 1983). Since there were MCA that reacted with one strain only there are specific epitopes on each MAV and RPV. In addition there are epitopes common to all 3 strains and others common to 2 of the strains, to MAV and RPV, or to MAV and PAV. Four epitopes have been found so far on MAV, 3 on RPV, and 2 on PAV. The presence of the latter epitope 1983). was confirmed by MCA in a competition ELISA (Diaco etal., Most Luteo viruses are serologically related (Rochow and Duffus, 1981;

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EVAMARIE SANDER AND RALF G. DIETZGEN

van Regenmortel, 1982). The strains MAV and PAV of BYDV showed identical serological reactivity with polyclonal antisera directed against different Luteo viruses, but did not appear related to RPV. However, with the MCA evidence was obtained that these 3 strains have one epitope in common. The strains MAV, RPV, and PAV are defined by the aphid species by which they are transmitted, MAV and RPV each by a different and PAV by both of these species (Diaco etal., 1983). Since each MCA is specific for a particular epitope it would be interesting to find if there is a region on the virus coat protein or an additional capsid protein responsible for the transmission by a specific aphid which can be detected with a MCA. A region of this nature could perhaps be postulated analogous to the results of Adam et al. (1979) where the transmission of pea enation mosaic virus by an apid species was found to be due to an additional capsid protein since it was lacking in the nontransmissible strain. Even though several authors (Table I) have produced MCA directed against PLRV there is very little information available on their properties as yet. The MCA obtained by Martin and Stace-Smith (1983) did not cross react with beet western yellows virus which is known to be serologically related to PLRV as established with polyclonal antisera. Thus with these MCA these 2 viruses can be differentiated.

D. Nepo Viruses Powell and Marquez (1983) selected from their hybridoma clones secreting MCA against TomRV one clone and investigated its reactivity with several isolates of this virus and with TobRV, a member of another Nepo virus subgroup. With this MCA a common epitope was detected on some TomRV isolates, but not on TobRV, found to be nonrelated by the use of polyclonal antisera. As determined by polyclonal antisera within the Nepo virus group there are serologically distinct subgroups, but within each subgroup the members are serologically related (Murant, 1981;van Regenmortel, 1982). On the basis of the reactivity of 29 MCA directed against ArMV with 4 strains of the same subgroup and the coat protein of the immunizing ArMV-AB 10 the presence of 6 distinct epitopes on the virion and/or the coat protein of ArMV was discerned (Dietzgen, 1983). No strain-specific epitope could be detected. Of the common epitopes one is present on all strains tested. One epitope is common to ArMV-AB 10 and ArMV-ivy, another epitope is common to ArMV-AB 10 and ArMV-S, thus the respective MCA discriminates between strains ivy and S. Two more epitopes are

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common to 3 different strains but not to ArMV-S or -E, respectively. By comparing the reactivity of the respective MCA a discrimination between strains S and E is possible. Another epitope, common to all 4 strains tested, is a neotope, only exposed on the surface of the intact virion and not on the isolated coat protein. Among the 29 MCA there were several showing an identical reactivity pattern, but different strength in the indirect ELISA. From them those with the strongest reactivity with the immunizing strain were selected for the determination of antigenic properties. According to preliminary results all MCA directed against ArMV cross reacted with several PLRV isolates and with TMV. Apparently, a cross reactivity as detected with these MCA does not necessarily depict a relationship between these viruses (Yewdell and Gerhard, 1981).

E. Poty Viruses The antigenic properties of 4 members of the Poty virus group, SMV, 1984), and PVY (Gugerli, 1982; Gugerli and LMV, MDMV (Hill etal., Fries, 1983), have been investigated so far with MCA. On each SMV and MDMV strains Ap and B one strain-specific epitope was found, since the respective MCA reacted only with the virus against which it was raised. MCA raised against LMV reacted not only with the immunizingvirus but with SMV and MDMV strains as well even though to a lesser extent. This indicates an epitope common to these Poty viruses, this epitope not being identical, but perhaps of slightly modified amino acid sequence or tertiary structure. No cross reactivity of the MCA was observed with nonrelated viruses like TMV and cowpea mosaic virus. When isolates belonging to the PVY strain groups N (tobacco veinal necrosis), 0 (common), and C (stipple streak) were assayed for antigenic properties by the reactivity of MCA, epitopes were recognized common to all 3 strain groups. Also epitopes strictly specific for the N strain group were discerned, since the respective MCA did not react with any member of the 2 other strain groups. According to the results presented by Gugerli and Fries (1983) several MCA reacted with one or more isolate(s) of one or more strain group(s), indicating for the first time well defined antigenic differences between PVY isolates. This “intermediate specificity” seems to indicate epitopes common to several strains. Experiments with polyclonal antibodies have revealed that the nucleocapsids of the members of the Poty virus group are not as closely related as the respective capsomers (van Regenmortel, 1982), indicating a common

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cryptotope. Perhaps the incorporation of isolated coat protein into the assay for detection of antigenic properties could assist in discerning relationships among the Poty viruses.

F. Sobemo Viruses Information about antigenic properties of Sobemo viruses detected with MCA is still scanty. Tremaine and Ronald (1983) found epitopes specific for the B or C strain of SBMV. Several bean strains of SBMV have an epitope in common. None of the mentioned strains showed any cross reactivity with other Sobemo viruses, such as SoMV and TRosV. According to results obtained with polyclonal antibodies the strains B and C are serologically related, but not identical (Sehgal, 1981). With these MCA however, the strains can be differentiated. The results so far obtained with MCA are in accordance with those obtained with polyclonal antisera by which no serological relationship between different Sobemo viruses could be detected (Sehgal, 1981; van Regenmortel, 1982). J. H. Tremaine (personal communication) introduced an interesting variation into the methods for detection of antigenic properties by changing the conformation of the SBMV particle by swelling and denaturing with SDS or formaldehyde following the scheme described by Hsu et al. (1977). Some of the MCA reacting with the virion in the nascent state reacted also with the virion in its changed form. From such results one is tempted to conclude that there are conformation-independent epitopes determined mainly by the sequence of their amino acids. These epitopes were exposed on the surface of both the virion and the isolatedcoat protein.

G. Tobamo Viruses Since TMV was the first virus ever discovered it has become profoundly described and is frequently employed as a model. Many investigations with polyclonal antisera have been conducted to reveal the antigenic properties of the virus, and 8 epitopes on the TMV protein monomer are described (Altschuh and van Regenmortel, 1982;Altschuh et al., 1983;van Regenmortel et al., 1983). Therefore, it was of great interest to investigate if by the use of MCA the results obtained so far could be confirmed and if new aspects on the antigenic fine structure could be discovered. A1 Moudallal et al. (1982) and Briand et al. (1982) obtained hybridoma clones that secrete MCA directed against TMV vulgare (TMV). By the reactivity of these MCA, epitopes exposed on the surface of the virion only (neotopes) and epitopes on both the nucleocapsid and the coat protein monomers of TMV were discerned. The latter were recognized only after

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the sensitivity of the ELISA system was improved (A1 Moudallal etal., 1984). The fine structure of epitopes on the TMV protein was discovered by the reactivity of 9 TMV-specific MCA with several TMV strains (Briand etal., 1982) and TMV mutants with single or double amino acid substitutions on defined loci in the coat protein (A1 Moudallal etal., 1982). Briand etal. (1982) concluded from their observations that all MCA obtained are fairly strain specific as they reacted mainly with strains that differed from the immunizing strain only by a few amino acid substitutions. This is illustrated by the reactivity of 2 MCA which have been selected as examples (Fig. 2A and B). No cr’oss reactivity with strains distantly related to TMV, for instance HR and U2, was observed in the indirect ELISA. Therefore, there are common epitopes on several closely related TMV strains. Some are identical or resemble each other so closely that they cannot be differentiated by the respective MCA (Fig. 2A: TMV, 03, 01, OM). Other epitopes with minor amino acid substitutions can be differentiated by some MCA because they react less (Fig 2B: 0 3 , 0 1 , OM) or more strongly (Fig. 2B: 0 6 ) with the changed epitopes than with the original epitope on the immunizing virus. The TMV strain 0 6 containing 3 amino acid substitutions as compared to TMV, as well as the TMV mutant 414 containing a single amino acid substitution which is also present in strain 06, both expose an epitope which causes the same MCA to bind stronger than to the corresponding epitope on the immunizing TMV (heterospecific MCA). The amino acid in question is substituted in strain 0 6 and mutant 414 in the same position. It is located in an epitope that has been formerly detected by polyclonal antisera to be within the amino acid sequence between residues 62 and 68 on the TMV protein (Fig. 3). The reactivity of the MCA with this particular epitope seems to indicate that this amino acid substitution is responsible for the stronger binding. Thus, by means of a MCA, already changes in the protein fine structure in the order of one amino acid can be detected. Van Regenmortel etal. (1983)mentioned that a substitution of a single amino acid can even alter an epitope so that the reactivity with the MCA which could bind there before is annulled. A1 Moudallal etal. (1982), who employeda wide variety of TMV mutants with known number and location of amino acid substitutions, contributed results in the same vein as those reported by Briand etal. (1982) with closely related strains. In addition, they presented evidence that amino acid substitutions in regions below the capsomer surface can create longrange conformational changes expressed then in epitopes on the protein surface that can be detected by different reactivity of MCA. These authors also found that some epitopes altered by amino acid substitutions could not be differentiated by polyclonal antisera, but that there were

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FIG. 2. (A) Detection of Tobamo viruses by indirect ELISA, using clone 20 diluted l/SOOO. (B) Detection of Tobamo viruses by indirect ELISA, using clone 19 diluted 1/1500. From Briand et02. (1982), with permission of the authors and Elsevier Biomedical Press, Amsterdam.

MCA which succeeded in the differentiation, thus proving the superior faculty of MCA to discern fine structural antigenic properties. Since the first report of MCA directed against a plant virus (Dietzgen and Sander, 1981) the authors selected from the hybridomas resulting from 10 cell fusions 76 clones that proved to be stable in growth and secretion of MCA directed against the nucleocapsid and/or the coat pro-

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32

FIG.3. Diagrammatic presentation of the folding of the TMV protein molecule, based on the X-ray crystallographic data of Bloomer etal. (1978). The radius scale starts at the center of the hole in the assembled virion. The helices are presented as cylinders. From Milton et al.(1980), with permission of Pergamon, New York.

tein monomers of TMV (Dietzgen and Sander, 1982;Dietzgen, 1983). Not only epitopes exposed on the surface of both nucleocapsid and protein monomers of TMV could be detected, but also neotopes and cryptotopes. The finding of cryptotopes is attributed to the fact that not only the nucleocapsid was used for the screening for antibody-secreting hybridomas, but that also the protein monomers were included in the screening for positive clones. The result that MCA were raised against cryptotopes and, therefore, reacted with protein monomers only, in spite of the mice being immunized with intact TMV, may be due to the presence of protein monomers even in

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virus preparations purified by several cycles of CsCl equilibrium centrifugation. As observed by Milton and van Regenmortel(l979) in such highly purified TMV preparations protein monomers are released spontaneously from the nucleocapsid. According to this observation and a probable degradation of the nucleocapsid in the mouse, antigenically reactive protein monomers may occur. The MCA directed against cryptotopes together with those against neotopes and epitopes on both nucleocapsid and protein monomers will allow a comprehensive analysis of antigenic properties. Because of the different reactivity of several MCA with the nucleocapsid and the protein monomers of the TMV strains vulgare (TMV), dahlemense (d), and Holmes’ Ribgrass (HR), epitopes were discerned common to the nucleocapsid and/or protein monomers of more than one strain. As measured by the indirect ELISA the strength of reactivity could differ indicating similar, but not identical epitopes which can thus be differentiated with MCA. For example the MCA TMV-92 reacted with nucleocapsid and protein monomer of TMV and TMV-d with the same strength, but the reaction with the TMV-HR nucleocapsid and protein monomers was of higher strength. This indicates a common epitope exposed on both nucleocapsid and protein monomers of all 3 strains, which is slightly modified on TMV-HR, thus permitting a better binding of the MCA. The MCA TMV-28 directed against an epitope exposed on both nucleocapsid and protein monomers of TMV did not react with TMV-d and reacted less strongly with TMV-HR. This indicates a similar epitope on both TMV and TMV-HR, not present on TMV-d. In comparison with the TMV-specific MCA obtained by Briand etal. (1982) which could detect epitopes common to closely related strains only, with the MCA obtained by Dietzgen (1983) even epitopes on more distantly related strains like HR could be detected. These different panels of MCA raised against the same TMV strain may be interpreted as resulting from different immunization schemes and different amounts of virus injected at different loci. Both working groups did not find as yet any strictly strain-specific MCA directed against an epitope only present on one Tobamo virus. The structure-dependent function of an epitope could be revealed by the characterization of several properties of the respective MCA (Dietzgen and Sander, 1983). By means of the indirect ELISA it was shown that this particular epitope is exposed on the surface of both the nucleocapsid and the protein monomers of 3 strains, TMV, TMV-d, and TMV-HR. The physical structure of the epitope was characterized with a chemically synthesized peptide. The peptide was made up of the sequence Gly-Pro-Ala-

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Thr of the C-terminal positions 154-158 of the coat protein of TMV vulgare (Fig. 3). This tetrapeptide was conjugated in the nascent sequential order with a carrier protein and used for coating of microtiter plates in the indirect ELISA. This peptide was chosen on the basis of data obtained with polyclonal antisera which describe the C-terminal end of TMV protein as containing an epitope. With the MCA that bound specifically to this tetrapeptide the sequence sufficient to define the epitope was elucidated and the position on the coat protein was confirmed. This epitope was still recognized by the MCA when one (TMV-HR) or two (TMV-d) amino acid(s) were substituted. This can perhaps be explained by the original amino acids being replaced by residues of similar polarity and size with identical functional groups and also by the proposed flexible conformation of the C-terminal region of TMV protein. Because of its high specific activity, measured as increase in Ext.405nm/ hour/100 pg MCA by the indirect ELISA (Engvall and Perlmann, 1971), the MCA firmly bound to the C-terminal epitope, thereby neutralizing the infectivity of TMV as assaysed by biotest on Nicotiana tabacum cv Xanthi (Table 111). This may be attributed to inhibition of the uncoating of the virus caused by a stabilization of the viral capsid in the sense of the allosteric transition model put forward by Mandel (1979). The C-terminal TABLE I11 NEUTRALIZATION OF TMV INFECTIVITY WITH MONOCLONAL AND POLYCLONAL ANTIBODIES Purified antibody sample (1 X g/ml) MCA TMV-95 MCA TMV-62 Monospecific polyclonal rabbit antibodies against TMV Polyclonal antibodies of nonimmunized mice Molecule ratio antibody:TMV

Inhibition of infectivity(%)" TMV concentration (g/ml) 1 x 10-6

1 x 10-6

93.6 43.9

75.6 Not done

93.1

81.0

35.2

31.0

2.67 x 104: 1

2.67 x 103: 1

Inhibition of infectivity (%) = 100 - {([lesions/halfleaf (TMV + antibody)j/[lesions/half-leaf (TMV)]) X 100). Bioassay on Nicotiana tabacum cv Xanthi nc; Dietzgen and Sander (1983).

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l lllltlllll 0 I00A FIG.4. Drawing of the structure of tobacco mosaic virus. From Klug and Caspar (1960), with permission of the authors and Academic Press, New York.

epitope of TMV protein containing the identical amino acid sequence as the chemically synthesized tetrapeptide appears to have a biological function with regard to the process of virus infection (Dietzgen and Sander, 1983). Considering the structure of TMV (Fig. 4) and according to the data obtained by Anderer and Schlumberger (1965) with polyclonal antisera and Dietzgen (1983) with MCA, for virus neutralization a surplus of antibodies is required with regard to the number of respective binding sites on the capsomers of each virus particle. For the first time known quantities of both parameters, purified MCA and virus particles, were employed to achieve neutralization under quantitative aspects. The presented data (Table 111)with MCA TMV-95 show an inhibition of TMV infectivity of nearly 100%which is 45-58% above the background of nonspecific inactivation of TMV caused by polyclonal antibodies of nonimmunized rabbits.

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V. APPLICATIONOF MONOCLONAL ANTIBODIESFORVIRUSDIAGNOSIS Plant virus diagnosis can be impaired by virus-unspecific reactions due to the different quality of polyclonal antisera. Monoclonal antibodies promise considerable improvement of virus diagnosis with regard to specificity, sensitivity, and reproducibility. In order to increase the efficiency of serological plant virus diagnosis the use of MCA with highly characterized properties is required. For each diagnostic purpose the MCA best suited should be chosen. This means in general the selection of the MCA with the highest affinity for the virus to be detected and the desired specificity. Depending on whether one virus strain or a defined group of strains is intended to be diagnosed strain specific or group specific MCA should be applied. If there is no MCA available which combines high affinity and the desired specificity a cocktail of several MCA may serve the purpose. A . Prunus Necrotic Ringspot and AppleMosaicVirus

For the application in the diagnosis of PNRV and ApMV in plants, Halk (1983) selected several MCA with the widest spectrum of reactivity to isolates of these two viruses. He tried several combinations of coating antibody and antibody - enzyme conjugate using polyclonal antisera and MCA in the DAS-ELISA. With all combinations PNRV could be detected in samples of rose and cucumber. The MCA showed comparable or better detection of the virus in the DAS -ELISA than polyclonal antisera. Purified MCA were used as cocktail of 2-3 for coating and for the enzyme conjugate. With a cocktail of 3 MCA as enzyme conjugate, 2 specific for ApMV and one reacting with both, ApMV and PNRV, no ApMV could be detected in infected plants or only when the virus concentration exceeded 100 ng. These MCA were later found to detect only disrupted virus, i.e., perhaps cryptotopes, and are, therefore, not suited to detect intact virus in infected plants by the DAS-ELISA.

B. SoybeanMosaicVirus For diagnostic purposes Hill etal. (1984) compared two methods involving tritium-labeled antibodies, the SPRIA (solid-phase radioimmunoassay), and the competition RIA. The diagnosis of SMV using SPRIA with the same MCA for coating and tritium-labeling lacked sensitivity when compared to the use of polyclonal antibodies. The authors presume this to be due to the competition of the MCA for the same epitope. The respective

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MCA was selected for an affinity to the virus superior to that of polyclonal antibodies. The sensitivity of the SPRIA could be improved when the solid phase was coated with the MCA and the polyclonal antibodies were used for labeling. This improvement was not achieved when the combination was reversed because the polyclonal antibodies bind also to the epitope to which the MCA specifically binds, thereby competing with the latter. (1983) designed a SPRIA in which On the basis of these results Hill etal. two MCA were used, each specific for a different epitope on SMV. All SMV strains tested by the authors could be detected with this assay, the sensitivity of which proved comparable to that with polyclonal antibodies. In a competition RIA in which only a single MCA is required, besides SMV also LMV and the MDMV strains Ap and B could be detected by their respective MCA. The sensitivity of this assay ranged from 10 to 50 ng purified virus/ml and was comparable to that with polyclonal antibodies and the SPRIA. Even when SMV-infected soybean seed extracts were assayed the sensitivity of the competition RIA was retained (Hill, 1983).

C. Potato Virus Y Extensive information is already available on the use of MCA in the diagnosis of PVY. For this purpose a MCA with high avidity toward the PVY isolates used for its characterization was applied in the DAS-ELISA (Gugerli, 1982). With this MCA which is directed against an epitope common to strains belonging to the prominent groups C, N, and 0, of 28 world-wide collected isolates 24 could be diagnosed (Gugerli and Fries, 1983). Perhaps all these PVY strains contain an evolutionary conserved antigenic region. The remaining 4 isolates were found to be members of a rare distinct serotype of PVY which also polyclonal antisera directed against common PVY could not detect. Since, with the MCA used, no reactivity with healthy plants occurred, the respective DAS -ELISA for PVY diagnosis can be considered strictly virus specific. This MCA is meanwhile commercially available and used for large scale diagnosis of seed potatoes (Gugerli, 1983a,b). A comparison of the detection efficiency and sensitivity with this MCA and polyclonal antisera for PVY in leaves of tobacco and potato and potato tubers in the DAS-ELISA was made by Torrance (1983). The sensitivity for virus detection with the MCA was 4-fold superior to that with polyclonal antisera. It appeared, however, that two isolates, one of the 0 and the other of the C strain group could not be detected with the MCA, but with polyclonal antisera. When 85 dormant potato tubers were assayed in the DAS -ELSA, with poly-

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clonal antisera 24, with MCA 38, and with biotest (symptoms) 83, tubers could be recognized as infected. The defined virus specificity of MCA and the absence of antihost antibodies improve the sensitivity and specificity of the ELISA. Because the MCA-enzyme conjugate can be used up to 10-fold more diluted without loss of sensitivity, the background reactions due to random absorption of the conjugates or plant contributed substances are reduced (Gugerli and Fries, 1983). VI. CONCLUSIONS Monoclonal antibodies raised against plant viruses are found to be specific for one strain only or a group of strains and to be different in their affinity to the epitope for which they are specific. According to the present data there are several epitopes on each virus particle, one or some strain specific or strain group specific. For the study of virus coat protein fine structure and epitope mapping a MCA of strain specificity would serve the best purpose. For virus diagnosis in crop plants a MCA with specificity for an epitope common to one or several strain group(s) would be chosen, and for epidemiological studies several MCA, each with a specificity for a different strain. For all three purposes MCA of high affinity are desirable. Whereas for virus purification by affinity chromatography a MCA specific for the respective virus but of lesser affinity is desired. The MCA exactly suited for the purpose are reported to be rare. In order to increase the chance of finding MCA of the desired properties several authors recommend (1) using a high concentration of purified antigen for immunization (Dietzgen and Sander, 1982), (2) using a variety of immunization schemes, loci of injection, organ source of the B-lymphoctes for fusion, (3) conducting many fusions (Reading, 1982; Yewdell and Gerhard, 1981), and (4) using the Western blotting technique for improved detection of MCA epitope specificity (Hohmann and Faulkner, 1983). Beyond the generally employed procedures for this purpose there have been several innovations put forward such as a method for immunization in uitro (Reading, 1982), the introduction of the genetic information for production of virus-specific antibodies into the myeloma cell by transfection (Davis, 1983), and the high grade purification of the immunizing virus by high-performance liquid chromatography (Anderson, 1983). When the properties desired to best suit the purpose of, for instance, virus diagnosis are not found in one MCA, a cocktail of MCA with defined specificity for certain epitopes and of defined MCA proportions can be-

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come an artificially composed defined polyclonal antiserum to recognize a group of related viruses, i.e., members of the PNRV/ApMV group or even of nonrelated viruses not wanted in a crop plant, i.e., potato virus X, Y, A, S, M, and PLRV in potato. Also conventional polyclonal antisera can be used for the same purpose, but by the mixing the sensitivity of the assay is frequently lost since the virus-specific antibodies in each antiserum become diluted, but the nonspecific reaction causing antibodies they may contain do not; this background reaction is largely circumvented with high titer MCA cocktails (Gugerli and Fries, 1983; Oxford, 1982). The ultimate goal should be to achieve by use of MCA a complete map of epitopes on the protein coat of a virus, to detect the fine structure of each epitope, and to ascertain the same reactivity of the MCA with the corresponding epitope obtained by peptide digest of the capsomers and with a chemically synthesized replica of this epitope in order to eventually raise the appropriate MCA by way of transfection. In case the conformation of the epitope is decisive for recognition by a MCA, but is lost in isolated peptides, the MCA specificity has t o be assayed with mutants (A1Moudalla1 etal., 1982). Since any serological assay gives information only on the presence of antigen, but not on its biological properties, a biological function of the epitope should be investigated by, for instance, inhibition of virus infectivity in a bioassay by binding of a MCA. Not only the properties of a MCA desired for investigation, but also the method of assay has to be suitable. Several MCA have proved as nonprecipitating the virus in an agar double diffusion test and binary binding tests require MCA of different specificity for coating and labeling (Hsu etal., 1983b;Halk, 1983). It is obvious that before assay procedures are designed the knowledge of the properties of MCA and of the location and fine structure of epitopes with which the MCA reacts on the virus coat protein is highly desirable. This demand is consistent with observations made in the field of vertebrate virology (Yolken, 1983; Yewdell and Gerhard, 1981). Several MCA show cross reaction not only with members of the virus group to which the immunizing virus belonged, but also with members of other virus taxa or even with host cell constituents (Yewdell and Gerhard, 1981; Oxford, 1982; Dietzgen, 1983). This is certainly a disadvantage of some MCA. Therefore, it is suggested that in the determination of the specificity of a MCA besides the assay with the immunizing virus and related strains, members of other virus taxa should be included before using the MCA for virus diagnosis. The selection of such nonrelated viruses coud be helped by the consideration which other viruses may infect the same host plant in which the virus is to be detected by the MCA. From such a phenomenon as the cross reaction of MCA with related epitopes alien to the immunizing virus, even though of different strength,

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Jerne (1960) speaks of an epitypic family the size of which is not known. Here again the request for the physical determination of epitopes becomes enhanced. At present, MCA are considered to have the highest degree of monospecificity available among immunological reagents (Yewdell and Gerhard, 1981) and virus assays with MCA are reported to be at least as sensitive as with polyclonal antisera, if not better (Yolken, 1983; Gugerli and Fries, 1983). With MCA the difficulty of inconsistent immune response of each animal can be overcome as they can be produced continuously once a hybridoma clone secreting them has been established. This production in cell culture is less laborious, time consuming, and space requiring as the raising of antisera in animals, thus reducing expense which is desirable considering the vast number of plant samples to be assayed for virus in seed or planting stock certification. Also with regard to this certification the known properties and measurable quantity of monoclonal virus-specific antibodies will facilitate the establishment of a standardized test for world-wide required virus assays of agricultural or horticultural certified plant material. The same applies for epidemiological investigations. Another advantage of the application of MCA for virus diagnosis is the improvement of the sensitivity of diagnostic assays because of a reduction of virus-unspecific reactions, and reduced label quantity required without loss in sensitivity of the assay. With regard to the analysis of epitope fine structure and investigations of virus taxonomy MCA of such specificity as to recognize the substitution of one amino acid in an epitope by better, less, or no reaction with this site makes them a highly desirable tool. MCA may also prove a useful tool for the investigation of the biological function of certain viral epitopes in virus pathogenesis.

ACKNOWLEDGMENTS The authors wish to express their appreciation to Drs. Halk, Hill, Hsu, Gugerli, Tremaine, and van Regenmortel for generously supplying manuscripts and preprints of their most recent work and Drs. Briand, Klug, and Milton for kind permission to reproduce figures from their publications.

REFERENCES Adam, G.,Sander, E., and Shepherd, R. J. (1979). Virology 92, 1-14. A1 Moudallal, Z., Briand, J. P., and van Regenmortel, M. H. V. (1982). EMBO J . 1,1005 1010.

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A1 Moudallal, Z., Altschuh, D., Briand, J. P., and van Regenmortel, M. H. V. (1984). J. Immunol. Methods 6 8 , 3 5 4 3 . Altschuh, D., and van Regenmortel, M. H. V. (1982). J. Immunol. Methods 50,99-108. Altschuh, D., Hartman, D., Reinboldt, J., and van Regenmortel, M. H. V. (1983). Mol. Immunol. 20, 271 -278. Anderer, F. A. (1963). Adu. Protein Chem. 18, 35. Anderer, F. A., and Schlumberger, H. D. (1965). Biochim. Biophys. Actu 97, 503-509. Anderson, N. G. (1983). Curr. Top. Microbiol. Immunol. 104, 197-213. Barbara, D. J., Clark, M. F., Thresh, J. M., and Casper, R. (1978). Ann. Appl. Biol. 90, 395-399. Beale, H. P. (1931). Contrib. B o p ThompsonInst. 3, 529. Bloomer, A. C., Champness, J. N., Bricogne, G., Staden, R., and Klug, A. (1978). Nature (London)276,362-368. Briand, J. P., A1 Moudallal, Z., and van Regenmortel, M. H. V. (1982). J. Virol. Methods 5, 293- 300. Claflin, L., and Williams, K. (1978). Curr. Top. Microbiol. Immunol. 81, 107-109. Clark, M. F., and Adams, A. N. (1977). J. Gen.Virol. 34, 475-483. Dangl, J. L., and Herzenberg, L. A. (1982). J. Immunol. Methods 52,l- 14. Davis, J. M., Pennington, J. E., Kubler, A.-M., and Conscience, J.-F. (1982). J. Immunol. Methods 50, 161-171. Davis, W. (1983). In “Hybridoma Technology in Agricultural and Veterinary Research” (N. J. Stern and H. R. Gamble, eds.). Rowman & Allanheld, New Jersey. Diaco, R., Lister, R. M., Durand, D. P., and Hill, J. H. (1983). Phytoputhology 73,788. Dietzgen, R. G. (1983). Dr. Dissertation, Tubingen University. Dietzgen, R. G., and Sander, E. (1981). Proc. Int. Congr. Virol., 5th 1981 p. 244. Dietzgen, R. G., and Sander, E. (1982). Arch. Virol. 74, 197-204. Dietzgen, R. G., and Sander, E. (1983). In “Hybridoma Technology in Agricultural and Veterinary Research” (N. J. Stern and H. R. Gamble, eds.). Rowman & Allanheld, New Jersey. Douillard, J. Y., and Hoffman, T. (1983). In “Methods in Enzymology,” Vol. 92, pp. 168174. Academic Press, New York. Dulbecco, R., and Freeman, G. (1959). ViroEogy 8,396-397. Dulbecco, R., andVogt, M. (1954). J.Exp.Med. 9 9 , 167. Dvorak, M. (1927). J. Infect. Dis.41, 215. Engvall, E., and Perlmann, P. (1971). Immunochemistry 8,871-874. Fazekas de St. Groth, S.(1983). J. Immunol. Methods 57, 121- 136. Fazekas de St. Groth, S.,and Scheidegger, D. (1980). J. Immunol. Methods 35,l-21. Feder, J., andTolbert, W. R. (1983). Sci. Am. 248,24-31. Fox, C. P., Berenstein, E. H., and Siraganian, R. P. (1981). Eur.J. Immunol. 11,431-434. Fulton, R. W. (1981). In “Handbook of Plant Virus Infections and Comparative Diagnosis” (E. Kurstak, ed.), pp. 377-413. Elsevier/North Holland Biomedical Press, Amsterdam. Galfr6, G., and Milstein, C. (1981). In “Methods in Enzymology” (J.J. Langone and H. Van Vunakis, eds.), Vol. 73, pp. 1-49. Academic Press, New York. Galfr6, G., Howe, S.C., Milstein, C., Butcher, G. W., and Howard, J. C. (1977). Nature (London)266,550-552. Goding, J. W. (1980). J . Immmunol. Methods 39,285-308. Gugerli, P. (1982). Proc. Int. Congr. “Research for the Potato in the Year 2000,”pp. 91-92. Gugerli, P. (1983a). In “Immunoenzymatic Techniques” (S.Avrameas et al., eds.), pp. 369 - 384. Elsevier/North Holland Biomedical Press, Amsterdam. Gugerli, P. (1983b). Proc. Int. Plant Pathol. Congr., 4th, 1983 p. 98.

MONOCLONAL ANTIBODIES AGAINST PLANT VIRUSES

167

Gugerli, P., and Fries, P. (1983). J. Gen.Virol. 64, 2471-2477. Halk, E. L. (1983). I n “Hybridoma Technology in Agricultural and Veterinary Research” (N. J. Stern and H. R. Gamble, eds.). Rowman & Allanheld, New Jersey. Halk, E. L., and Franke, J. (1983). Phytopathology 7 3 , 789. Halk, E. L., Hsu, H. T., Aebig, J., and Chang, K. (1982a). Phytopathology 7 2 , 707. Halk, E. L., Hsu, H. T., Aebig, J., and Chang, K. (1982b). Phytopathology 72,938. Halk, E. L., Hsu, H. T., and Aebig, J. (1982~). Phytopathology 7 2 , 953. Halk, E. L., Hsu, H. T., Aebig, J., and Franke, J . (1984). Phytopathology 74, 367-372. Hlimmerling, G. J . (1977). Eur. J. Imrnunol. 7,743-746. Hammerling, G. J., and Hammerling, U. (1981). Res.Monogr. Irnrnunol. 3, 563-587. Hewish, D. R., Shukla, D. D., Johnstone, G. R., and Sward, R. J. (1983). Proc. Int. Plant Pathol. Congr., 4th,1983 p. 463. Hill, E. K., Durand, D. P., and Hill, J. H. (1983). Proc. Am. SOC.Microbiol., p. 299. Hill, E. K., Hill, J. H., and Durand, D. P. (1984). J. Gen.Virol. 65,525-532. Hill, J. H. (1983). Proc. Int. Plant Pathol. Congr., 4th, 1983 p. 390. Hohmann, A. W., and Faulkner, P. (1983). Virology 125, 432-444. Hoogenraad, N., Helman, T., and Hoogenraad, J. (1983). J . Irnrnunol. Methods 61, 317320. Hsu, C. H., White, J. A., and Sehgal, 0. P. (1977). Virology 81,471-475. Hsu, H. T., Aebig, J., Rochow, W. F., and Lawson, R. H. (1983a). Phytopathology 73, 790. Hsu, H. T., Halk, E. T., and Lawson, R. H. (1983b). Proc. Int. Plant Pathol. Congr., 4th, 1983 p. 25. Hsu, H. T., Lawson, R. H., Beijersbergen, J. C. M., and Derks, A. F. L. M. (1983~).Proc. Znt. Plant Pathol. Congr., 4th,1983 p. 117. Jerne, N . K. (1960). Annu.Reu.Microbiol. 14, 341-358. Kearney, J. F., Radbruch, A., Liesegang, B., and Rajewski, K. (1979). J. Irnrnunol. 123, 1548-1550. KIug, A., and Caspar, D. L. D. (1960). Adu.Virus Res.7,225 -325. Koenig, R. (1981) J. Gen.Virol. 55, 53-62. Kohler, G., and Milstein, C. (1975). Nature (London) 256,495-497. Littlefield, J. W. (1964). Science 1 4 5 , 709-710. McCullough, K. C., Butcher, R. N., and Parkinson, D. (1983). J. Biol.Stand. 11,171- 194. Mandel, B. (1979). Compr. Virol. 15, 37-121. Martin, R. R., and Stace-Smith, R. (1983). Phytopathology 73, 792. Milton, R. C. de L., and van Regenmortel, M. H. V. (1979). Mol. Irnrnunol. 16,179-184. Milton, R. C. de L., Milton, S. C. F., von Wechmar, M. B., and van Regenmortel, M. H. V. (1980). Mol. Irnmunol. 17, 1205-1212. Moore, G. E., Gerner, R. E., and Franklin, H. A. (1967). J.Am. Med.Assoc. 199.519-524. Murant, A. F. (1981). In “Handbook of Plant Virus Infections and Comparative Diagnosis” (E. Kurstak, ed.), pp. 197-238. Elsevier/North Holland Biomedical Press, Amsterdam. Nowinski, R. C., Lostrom, M. E., Tam, M. R., Stone, M. R., and Burnette, W. N. (1979). Virology 93, 111-126. Oxford, J. (1982). J. Hyg. 88, 361-368. Pearson, T. (1983). I n “Hybridoma Technology in Agricultural and Veterinary Research” (N. J. Stern and H. R. Gamble, eds.). Rowman & Allanheld, New Jersey. Powell, C. A., and Marquez, E. D. (1983). Proc. Int. Plant Pathol. Congr., 4th,1983 p. 483. Purdy, H. A. (1928). Proc. SOC.Exp.Bid. Med. 2 5 , 702. Purdy, H. A. (1929). J . Exp.Med.49, 919. Reading, C. L. (1982). J. Irnmunol. Methods 53,261-291. Rochow, W. F., and Duffus, J. E. (1981). In “Handbook of Plant Virus Infections and

168

EVAMARIE SANDER AND RALF G. DIETZGEN

Comparative Diagnosis” (E. Kurstak, ed.), pp. 147 - 170. Elsevier/North Holland Biomedical Press, Amsterdam. Schafer, W. (1979). Z. Naturforsch. C: Biosci. 34c, 306-309. Schramm, G., and Friedrich-Freksa, H. (1941). Hoppe-Seykrs Z. Physwl. Chem.270,233. Schreier, M., Kohler, G., Hengartner, H., Berek, C. Trucco, M., Forni, L., Staehelin, T., Stocker, J., and Takacs, B. (1980). “Hybridoma Techniques.” EMBO, SKMB Course, Basel. Sehgal, 0. P. (1981). In“Handbook of Plant Virus Infections and Comparative Diagnosis” (E. Kurstak, ed.), pp. 91 - 121. Elsevier/North Holland Biomedical Press, Amsterdam. Siraganian, R. P., Fox, P. C., and Berenstein, E. H. (1983). In“Methods in Enzymology,” Vol. 92, pp. 17-26. Academic Press, New York. Stahli, C., Staehelin, T., and Miggiano, V. (1983). In“Methods in Enzymology,” Vol. 92, pp. 26-36. Academic Press, New York. Torrance, L. (1983). Proc. Eur.Assoc. Potato Res., Sect. Virol., 1983. Tremaine, J. H., and Ronald, W. P. (1983). Phytopathobgy 73, 794. Tsugita, A., Gish, D. T., Young, J., Fraenkel-Conrat, H., Knight, C. A., and Stanley, W. M. (1960). Proc. Natl. Acad. Sci. U.S.A. 4 6 , 1463-1469. Underwood, P. A., Kelly, J. F., Harmann, D. F., and McMillan, H. M. (1983). J.Immunol. Methods60,33-45. van Deusen, R. A., and Whetstone, C. A. (1981). 24th Annu.Proc. Am. Assoc. Vet. Lab. Diagn. pp. 211-228. van Regenmortel, M. H. V. (1982). “Serology and Immunochemistry of Plant Viruses.” Academic Press, New York. van Regenmortel, M. H. V., Altschuh, D., and Briand, J. P. (1983). In“Protein Conformation as an Immunological Signal” (F. Celada, V. N. Schumaker, and E. E. Sercerz, eds.), pp. 181- 190. Plenum, New York. van Vloten-Doting, L., Francki, R. I. B., Fulton, R. W., Kaper, J. M. K., and Lane, L. C. (1981). Intervirology 16, 198-203. Williams, A. R. (1978). In“Handbook of Experimental Immunology” (D. M. Weir, ed.), 3rd ed., Vol. 111, p. 9. Lippincott, Philadelphia, Pennsylvania. Yewdell, J. W., and Gerhard, W. (1981). Annu.Rev.Microbiol. 35, 185-206. Yolken, R. H. (1983). Curr.Top.Microbiol. Immunol.104, 117-135. Zimmerman, U. (1982). Biochim. Biophys. Acta694,227-277.

NOTEADDEDIN PROOF

Note to Section IV,C: for the first time specificity of MCA was characterized by a direct and indirect ELISA in parallel and by virus neutralization assays inuitro and in aphid species transmitting some of the BYDV strains [Hsu, H. T., Aebig, J., and Rochow, W. F. (1984). Phytopathobm 74,600-6051.

ADVANCES IN VIRUS RESEARCH. VOL. 29

IMMUNOSORBENT ELECTRON MICROSCOPY FOR DETECTION OF VIRUSES David Katz and Alexander Kohn Department of Virology Israel Institute for Biological Research Ness Ziona, Israel

I. Introduction.

............................

........................... A. The Antibody-Coated Grid Technique (AB-CGT) . . . . . . . . . . B. The Protein A-Coated Grid Technique (PA-CGT) . . . . . . . . . . C. The Protein A-CoatedBacteria Technique (PA-CBT) . . . . . . . . D. The Antigen-Coated Grid Technique (AG-CGT) . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. ISEM Methods

111.

169 170 172 177 182 187 188 193

I. INTRODUCTION There is a wealth of methods in immunoelectron microscopy (IEM) as a diagnostic tool in virology. In all these methods the main step is the observation in an electron microscope of the interaction between viruses and their antibodies. This procedure permits, therefore, not only the direct visual recognition of the virion by its morphology but it also permits a specific identification of the virus via the antibody reacting with the virus. This combination of immunological and morphological method has an advantage over the other methods, where immunological reactions provide only a clue to the identity of the virus. In this review, we shall only deal with immunoelectron microscopy (IEM) of viruses in suspension (or body fluids); we shall not consider the identification of viruses in histological sections by means of antibodies labeled with ferritin (Rifkind, 1976) or enzymes (Kraehenbuhl and Jamieson, 1976; Kurstak etal., 1977). Clinical application of electron microscopy in medical virology has been reviewed by Field (1982). The first IEM observation of a virus-antibody interaction was demonstrated by Anderson and Stanley in 1941 who used tobacco mosaic virus (TMV). The popularity of this method increased when Brenner and Horne (1959) introduced the simple principle of negative staining. In 1969 Almeida and Waterson (1969a) showed how agglutination of viruses by their antibodies could be demonstrated in the electron microscope and 169

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be used in the specific diagnosis of the virus. They called this phenomenon “Clumping.” Milne and Luisoni (1975) showed the possibility of specific viral diagnosis based on the halo that antibodies formed around individual viruses or clumps of viruses and called this type of reaction “Decoration.” These two main methods were employed in a number of variations: Optimal conditions for the reactions were found by varying the relative concentrations of virions and antibodies and by removing impurities from the reaction mixture either by centrifugation or by deposition of the complexes on agar (Kelen etal., 1971; Anderson and Doane, 1973). These methods permitted the detection and identification of the viruses of rubella, corona, rhino, hepatitis A and B, rota, adeno, Norwalk, papilloma, etc. (Best etal., 1967; Pensaert etal., 1981; Kapikian etal., 1972a,b; Feiastone etal., 1973; Almeida et al., 1971; Flewett and Boxall, 1976; Almeida and Waterson, 1969a). These “classical methods,’’ including the agar technique, were amply reviewed by Almeida (1980), Doane etal. (1974), Flewett and Boxall (1976), Doane and Anderson (1977), Almeida and Waterson (1969a), Milne and Luisoni (1977a,b), and van Regenmortel(1981a,b) and will not be described here. We shall, however, describe in detail IEM methods that are based on the principle introduced by Derrick (1973). Derrick coated the electron microscope grid with antibodies so as to specifically trap from the suspension the viruses deposited on the grid. Since Derrick’s method resembles solid phase immunoassays such as SPRIA (Catt, 1969) and ELISA (Engvall and Perlmann, 1971),it has been suggested that his method be called immunosorbent electron microscopy (ISEM) (Roberts etal., 1982). In our opinion, Roberts’ definition of ISEM is too narrow and should include not only methods with antibody-coated grids, but all other methods where a solid absorbent participates in the antibody-virus interaction, like the method of Milne and Luisoni (1975), where the virus is absorbed on the grid, or our own method (Katz etal., 1980) in which the virus is absorbed to antibody-coated Staphylococcus aureus. ISEM methods were used relatively more in plant virology than in animal virology. In this review, we shall discuss the newer modifications of ISEM methods in both plant and animal virology that were not covered by the review of Milne and Luisoni (1977b);we shall also present some of our own unpublished results in this field.

11. ISEM METHODS ISEM methods in this review include all the techniques where the “solid phase principle” is essential in a way similar to other solid phase immun-

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oassays. For the sake of simplicity and uniformity, we propose to replace the older names and acronyms by new ones (see Table I). Thus the Derrick technique (Derrick, 1973) will be named the antibodycoated grid technique (AB-CGT),the method of Shukla and Gough (1979) the protein A-coated grid technique (PA-CGT), the method of Katz etal. (1980) using protein A containing S. aureusbacteria the protein A-coated bacteria technique (PA-CBT), and the “decoration” technique as proposed by Milne and Luisoni (1975)the antigen-coated grid technique (AGCGT).

TABLE I

PROPOSED NEWNAMES FOR IMMUNOSORBENT ELECTRON MICROSCOPY (ISEM) TECHNIQUES Method first described by Derrick (1973)

“Old” names Derrick’s method

SSEM

Milne and Luisoni (1975) Shukla and Gough (1979)

ISEM STREM Trapping method D-method SPIEM-DAT On grid IEM technique Decoration

Shukla’s method Serological trapping STREM ISEM D A method SPIEM SPIEM

+

Katz etal. (1980)

ISEM

References“ 2,5,8,9 13 6,16,22 10 11,12 13 18 20 2,4,5,22

7,14 12 10 17 13 19 15 21

Proposed new name Antibody-coated grid technique (AB-CGT)

Antigen-coated grid technique (AG C G T ) Protein A-coated grid technique (PA-CGT)

Protein A-coated bacteria technique (PA-CBT)

a (1) Derrick (1973); (2) Milne and Luisoni (1975); (3) Derrick and Brlansky (1976); (4) Milne and Luisoni (1977a); (5) Milne andLesemann (1978); (6) Roberts and Harrison (1979); (7) Shukla and Gough (1979); (8) Lesemann etal. (1980); (9) Lesemann and Paul (1980); (10) van Regenmortel etal. (1980); (11)Nicolaieff and van Regenmortel(l980); (12) Nicolaieff etal. (1980); (13) Milne (1980); (14) Gough and Shukla (1980); (15)Katz etal. (1980); (16) Kerlan etal. (1981); (17) Obert etal. (1981); (18) Giraldo etal. (1982); (19) Kjeldsberg and Mortensson-Egnund (1982); (20) Rubinstein and Miller (1982); (21) Nicolaieff etal. (1982); (22) Roberts et~ l . (1982).

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A. TheAntibody-Coated Grid Technique (AB-CGT) This technique, described by Derrick in 1973, was the first application of the principle of solid phase immunoassays to immunoelectron microscopy. Parlodion -carbon-coated grids were floated on drops of anti-tobacco mosaic virus (TMV) and anti-potato virus Y (PVY) (1: 10 dilution) rabbit antisera. The grids were then washed to remove residual unattached antibodies and incubated for 1 hour with homologous and heterologous viruses derived from crude leaf extracts. Free viruses, as well as impurities, were washed off. The grids were dried and shadowed with metals. It was shown that 40 to 50 times more TMV particles were "trapped" on anti-TMV coated grids as compared to anti-PVY coated grids, while PVY was trapped 20 times more efficiently on anti-PVY coated grids than on anti-TMV coated grids. This AB-CGT was also shown to be suitable for quantitation, since the log of the number of virus particles specifically absorbed to grids decreased linearly with the virus dilution. Longer incubation times and higher temperature were found to increase the sensitivity of the AB-CGT. Derrick and Brlansky (1976) applied the AB-CGT to the diagnosis of other plant viruses as well as to the corn stunt mycoplasma. In this work, the grids were positively stained with 1%uranyl acetate in 50% ethanol. The authors claimed that this stain was superior to phosphotungstate or ammonium molybdate negative staining. They also noticed that Formvar-coated grids with or without carbon coating were not suitable for the AB-CGT, since proteins did not absorb to Formvar. They obtained best results in their work with carbon fronted parlodion-coated grids. Derrick and Brlansky also noticed that the addition of 0.4 M sucrose to the washing buffer (Tris buffer) markedly reduced the amount of debris on the grids. In distinction from the previous work (Derrick, 1973), Derrick and Brlansky (1976) coated grids with high dilutions of antisera. The degree of dilution of the antiserum (except at very high dilution) had no effect on the number of virions trapped on the antibody-coated grids. In their review, Milne and Luisoni (1977b) described not only the original papers of Derrick and Brlansky but proposed a few modifications, such as using carbon-fronted Formvar-coated grids and the use of shorter incubation times of antiserum and virus. Diluted antisera (1 :10 or 1 :100) in phosphate buffer (PB)were incubated on grids for 5 minutes. After washing with PB, drops of virus were incubated for 15 minutes on grids, washed with water, and stained with aqueous uranyl acetate. They preferred this negative staining upon the positive staining, obtained with ethanolic uranyl acetate that was used by Derrick and Brlansky (1976). With the

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negative staining technique, better resolution was obtained and the viral capsids were well preserved, though better contrast was obtained by positive staining. Another modification proposed was an improved way for preparing and storing antiserum-coated grids. In this procedure, grids were adsorbed with antiserum, washed with PB and water, and then dried and stored (temperature not stated). Before use, the grids were wetted with PB, drained, and incubated with the virus. Results were only slightly better with “fresh” as compared to the stored grids. In the same review, Milne and Luisoni proposed to combine the ABCGT with “decoration.” In this procedure, the trapped viruses (by the AB-CGT) were incubated with antiserum diluted 1:100 for 15 minutes, washed with PB and then with water, and stained with uranyl acetate. A virus was considered specifically trapped only if it was also “decorated” by the second layer of antibodies. In the authors’ view, decoration is the best proof for a specific immune reaction, since with all other methods, clumping or trapping may occur nonspecifically, due to factors not entirely understood. Milne and Lesemann (1978) confirmed the data of Derrick (1973) and Derrick and Brlansky (1976) that larger numbers of viruses were trapped on antibody-coated grids than on untreated grids or on control grids treated with normal serum. However, in disagreement with Derrick and Brlansky, Milne and Lesemann stated that in order to obtain maximal trapping, optimal dilutions of sera had to be used. The most effective dilutions were between 1:800 and 1:3200. At low dilutions of serum an inhibition of trapping occurred due to serum proteins competing with the antibodies for sites on the grid. This assumption was elegantly confirmed The addition of increasing amounts of normal serum or bovine serum albumin (BSA) to an antiserum diluted 1:600 progressively inhibited the trapping of the homologous viral particles. Roberts and Harrison (1979) used the AB-CGT for the detection of potato leafroll and potato mop-top viruses (PLRV and PMTV, respectively). The grids freshly coated with carbon only were incubated for 1 hour at 37°C with diluted antiserum (dilution 1:lOOO in PB pH 6.5), washed, and further incubated on drops of virus (leaf extracts or aphid extracts) at 4°C for 1 to 72 hours. PMTV were stained with 1 or 2% sodium phosphotungstate or 2% ammonium molybdate. These stains were unsatisfactory for PLRV. The only stain with which good contrasts were obtained and did not disrupt PLRV was uranyl formate sodium hydroxide at pH 4.8, diluted 1:3 with distilled water. With this modified AB-CGT of Roberts and Harrison (1979), a t least a thousand times more virus particles were trapped on antibody-coated grids than on untreated grids. The viruses were more evenly distributed when

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incubated a t 4°C than at higher temperatures. To confirm the specificity of the AB-CGT, viruses bound to the grids were incubated on drops of antibody for 1to 3 hours a t room temperature, stained, and observed for antibody coating (“decoration”). The AB-CGT of Roberts and Harrison thus enables examinations and measurements of fragile viruses (PMTV) which are found in low amounts in fresh leaf extracts, without the need of purification and centrifugation steps. Lesemann etal. (1980) studied various parameters of the AB-CGT which influence specific (serological) and nonspecific binding of tymovirus particles to electron microscope grids. Carbon -Formvar-coated grids were treated by the glow discharge procedure, and floated for 5 minutes on antiserum, normal serum, or buffer (PB). Grids were then washed with PB and floated for 15 minutes on virus drops. Nontrapped viruses were washed away with water. The grids were then stained with 2% aqueous uranyl acetate. With this procedure, purified viruses adsorbed to buffertreated grids to the same degree as to antibody-coated grids. Different viruses exhibited different degrees of nonspecific binding. The nonspecific binding was inhibited by coating grids with normal serum or by diluting the virus with crude plant sap. Specific binding, however, was not inhibitedby the plant sap. In agreement with Milne and Lesemann (1978) as well as with Roberts and Harrison (1979), Lesemann etal. (1980) declared that optimal antiserum dilutions have to be found for maximal trapping efficiency. There was a linear relationship between the log virus concentration and the log virus particle count, up to a virus concentration of 10 ,ug/ml. At higher concentrations, the grids were saturated. Top and bottom components of 10- 40% sucrose gradients, used for the purification of the virus, were used to determine the strength of binding of the virus onto buffer and antiserum-treated grids. They were distinguished in the electron microscope (EM)by their different staining. Nonspecifically bound components (on buffer-treated grids) could be easily removed, while specifically bound particles on antiserum-coated grids were tightly bound. Heterologous viruses could not be replaced by homologous viruses on antiserum-coated grids (and vice versa). Nicolaieff and van Regenmortel (1980) determined the conditions of specific trapping using the AB-CGT for three isometric viruses [turnip yellow mosaic virus (TMYV), tomato bushy stunt virus (TBSV), and cauliflower mosaic virus (CaMV)] and five strains of TMV. They used Formvar - carbon-coated grids and PB for the dilution of antisera and viruses. Grids were floated for 4 minutes on diluted antiserum, washed with PB, and again floated for various periods of time on drops of purified viral preparations. Visualization of the adsorbed virions was done either

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by platinum shadowing or by positive staining with 1%uranyl acetate in 45% ethanol. In agreement with the results of others, Nicolaieff and van Regenmortel showed that optimal conditions for the specific serological trapping should be defined for each virus. High serum concentrations were inhibitory, but the degree of inhibition was different with different viruses. Crude sap inhibited adsorption of viruses to untreated grids, but not to antibodycoated grids. By using the AB-CGT at carefully defined conditions, the serological distance between TMV strains could be determined since the extent of serological trapping on the grid was proportional to the serological distance between the strains. For the determination of weak cross reactions of distant viruses, purified antibody preparations, diluted in 1: 1000 normal serum or in 5% of BSA solution, were used for coating grids. The suitability of the AB-CGT for the differentiation of TMV strains was examined and compared to an indirect ELISA in another work (van Regenmortel etal., 1980). The best method for the detection of serologically distant strains of TMV using one single antiserum is the indirect ELISA. With the AB-CGT, several serotypes could also be detected. However, fewer strains could be detected by the AB-CGT than by indirect ELISA. The authors conclude that the AB-CGT is highly sensitive and offers many advantages over other diagnostic procedures. The need of optimal, usually high, dilutions of antiserum for use in the AB-CGT stressed by Milne (1980) has been confirmed by others. However, Milne reported that out of 15 plant viruses tested, the AB-CGT did not work for 8 viruses. These viruses were representatives of three main groups: potyviruses, cucumoviruses, and nepoviruses. Milne suggested that their specific trapping on the antibody-coated grids was blocked by soluble coat viral antigens. Kerlan etal. (1981) applied the AB-CGT for the detection of plum pox virus (PPV) and chlorotic leaf spot virus (CLSV). Carbon-coated grids were coated by floating, for 5 minutes, on antisera, diluted 1:100, washed, and floated for 15 minutes on extracts from infected plants. Staining was with aqueous 2% uranyl acetate. In most experiments, grids before staining were further incubated with homologous rabbit antiserum for decoration, as proposed by Milne and Luisoni (1977b) and in some experiments, a double decoration was performed by a second incubation of the grids with sheep anti-rabbit immunoglobulin (IgG). For simple and double decorations antisera were diluted 1: 100. The AB-CGT that was even more sensitive than ELISA detected 5 to 10 ng/ml of the viruses. The sensitivity was attributed to the enhanced trapping efficiency of the antibody-coated grids. The single decoration

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step was used for confirmation of specificity while the second decoration (with sheep anti-rabbit IgG) increased the sensitivity of the AB-CGT. With double decoration, lower magnification in the EM could be employed, since the width of the virions was increased 3-fold as compared to the untreated viruses. The authors concluded that the AB-CGT provided a useful diagnostic tool and could be used as an alternative for ELISA. Cohen etal.(1982) studied the effect of the pH of virus extracts and antiserum on the trapping efficiency in the AB-CGT. Four plant viruses were examined cucumber mosaic virus (CMV), lily symptomless virus (LSV), potato virus Y (PVY), and carnation mottle virus (CarMV). The technique was as described by Milne and Luisoni (1977a). The carbonfronted Formvar-coated grids were incubated with optimally diluted antisera at different pH values, and then reacted with viruses extracted from infected leaves with PB at different pH values. The efficiency of trapping of the AB-CGT was compared to the trapping of viruses on untreated grids, and to control grids that were coated with normal sera. The results of Cohen etal. (1982) indicated that the pH of the virus extract had a marked effect on the efficiency of trapping, yet each of the viruses behaved differently. The optimal pH for LSV and for CarMV was 7.0. LSV had one sharp peak at pH 7.0 while CarMV was trapped only slightly less efficiently at pH values of 5.0 and 6.0; pH 8.0 was not satisfactory for both viruses. CMV was trapped most efficiently at pH 8.0 and about 2-fold less at pH 5.0 and 7.0; the worst pH was 6.0. PVY had two optimal pH values for trapping: 6.0 and 8.0, while pH values of 5.0 and 7.0 were less satisfactory. The effect of the pH of the antiserum on the trapping efficiency was smaller though still significant. The reasons for the pH dependence of trapping that was demonstrated with the AB-CGT in this work are not understood and the conclusion is that optimal pH conditions should be determined for each virus separately. The first to apply the AB-CGT to the diagnosis of an animal virus (rotavirus) were Nicolaieff etal. in 1980. The results of their work with AB-CGT as compared to the PA-CGT, are reviewed in Section I1,B. Giraldo etal. (1982) applied the modified AB-CGT combined with decoration of Milne and Luisoni (1977b)for the detection of BK virus (BKV), a member of the human papovaviruses. In their optimal procedure they incubated for 5 minutes a drop of 1:500 dilution of rabbit antiserum, on a Formvar-carbon-coated grid. After washing with PB saline (PBS) a drop of virus was incubated on the grid for 15 minutes, washed again, and incubated for decoration with a drop of an 1:2500 antiserum dilution. After a fixation step by 0.8% glutaraldehyde the grid was again washed

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with PBS and water and stained with 1%aqueous uranyl acetate. The specificity of the reaction was confirmed by cross experiments with polyoma virus. Similar number of viruses were trapped with a wide range of capture-antibody dilutions (1:500 to 1:10,000). The decoration step facilitated viewing of the virions. Antibody-coated grids trapped 17 to 28 more viruses as compared to untreated grids. As few as lo2to lo3plaque forming units per ml (PFU/ml) were detected specifically within 1 hour. The method is regarded by the authors as rapid, sensitive, and specific and is recommended for the detection of viruses in clinical specimens. Rubinstein and Miller (1982) compared as ELISA method to an EM procedure and to the AB-CGT for the detection of rotaviruses. In their AB-CGT, Formvar - carbon-backed grids were incubated for 15 minutes on drops of antiserum diluted 1:2000 in PBS, washed on three drops of PBS, further incubated for 15 minutes on virus drops, and washed again. Negative staining was done with 2%phosphotungstic acid. The ELISA and AB-CGT were equally sensitive while the classical EM procedure was at least 9 times less sensitive. It was estimated that the ELISA and AB-CGT were able to detect approximately lo6simian rotavirions and lo’ particles of human rotavirions. Out of 455 clinical specimens (stools from children with diarrheal diseases) 197 were positive by the AB-CGT while 193 of the 197 were also positive by ELISA. Of the 258 negative samples by the AB-CGT, 238 were negative by ELISA. For 18specimens that were positive by ELISA and negative by the AB-CGT a confirmatory blocking test showed that all of them were “true” positives. The failure to detect the viruses in the ELISA positive samples by the AB-CGT can be partially explained by the presence of viral debris which may block the adsorption of intact particles to the antibody-coated grids. However, a few samples that were positive by the AB-CGT were negative by ELISA. The reason for this phenomenon is not quite understood.

B. TheProtein A-Coated GridTechnique (PA-CGT) In 1979 Shukla and Gough suggested coating of grids with protein A before coating them with specific antiserum to improve the trapping capacity of such grids. They found that with this PA-CGT they could detect 339(!) times more sugarcane mosaic virus (SCMV) and 5 times more TMV than on untreated grids, and 67 and 7 times more, respectively, with grids treated with antiserum alone (AB-CGT). The optimal procedure in their report was as follows: 5 pl of 0.1 mg/ml protein A was deposited on grids for 10 minutes followed by 1:20 dilution of TMV antiserum for 10 minutes. Five microliters of infected plant sap was then added to grids for 10 minutes. This was followed by “decorating” antibody (anti-TMV l :100,

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anti-SCMV 1:5) for 10 minutes and staining with 2% aqueous uranyl acetate, pH 4.5. After each step (except the first) the grids were washed with 20- 30 drops of buffer. It was important not to let the grids dry except after staining. In a later paper Gough and Shukla (1980)became aware that the results of their PA-CGT were sensitive to the serum concentration and found that the optimal serum concentration for protein A-coated grids was 1:100 or less, while with grids coated with antiserum alone, the serum at 1:10001:2000 gave best results. With this modified method the increase in the number of particles on grids treated with protein A and antiserum over those treated with antiserum alone (each at its optimal concentration) was 25-fold for SCMV and about 2-fold for erysimum latent virus and for TMV. Gough and Shukla also found that protein A and antiserum-coated grids could be stored up to 6 months at 4°C while still retaining 25% of their trapping activity. Storage a t room temperature did not give good results. Protein A could be stored at least 18 months in a frozen state. Milne (1980) compared the performances of the AB-CGT and the PACGT for the trapping of two plant viruses (ryegrass cryptic virus (RCV) and grapevine stem pitting-associated virus (GSP-AV). For both techniques carbon -Formvar-coated grids were used. The carbon coating was performed immediately before use. For the AB-CGT a drop of serum was placed on the grid for 10 minutes, rinsed with 20 drops of PB, and drained. A drop of the virus preparation was then placed on the grid for 15 minutes. The grid was then rinsed with 20 drops of water and stained with 5 drops of 2% aqueous uranyl acetate.For the PA-CGT, grids were first covered with a drop of 0.1 mg/ml protein A for 10 minutes, rinsed with PB, and processed as for the AB-CGT. Milne concluded that an optimum serum dilution is required in both methods. High antiserum concentrations were inhibitory, although somewhat less in the PA-CGT than in the AB-CGT. Protein A treatment of grids did not lead to a dramatic increase of the number of virions trapped over grids treated with antiserum alone. At optimal conditions for both techniques, PA-CGT trapped only 2 to 3 times more virions than the AB-CGT. However, at low virus concentrations similar numbers of virions were trapped on antiserum coated grids with or without protein A. Protein A may thus be of advantage in cases where the number of antibodies is a limiting factor but not when the numer of virions is limited. Lesemann and Paul (1980) studied the effect of various conditions for the use of protein A in the PA-CGT and compared the results with the AB-CGT. They used pioloform- carbon-coated grids after exposure to glow discharge. The grids were floated for 5 minutes on drops of protein A

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(10 pg/ml or more) diluted in PB, washed with 20 drops of PB, drained on filter paper, and floated again for 5 minutes on drops of diluted antiserum or normal serum. After washing with 20 drops of PB, the grids were transferred for 15 minutes onto drops of virus. The grids were then washed with 40 drops of water and stained with 2% aqueous uranyl acetate. The work was done with a purified preparation of maize chlorotic mottle virus (MCMV) and with eggplant mottled crinkle virus (EMCV) in the form of crude plant extracts. Lesemann and Paul concluded that protein A a t concentrations less than 10pg/ml was not sufficient to bind antibodies in saturating amounts, and that higher concentrations of protein A did not improve the test. In their opinion the main advantage of the protein A in the PA-CGT is that it allows the use of sera at high concentrations which are inhibitory in the AB-CGT. The use of higher antiserum concentrations enhances trapping capacity of the grids. Under these conditions six times more particles (from high virus concentrations) were trapped in the PA-CGT as compared to the number trapped in an optimized AB-CGT. Other advantages of using high serum concentrations in the PA-CGT are (1)nonspecific binding on normal serum coated grids is depressed; (2) low titered antisera can be used; and (3) high concentration of antibody on the grid permits the detection of weak heterologous reactions. However, when the trapping efficiency of the two tests was compared under conditions where the virus was present at low concentrations the PA-CGT did not show any advantage over the AB-CGT (see also Milne, 1980). Two explanations were proposed for these results: (1)that not all of the immunoglobulins are absorbed (like IgM) on protein A-coated grids, and thereby some antibody activity is lost; and (2) grids coated with diluted antiserum alone are not completely covered and some of the virions attach nonspecifically. Nicolaieff etal. (1980) used the PA-CGT for the trapping of animal viruses such as rotaviruses. In their study the virus specimens were fecal extracts from infants suffering from diarrhea. The grids were first coated by flotation on 50 p1 drops of 25pg/ml of protein A for 4 minutes, and after transfer through drops of PB the grids were floated on 50 pl drops of 1:500 diluted antiserum (10 minutes), followed by rinsing in PB. Such grids were put overnight on drops of stool extracts diluted in PB. The grids were then washed in distilled water and stained with 1%uranyl acetate in 45% ethanol for 2 minutes. Using that method, Nicolaieff etal. (1980) found virus particles in 71% of specimens as compared to only 20% on uncoated grids. The protein A layer improved the trapping 5- to 10-fold as compared to grids coated only with rabbit antiserum. Equivalent results were obtained on grids coated

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with purified immunoglobulins from immunized chickens at optimal concentration of 0.1 mg/ml. The trapping of rotavirus by the PA-CGT was highly specific in that coronavirus and other 27 nm particles detected by standard electron microscopy were not seen on the protein A-treated grid. 1981), In a more extensive work from the same laboratory (Obert etal., the sensitivity of detection of rotavirus in human stools by the PA-CGT was compared to direct EM, counterimmunoelectrophoresis (CIEP),and ELISA. While EM and CIEP detected rotaviruses in 36 and 38%,respectively, of the 63 specimens tested, ELISA and PA-CGT detected 59 and 6176, respectively. Both ELISA and PA-CGT were equally sensitive and could detect about 2 ng/ml of the virus. However, the direct visualization of particles in the PA-CGT provided an advantage over ELISA, since no confirmatory tests were necessary. With most samples, particles were visible by the PA-CGT after 60 minutes incubation on grids, yet for maximum sensitivity the overnight incubation was routinely used. The authors pointed out that with PA-CGT, about 80 samples a day could be handled by one person. This method, extensively used in plant virology, is also likely to find many applications in the diagnosis of animal viruses. Another detailed comparison of direct EM, PA-CGT, and ELISA for the detection of rotaviruses was performed by Kjeldsberg and MortenssonEgnund (1982) on 115 fecal samples from children with gastroenteritis. For optimal results in the PA-CGT they used grids coated with 10pg/ml of protein A and antiserum at a dilution of 1 :640- 1 :2560. The coated grids were then incubated on the specimen drops for 18 hours at room temperature. Rotavirus was found in 36%of samples by both the PA-CGT andELISA without false positives, while by direct EM the virus was found in only 30%. The advantage of direct EM in this study was that (1)in addition to rotavirus, in 8 samples also adenovirus, astrovirus, and calicivirus particles were observed, and (2) the examination time was shorter. On the other hand this is the least sensitive method and usually requires centrifugation of the fecal extract. Though PA-CGT and ELISA were equally sensitive, ELISA lends itself better to mass screening than electron microscopy. In one microplate up to 22 crude fecal extracts can be set up, and several specimens may be examined in a single test, whereas in electron microscopy each specimen has to be observed separately; therefore assuming even that the time allowed for one specimen is 4 - 5 minutes, it would require about 2 hours to achieve a result equivalent to 1- 2 minutes reading time of ELISA. The PA-CGT technique has also been used by us for the detection of

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Sindbis virus as a model for arboviruses. One of us (DK) has determined the optimal conditions for trapping Sindbis virus and for its visualization by electron microscopy (D. Katz and Y. Straussman, unpublished results, 1982). Best results were obtained with commercial 400 mesh carbon-coated grids (Polaron Equipment Ltd Watford, England). For trapping Sindbis virus these grids were first coated with 1pg/ml of protein A in PBS for 15 minutes and then after washing with PBS, with Sindbis rabbit antiserum at a dilution of 1:500 for 15 minutes. Grids were then washed with 0.1% bovine serum albumin (BSA) in PBS and incubated for 1-3 hours on droplets of virus suspension. At no time in the procedure until after staining were the grids allowed to dry. The grids were positively stained for 3 minutes with 2% uranyl acetate in 47% ethanol. This stain gave better results than an aqueous solution of uranyl acetate or phosphotungstate. Our results showed, in agreement with other investigators, that significantly more virus particles were trapped on grids treated according to the PA-CGT as compared to the number trapped on nontreated grids. We found that 1fig/ml of protein A used for coating grids was as good as 5 and 25 ,ug/ml. However, grids that were coated with antiserum only trapped less virions than grids coated with protein A a t any of the concentrations tested at antiserum (dilutions 1:100 and 1:500). At antiserum dilution 1:2500 the differences were not significant. Under best conditions the PA-CGT trapped about 1.5 times more viruses than the AB-CGT (grids treated with antiserum only). In accordance with Lesemann and Paul (1980)we also found that the main advantage of the PA-CGT over the AB-CGT is that the former is less dependent on antiserum dilution. An important finding was that the washing of the grids in PBS alone (after incubation with antiserum and with virus) contributed to nonspecific trapping of virions. When, however, the washing solution was replaced by PBS-BSA (BSA, 0.1%) the test became very specific and the ratio of specific to nonspecific counts on the grids was about 40 :1. These results are summarized in Table 11. Time and temperature influence the amount of virus trapped on grids. This was concluded from an experiment in which a 1:150 dilution of Sindbis virus (1.3 X lo7PFU/ml) was incubated with coated grids for 1,2, 3, and 5.5 hours at 24°C (room) and 37°C. The optimal time of incubation was 3 hours; longer or shorter incubation times were less efficient (Fig. 1). At 37°C (3 hours incubation) about 1.5 more viruses were trapped as compared to the amount trapped at 24°C. However, 3 hours of incubation a t 37°C caused damage to the virions and the surfaces of the grids were covered with debris (Fig. 2).

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DAVID KATZ AND ALEXANDER KOHN TABLE I1

PRESENCE OF BSA IN BUFFER AFFECTS THE SPECIFITY OF VIRUSTRAPPING Grids washed after serum coating with Serum dilution" Antiserum Normal serum

1:500 1:2500 1:500

PBS

PBS-BSA

NDb

241f43c 193 k 15 6 & 9.6 17k-7

140 fz 39

ND 178fz48

1:2500

Serum incubated on grids previously coated with protein A (1,ug/ml). * Not done. Number of viruses trapped per unit area.

Figure 3 shows that the log,, number of virus trapped on grids was proportional to the virus concentration. The minimum detectable amount of virus was at about lo6 PFU/ml.

C. The Protein A-Coated Bacteria Technique (PA-CBT) In the last 2 years we have described (Katz etal., 1980; Nicolaieff etal., 1982) another ISEM method where the trapping device is a strain of

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FIG.2. Trapping of Sindbis virus (1:50 diln) on grids treated as described in Fig. 1. (a) Incubation of virus on grids at 24°C; (b) incubation of virus at 37°C. Note that the virions in (h) are damaged and there are many debris. Bars = 200 nm.

Staphylococcus aureus that contains protein A on its surface and can therefore easily be coated with specific antibody directed against a given virus. Such coated bacteria can be then used to “collect” the target virus from a suspension. The virions trapped on the surface of the bacteria can then be visualized in the electron microscope. We have used this technique (PACBT) for trapping of Sindbis virus (Katz et al., 1980) and of plant viruses such as tomato bushy stunt (TBSV), turnip yellow mosaic (TYMV), and tobacco mosaic (TMV) viruses (Nicolaieff et al., 1982). The procedure for Sindbis virus was as follows: A suspension of S. aureus (3 X lo8cells/ml in PBS)was mixed with rabbit anti-Sindbis virus diluted 1: 10 in PBS containing 0.02% sodium azide (PBS Az) and incubated at 37°C for 15 minutes. The suspension was then centrifuged in an Eppendorf centrifuge for 1.5 minutes (3200 g) and washed similarly in PBS Az. The pellet was resuspended in 1 ml of a target virus suspension for 40 minutes in a 37°C bath (or incubator) with shaking; after centrifugation for 2 minutes the pellet was resuspended in 0.1 ml of

+

+

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DAVID KATZ AND ALEXANDER KOHN

10

50

Reciprocal

150

450

1800

virus dilution

FIG.3. Trapping of Sindbis virus at 24 and 37°C by the PA-CGT. Grids coated with 1 ,ug/ml of protein A and antiserum (1 :500) were incubated on droplets of virus suspension at 24 and 37°C for 1 hour. The virus was applied to grids at various dilutions. Note the linear relationship between the log number of virions observedper unit area and their concentration in the suspension.

+

PBS Az by vigorous mixing (vortex). A drop of the suspension was applied to Formvar-carbon-coated grids, drained, and stained for 1minute with 2% phosphotungstate pH 7.3. The virions could be seen attached to the surface of the bacterium as single particles or as a continuous layer according to the virus concentration (Fig. 4a). The minimum concentration of Sindbis virus that could be detected as single virions per cell was lo6- lo6 PFU/ml.

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FIG.4. Trapping of viruses on Staphylococcus aureus. S. aureus suspensions were coated with specific antiserum (PA-CBT) against (a) Sindbis virus (lOs/ml), (b) tobacco mosaic virus (TMV) (100 ng/ml, and (c) TYMV (turnip yellow mosaic virus) (500 ng/ml). Bars = 200 nm.

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A similar procedure of the PA-CBT was used for plant viruses (Nicolaieff etal., 1982), however, it was found that higher dilutions of antiserum (1:10,000) were preferable for coating of bacteria (Fig. 4b and c). By this method (method 1)20-50ng(2-5 X 109virusparticles)/mlofTYMVand TBSV could be detected. The specificity of the PA-CBT expressed as a ratio between the number of homologous virions to heterologous ones was 20 :1 for TBSV/TYMV and 43 :1 for TYMV/TBSV. The sensitivity of the PA-CBT was increased about 4-fold when a smaller number of coated bacteria was used for trapping (2 X 106/ml),the final centrifugation step was omitted, and the suspension allowed to settle on the microscope grid for 4.5 hours (method 2). Both techniques, PA-CGT and the PA-CBT, have similar sensitivities, however the PA-CBT is less reproducible because of various technical problems such as bacterial clumping and heterogeneity. The PA-CBT was used by Lee etal. (1981) for serotyping of herpes virus and for the demonstration of adenovirus - antibody immunocomplexes. For serotyping, 0.1 ml of packed, heat killed, formalin-fixed, S. aureus containing protein A bacteria was mixed with 0.4 ml of a 1:400 dilution of rabbit anti-herpes virus antiserum and incubated at 37°C for 1hour. The cells were spun down at 2000 g for 10 minutes, washed twice with PBS, and resuspended in 12 ml PBS of the herpes virus suspension, incubated, and layered on 2 ml of a 5% sucrose layer for centrifugation at 2000 g for 15 minutes. The pellet was resuspended in 1 ml and processed for electron microscopy by the pseudoreplica technique. As in the PA-CBT described by us, in case of a positive result viruses are seen adsorbed to the surface of the bacteria. For demonstration of immunocomplexes a model system was used in which radioactively labeled adenovirus was complexed with its antibody. The complexes were precipitated in the cold with 4% polyethylene glycol (PEG), pelleted at 2000 g for 20 minutes, washed again with 4% PEG, centrifuged as before, and resuspended in PBS. The immunocomplexes were then mixed with 3% of protein A-containing bacteria, incubated for 2 hours at 37°C and centrifuged on a 5% sucrose cushion, 2000g for 10 minutes at 4" C. The adsorbed viruses were then eluted from the pelleted bacteria with KCI- HC1 buffer, pH 2.5, and after another centrifugation at 2000 g for 10 minutes, the supernatant was processed for electron microscopy by the pseudoreplica technique. It was calculated that 60% of the radioactive-labeled virus was present in the final eluate. In this procedure the bacteria are used merely as an intermediate immunoadsorbent: a positive reaction is indicated by the appearance of single virions in the electron microscope.

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D. The Antigen-Coated GridTechnique (AG-CGT) In the AG-CGT the grid is directly coated with the virus (antigen) which is specifically identified by the decoration of a specific antibody. This technique was extensively used by Yanagida and Ahmad-Zadeh (1970) and Yanagida (1972) for the localization of gene products and identification of antigenic precursors in bacteriophage T4. Other authors used this technique for similar purposes. Wrigley etal. (1977) studied the binding sites of antibodies to isolated hemagglutinin and neuraminidase of influenza virus, Norrby (1969) identified antigen specificities at the surface of adenovirus, and Vernon etal. (1981) studied the localization of herpes simplex virus nucleocapsid polypeptides. The first who used the AG-CGT for plant viral diagnosis and not for morphological localization of antigens were Milne and Luisoni (1975). In their procedure carbon-fronted Formvar-coated grids were touched to the virus suspension for a few seconds, rinsed with 30 drops of PB, drained, and further incubated with a drop of diluted antiserum for 15 minutes a t room temperature in a humid chamber. The grids were then washed with 20 drops of PB and with 50 drops of distilled water and finally stained with 2% of aqueous uranyl acetate. Some viral suspensions were either purified preparations in dilute buffer, or preparations from CsCl or sucrose bands. Others were infected leaf extracts. The washing steps which were essential to remove efficiently salts, sugars, and many impurities did not detach the viruses. Once a homologous antiserum was used, the interaction with the virus was evident as a halo of antibodies surrounding the virus. All the viruses tested (TMV, TRV (tobacco rattle virus), CVMV (carnation vein mottle virus), TBSV, and CMV remained intact after the AG-CGT except for MRDV (maize rough dwarf virus), For this virus a fixation step with 2% glutaraldehyde was included before washing and proceeding to the antiserum coating step. In the same paper a short timedclumpingmethod was compared to the AG-CGT. The authors conclude that whenever adequate numbers of viruses are present, the AG-CGT is preferable because it is quick and simple: when the virus concentration is low, either the AB-CGT (Derrick, 1973) or the “clumping method’’ (Milne and Luisoni, 1977a) should be used. The AG-CGT was successfully used for a detailed serological analysis of fractions from MRDV virions and for the determination of the serological relationship to a cross reacting rice black streaked dwarf virus (Luisoni et al., 1975). Milne and Lesemann (1978) compared three methods, AG-CGT, clumping, and AB-CGT, in a study of oat sterile dwarf and related viruses.

188

DAVID KATZ AND ALEXANDER KOHN

Their conclusion was that the AB-CGT was the most reliable for the study of serological relationships between the viruses since in principle only one particle was sufficient to obtain reliable results.

111. DISCUSSION In all immunosorbent electron microscopical (ISEM) methods like in the ELISA or SPRIA methods, one of the components of the system is adsorbed to a solid phase. We have discussed in this review four methods. In three of them (AG-CGT, PA-CGT and AB-CGT) one of the reagents is adsorbed to an electron microscopic grid, while in the fourth (PA-CBT) protein A is naturally present on the surface of a bacterium, which serves as a solid support. For the sake of uniformity of nomenclature we have suggested that these methods be given new names, that would identify both the support as well as the adsorbed reagent. Inspection of Table I would indicate how urgent is the need to unify the nomenclature of the ISEM methods. The method of Derrick (1973) (AB-CGT) and its modifications are a real breakthrough in immunoelectron microscopy. At optimal conditions they give highly specific and reproducible results, and their sensitivity is similar to that of the “classical IEM methods” that were based on the phenomenon of clumping (Almeida and Waterson, 1969b). The AB-CGT method is attractive also because there is no need to purify the samples examined, and because it permits good identification of virions present in crude extracts of infected secretions or excretions. This fact is emphasized by Narang and Codd (1981) who studied acute nonbacterial gastroenteritis: differential centrifugation of fecal samples actually led to loss of some viral flora. While in untreated fecal samples examined by direct EM methods adenovirus, astrovirus, rotavirus, and “small round” viruses were seen, in the centrifuged samples some of these viruses were lost, most probably because some viruses, already clumped by indigenous antibodies in faeces, are removed by centrifugation. The presence of such clumps may be mistakenly interpreted as due to the effects of specific immune serum added during the process of preparation of the sample for IEM. In the ISEM methos viruses cannot only be identified but also counted and their concentration may be numerically expressed as number of virions per unit of area, and can therefore be statistically evaluated. With these methods, therefore, one can quantify the effects of quality of the supporting grid, the time of adsorption, the pH, the presence of salts, and the type of staining, and thus optimize the results of the test.

IEM FOR DETECTION OF VIRUSES

189

1. Grid Coating

It is now quite clear that the critical parameter in all ISEM methods is the quality of the grid and its coating. Various investigators employed in their studies carbon-backed Formvar-coated grids (Rubinstein and Miller, 1982), carbon-fronted Formvar grids (Milne and Luisoni, 1977b), carboncoated grids only (Roberts and Harrison, 1979),and numerous other variations. Lesemann and Paul (1980) as well as Milne and Luisoni (197713) advocate the pretreatment of grids with glow discharge, but Milne and Leseman (1978)state that in the case of the AB-CGT this treatment has no particular advantage. Each laboratory should, therefore, test the grids and their coating for the particular system and viruses which it is investigating. The same consideration also applies to staining: one stain may be suitable for one group of viruses, but deletorious to another (Roberts and Harrison, 1979). 2. Antisera and Buffers

Another parameter which deserves consideration is the dilution of the antiserum used for coating the grids. In some cases, undiluted serum may actually inhibit the adsorption of virions. This fact is also recognized in other solid phase immunoassays. One has, therefore, to determine the optimal serum dilution fm every virus (especially in the AB-CGT) (Nicolaieff et al., 1980, 1982; Lesemann et al., 1980; Milne, 1980). Other factors of importance in the ISEM methods are the quality of the antiserum and the composition of the adsorption and washing buffers. So, for instance, the addition of a protein (BSA) to the washing buffer and to the virus suspension improved the specificity of the results, i.e., increased the differential counts on antiserum-coated grids as compared to those obtained on grids coated with normal serum (control) (Table 11, Section 11,B). In many studies, however (Milne and Luisoni, 1977b; Nicolaieff et al., 1980), good specific results were obtained, though protein was not present in the washing buffer. In those cases the procedure involved washing with some 20-50 drops between each incubation step; when BSA was present in the buffer, 6 drops were sufficient to achieve an equivalent degree of specificity. 3. Incubation Time

The time of incubation of the virus sample on the antiserum-coated grid affects the number of virions trapped (Nicolaieff et al., 1982; Kjeldsberg and Mortensson-Egnund, 1982). We observed that the maximal number of Sindbis virions adsorbed after 3 hours; longer incubation periods resulted in a decrease of the number of virions on the grid. It is feasible that

190

DAVID KATZ AND ALEXANDER KOHN

this finding may be true for other sensitive viruses, but not necessarily for viruses which are refractory to prolonged incubation. 4. Temperature of Incubation

In most of the studies, reviewed here, the virus samples were incubated on the grids at room temperature. In our own studies with Sindbis virus, incubation at 37°C led to an increase in the number of virions seen on the grid, but their structure was impaired (Fig. 2, Section 11,B). 5. Effect of p H on Trapping of Virions The pH of the buffer may strongly affect the degree of adsorption of the virions to the grids in the AB-CGT (Cohen et al., 1982). This p H dependence may explain the failure of Milne (1980) to trap efficiently cucumber mosaic virus (CMV). Milne worked with buffers at pH 7.0; Cohen et al. (1982) finds that for CMV the optimal pH was 8.0. 6.Protein A Coating of Grids

There has been a general agreement among the investigators cited in this review that treatment of grids with protein A (Shukla and Gough, 1979)before their coating with antiserum improved the results in comparison to the AB-CGT. Coating of grids with protein A subsequently permits use of a wider range of dilutions of the antiserum, and the inhibition of binding with undiluted serum is avoided. Because of that, one may safely use undiluted sera of low titer. The number of virions trapped on protein A-antiserum-coated grids is greater than that with the AB-CGT by a factor of least 2 (Milne, 1980), provided that the tests are done each at its optimal performance. With samples with small number of virions both methods were similar in their trapping efficiency. The coating of grids with protein A obviates the need to find the optimal pH for the binding of immunoglobulins (antiserum) to the grid (Cohen et al., 1982), since binding to protein A occurs as well at the neutral pH of buffers usually employed in most laboratories. 7. Decoration Decoration was used in conjunction with AB-CGT (Milne and Luisoni, 1977b; Giraldo et al.,1982; Roberts and Harrison, 1979) or with PA-CGT (Shukla and Gough, 1979). Kerlan et al. (1981) even employed a double decoration method so as to make the virions in the image increase in size due to the double antibody coating on them. All investigators agree that decoration aids the specificity of the tests and makes the identification of the virus easier. In our experience with Sindbis virus, the specificity of PA-CGT was equally high with or without decoration, presumably because

IEM FOR DETECTION OF VIRUSES

191

of the use of BSA in the buffer. It seems thus that the decoration method would be advantageous for very small viruses, or when there is a problem of background and contrast. 8. S. aureus - Protein A

In 1980 we suggested to use as a trapping agent Staphylococcus aureus which has on its surface protein A; we now call this technique PA-CGT (Katz et al., 1980). We hoped that this method would be more sensitive than the other ISEM methods because (1)protein A is a natural product of the bacteria, and they adsorb large amounts of gamma globulins; (2) the bacteria added to a virus suspension trap viruses during their brownian movement in the suspension; (3) though freely moving, each bacterium performs as a solid phase; and (4)after the incubation of the bacteria with the viral suspension it is easy to spin down the bacteria even in a clinical centrifuge, and to resuspend them in a minute volume of buffer, suitable for the deposition on the EM grids. Though the efficiency of trapping with the PA-CBT is higher than that of PA-CGT (Nicolaieff et al., 1982), the sensitivity of PA-CBT is about equal to that of PA-CGT. The reason for this lack of improved sensitivity lies in the fact that in the EM one may see only the virions at the circumference of bacteria (Fig. 4), but not those adsorbed on top or underneath them (a calculation indicates that 45 times more bacteria are present on the bacterium than actually seen). In addition the bacteria tend to clump, and this clumping also impairs the observation in the EM. The use of bacteria, however, by the method of Lee et al. (1981) may avoid some of the problems that our method poses. The improvement suggested by Lee et al. (1981) is to “peel off” the viruses trapped on the bacteria by elution at low pH, and thus to obtain a concentrated suspension of the virions, free of bacteria. This technique that also facilitates the detection of immunocomplexes may be of importance in diseases such as hepatitis B (Almeida and Waterson, 1969b). 9. Direct Trapping of Viruses on Grids

It seems to us that the AG-CGT as described by Milne and Luisoni (1975) is useful only if the quantity of the virus in the sample is very high and therefore that this method is more useful for the study of the antigenic structure of a virus rather than for diagnosis. 10. I S E M and Other Solid Phase Immunoassays

The sensitivities of PA-CGT or AB-CGT are equivalent to those of ELISA (Rubinstein and Miller, 1982; Nicolaieff et al., 1982). The sensitivity of the methods is expressed either as limiting ng/ml or as the number

192

DAVID KATZ AND ALEXANDER KOHN

of infective units of virus (e.g., PFU/ml). ELISA is able to detect a few ng/ml of viral protein which is equivalent to lo7- lo8 virions. Katz etal. (1980) found that the limit of detection of Sindbis virus with PA-CBT is lo6- lo6 PFU/ml, while Giraldo etal. (1982),working with papova viruses, set the limit lo2-lo3 PFU/ml. As long as the exact number of virions necessary for 1PFU is not determined it is very misleading to compare various methods using PFU as a criterion. In respect to the Sindbis virus 1 PFeT contains approximately 30 virions (A. Shapira and S. Lustig, personal communication). Therefore, the limiting detectable number of Sindbis virions by the PA-CGT would be 3 X lo7. As to the data of Giraldo etal. (1982) concerningpapovavirus we do not know how many virions there are in 1 PFU, and therefore it is impossible to state whether his method is more or less sensitive. What are the comparative merits of ISEM methods in relation to SPRIA or ELISA? The relative disadvantages of ISEM lie, first, in the requirement for an expensive instrument (electron microscope), second, in the small number of samples that can be visually processed in EM, and third, in the fact that the presence of soluble antigens in the sample may decrease greatly the sensitivity of the method. On the other hand the advantages of ISEM are (1)direct and dependable identification of a virus based not only on the specific antigen-antibody reaction, but also on morphology; and (2) the preparation of samples for electron microscopy requires only 15-60 minutes for most of the ISEM methods. 11. Plantus Animal Viruses

The number of publications describing the use of ISEM methods for the diagnosis of animal viruses is small in relation to the number of studies on plant viruses. We assume that this state of affairs is due t o the historical fact that the ISEM methods were primarily developed by plant virologists and the transfer of methodology from the plant virus field to that of animal viruses might take some time. In distinction from animal viruses which are present only in scant numbers in body fluids and excretions, plant viruses are quite abundant in the tissues of infected plants, and their concentrations there may reach values as high as 1mg/ml (Lesemann et al., 1980). The finding and identification of plant viruses by EM is thus much easier than that of animal viruses. It is therefore understandable that quite a considerable effort has yet to be invested in the optimization of the ISEM methods for the diagnosis of animal viruses. Plant viruses are not infective to animals, while any infective virus of man presents a potential hazard for the laboratory worker (Field, 1982). The existing methods for the inactivation of virus infectivity for ISEM

IEM FOR DETECTION OF VIRUSES

193

(that would not affect the integrity and characteristic morphology of the virus in question) are still in the stage of development. Nevertheless, in those studies where ISEM methods had been applied to the diagnosis of animal viruses, satisfactory results have already been obtained. We believe therefore that diagnostic laboratories equipped with EM would do well to introduce and to perfect the ISEM methods, especially in those cases where other diagnostic methods are not yet satisfactory. The ISEM also permits a quite detailed study of antigenic variations in the same genus of virus, especially now when monoclonal antibodies could be used as the trapping or decorating y-globulins, and thus would visually pinpoint the type or strain differences. One has to bear in mind, however, that in the methods of the type of IEM, SPIRA, or ELISA, one examines at a time the presence of only one specific antigen. With the direct EM methods, one can distinguish several morphologically and antigenically distinct viruses (Kjeldsberg and Mortensson-Egnund, 1982). Berthiaume etal. (1981) suggest using commercial pools of y-globulin as clumping antibodies, since such pools contain antibodies to a large number of common animal viruses. Such pools would thus be very useful to pick up from feces not only rotaviruses but other viruses such as astrovirus, adenovirus, and calicivirus. Berthiaume’s method can be hopefully applied to the PA-CGT, as well as to other ISEM methods.

REFERENCES Almeida, J. D. (1980). YaleJ.Biol. Med. 6 3 , 5. Almeida, J. D., and Waterson, A. P. (1969a). A h . Virus Res.15, 307. Almeida, J. D., and Waterson, A. P. (1969b). Lancet 2,983. Almeida, J. D., Rubenstein, D., and Stott, E. J. (1971). Lancet 2, 1225. Anderson, N., andDoane, F. W. (1973). Can. J. Microbiol. 19,585-589. Anderson, T. F., and Stanley, W. M. (1941). J.Biol. Chern.139,339. Berthiaume, L., Alain, R., McLaughlin, B., Payment, P., and Trepanier, P. (1981). J. Gen. Virol. 55, 223. Best, J. M., Banatvala, J. E., Almeida, J. D., and Waterson, A. P. (1967). Lancet 2 , 237. Brenner, S., and Horne, R. W. (1959). Biochirn. Biophys. Acta 3 4 , 103. Catt, K. J. (1969). Endocrinology 6 3 , (Baltimore) Suppl. 142, 222. Cohen, J., Loebenstein, G., and Milne, R. G. (1982). J. Virol. Methods 4 , 323. Derrick, K. S. (1973). Virology 66, 652. Derrick, K. S., and Brlansky, R. H. (1976). Phytopathology 66,815. Doane, F. W., and Anderson, N. (1977). I n “Comparative Diagnosis of Viral Diseases” (E. Kurstack and C. Kurstack, eds.), Vol. 11, Part B, pp. 506-539. Academic Press, New York. Doane, F. W., Anderson, N., Chao, J., and Noonan, A. (1974). Appl. Microbial. 27,407. Engvall, E., and Perlmann, P. (1971). Zrnrnunochernistry 8 , 8 7 1 . Feinstone, S. M., Kapikian, A. Z., and Purcell, R. H. (1973). Science 182, 1026. Field, A. M. (1982). Ada Virus Res.27, 1.

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Flewett, T. H., and Boxall, E. (1976). Clin. Gastroenterol. 6,359. Giraldo, G., Beth, E., Lee, J., De Harven, E., and Chernesky, M. (1982). J. Clin. Microbiol. 15,517. Gough, K. H., and Shukla, D. D. (1980). J. Gen. Virol. 61, 415. Kapikian, A. Z., Almeida, J. D., and Stott, E. J. (1972a). J. Virol. 10, 142. Kapikian, A. Z., Wyatt, R. G., Dolin, R., Thornhill, T. S., Kalica, A. R., and Chanock, R. M. (1972b). J. Virol. 10, 1075. Katz, D., Straussman, Y., Shahar, A., and Kohn, A. (1980). J. Immunol. Methods 38,171. Kelen, A. E., Hathaway, A. E., and McLeod, D. A. (1971). Can. J. Microbiol. 17, 993. Kerlan, C., Mille, B., and Dunez, J. (1981). Phytoputhology 71,400. Kjeldsberg, E., and Mortensson-Egnund, K. (1982). J. Virol. Methods 4, 45. Kraehenbuhl, J. P., and Jamieson, J. D. (1976). Methods Immunol. Immunochem. 5,482495. Kurstak, E., Tyssen, P., and Kurstak, C. (1977). I n “Comparative Diagnosis of Viral Diseases” (E. Kurstak and c . Kurstak, eds.), Vol. 11, Part B, pp. 403-448. Academic Press, New York. Lee, F. K., Nahmias, D. G. E., andNahmias, A. J. (1981). Proc. Int. Congr. Virol., 5th, 1981 Abstract P 13/11, p. 169. Lesemann, D. E., and Paul, H. L. (1980). Actu Hortic. 110, 119. Lesemann, D. E., Bozarth, R. F., and Koenig, R. (1980). J. Gen. Virol. 48, 257. Luisoni, E., Milne, R. G., and Boccardo, G. (1975). Virology 68, 86. Madalinski, K., Sztachelska-Budkowska, A., and Brzosko, W. J. (1974). J. Infect. Dis. 129, 371. Milne, R. G .(1980). Actu Hortic. 110, 129. Milne, R. G., and Lesemann, D. E. (1978). Virology 90, 299. Milne, R. G., and Luisoni, E. (1975). Virology 68,270. Milne, R. G., and Luisoni, E. (1977a). Virology 80, 12. Milne, R. G., and Luisoni, E. (197713). Methods Virol. 6,265-281. Narang, H. K., and Codd, A. A. (1981). J. Clin. Microbiol. 5, 982. Nicolaieff, A., and van Regenmortel, M. H. V. (1980). Ann. Virol. (Znst. Pasteur) 131,95. Nicolaieff, A., Obert, G., andvan Regenmortel, M. H. V. (1980). J. Clin. Microbiol. 12,101. Nicolaieff, A., Katz, D., and van Regenmortel, M. H. V. (1982). J. Virol. Methods 4, 155. Norrby, E. (1969). Virology 37,565. Obert, G., Gloeckler, R., Burckard, J., and van Regenmortel, M. H. V. (1981). J. Virol. Methods 3, 99. Pensaert, M. B., Debouck, P., and Reynolds, D. J. (1981). Arch. Virol. 68,45. Rifkind, R. A. (1976). Methods Immunol. Immunochem. 5,458-450. Roberts, I. M., Milne, R. G., and van Regenmortel, M. H. V. (1982). Interuirology 18,147. Roberts, I. M., and Harrison, B. D. (1979). Ann. Appl. Biol. 93, 289. Rubinstein, A. S., and Miller, M. F. (1982). J . Clin. Microbiol. 16, 938. Shukla, D. D., and Gough, K. H. (1979). J. Gen. Virol. 45,533. van Regenmortel, M. H. V. (1981a). Compr. Virol. 17, 183. van Regenmortel, M. H. V. (1981b). “Serology and Immunochemistry of Plant Viruses.” Academic Press, New York. van Regenmortel, M. H. V., Nicolaieff, A., and Burckard, J. (1980). Actu Hortic. 110, 107. Vernon, S.K., Ponce De Leon, M., Cohen, G .H., Eisenberg, R. J., and Rubin, B. A. (1981). J. Gen. Virol. 64, 39. Wrigley, N. G., Laver, W. G., and Downie, J. C. (1977). J. Mol. Biol. 109, 405. Yanagida, M. (1972). J. Mol. Biol. 65, 501. Yanagida, M., and Ahmad-Zadeh, C. (1970). J . Mol. Biol. 61, 411.

ADVANCES IN VIRUS RESEARCH. VOL. 29

THE ENTOMOPOXVIRUSES Basil M. Arif Forest Pest Management Institute Canadian Forestry Service Department of the Environment Sault Ste. Marie, Ontario, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Host Range. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Structural Features . . . . . . . . . . . . . . . . . . . . . . . . . A. Thevirion. . . . . . . . . . . . . . . . . . . . . . . . . . . B. Spindles. . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Viral Components. . . . . . . . . . . . . . . . . . . . . . . . . . A. Structural Proteins . . . . . . . . . . . . . . . . . . . . . . . B. Enzymes of Virion and Inclusion Body. . . . . . . . . . . . . . . C. NucleicAcid . . . . . . . . . . . . . . . . . . . . . . . . . . V. Virus Infection and Multiplication. . . . . . . . . . . . . . . . . . . A. InLarvae . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Tissue Culture Cells. . . . . . . . . . . . . . . . . . . . . . VI. Is There a Potential for These Viruses in Pest Control? . . . . . . . . . VII. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195 198 198 198 200 200

201 203 205 206 207 208

209 210

211

I. INTRODUCTION

“A new type of insect virus,” a poxvirus of insects was originally described by Vago (1963), and since then these viruses have been isolated from approximately 31 species of insect in widespread geographical locations. The International Committee on Taxonomy of Viruses (ICTV) included the genus Entomopoxvirus (EPV) in the Poxviridae family (Matthews, 1979). The genus contains three probable subgenera represented by the type species Melolontha rnelolantha EPV (subgenus A, Coleoptera), Amsacta moorei and Choristoneura biennis EPV (subgenus B, Lepidoptera), and Chironomus luridus (subgenus C , Diptera; Matthews, 1979). In this review, the system for the identification of each EPV will be similar to that used to name nuclear polyhedrosis viruses (NPVs), i.e., a virus assumes the name of the host insect from which it was originally isolated. For example, an isolate from the eastern spruce budworm, Choristoneura fumiferana, will be called CfEPV. Compared to the other occluded insect viruses, the NPVs, the granulosis 195

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TABLE I MORPHOLOGICAL PROPERTIES OF CERTAXN ENTOMOPOXVIRLISES"

Host

+

8

Lepidoptera Am-sacta moorei (Arctiidae) Oreopsyche angustella (Psychidae) Operophtera brumata (Geometridae) ~ u x o auniliaris a (Noctuidae) Choristoneura biennis (Tortricidae) Choristoneura conflictana (Tortricidae) Choristoneura diversanu (Tortricidae) Coleoptera Melolontha melolonth (Scarabaeidae) Othnonius batesi (Scarabaeidae) Demodenaboranensis (Scarabaeidae)

Virus shape

Core shape vertical section

Virus size (nm)

Inclusion body (pm)

350 X 250

1-4

Oval

Rectangular

Few

360 X 260

3-10 X 2-7

Oval

Rectangular

Present

400 X 350

3- 15

Oval

Rectangular

Present

260 X 165

4-5

Oval

Rectangular

Absent

Weiser and Vago (1966); Weiser etal.(1970) Sutter (1972)

400 X 300

2-3

Oval

Rectangular

Present

Bird etal.(1971)

273-235

7x4

oval

Rectangular

Present

Cunningham et al. (1973)

280 X 220

7x5

Oval

Rectangular

Present

Katagiri (1973)

450 X 250

10-24

Oval

Unilaterally concave

Present

470 X 265

5-10

Oval

Present

420 X 230

8-11

Oval

Unilaterally concave Unilaterally concave

Hurpin and Vago (1963); Bergoin etal.(1968); Hurpin and Robert (1967) Goodwin and Filshie (1969); Goodwin and Roberts (1975) Vago etal. (1968a)

Spindles

Present

Fteferences Roberts and Granados (1968); Granados and Roberts (1970) Meynadier etal.(1968)

Geotrupes silvaticus (Scarabaeidae) Dermolepida alborhirtum (Scarabaeidae) Aphodius tasmaniae (Scarabaeidae) Anomalacuprea (Scarabaeidae) Phyllopertha horticola (Rutelidae) Figulus sublaevis (Lucanidae)

& . l

Diptera Chironomus luridus (Chironomidae) Chironomus attenuntus (Chironomidae) Camptochironomus tentans (Chironomidae) Goeldichironomus holoprasinus (Chironomidae) Aedesaegypti (Culicidae) Orthoptera Melanoplus sanguinipes (Acrididae)

366-416 X255-286 420-450 X 220 - 240 380-430 X250-300 440 X 250

3.5-11

Oval

3-5

Oval

5-12

Oval

5x8

Oval

400 X 240

6-25

Oval

330 X 290

1-5

Oval

330 X 230 XllO

4-7

Cuboidal

330 X 250 X 130

Up to 6

220-250 X 270 - 300 346 X 300 X 160

Present

Lipa and Bartkowski (1972)

Absent Present

Goodwin and Filshie (1975); Goodwin and Roberts (1975) Goodwin and Filshie (1975)

Present

Katagiri etal. (1975)

Present

Vago etal.(1969)

Present

Vago etal.(1968b)

Dumbell

Absent

Gotz etal. (1969); Huger etal.(1970)

Cuboidal

Dumbell

Absent

Stoltz and Summers (1972)

2-8

Cushion shape

Dumbell

Absent

Weiser (1969)

3x5

Cushion shape

Dumbell

Absent

Federici etal. (1974)

320 X 230

320 X 250

Modified from Kurstak and Garzon (1977).

Unilaterally concave Unilaterally concave Unilaterally concave Unilaterally concave Unilaterally concave Unilaterally concave

Dumbell

2-11

Rectangular or dumbell

Buchatskyi (1974)

Absent

Henry etal.(1969)

198

BASIL M. ARIF

viruses (GVs), and cytoplasmic polyhedrosis viruses (CPVs), the EPVs are not as well characterized probably due to the fact that they have not been considered as potential biological control agents. The object of this review is to bring together the current knowledge on EPVs, particularly the biochemical and biophysical aspects, and hopefully will complement, rather than repeat, the other excellent reviews written on this subject (Bergoin and Dales, 1971; Granados, 1973a, 1978,1981; Kurstak and Garzon, 1977).

11. HOST RANGE EPVs have so far been found in four insect orders, Lepidoptera, Coleoptera, Diptera, and Orthoptera. Most of the isolates originate from Coleoptera and Lepidoptera and the least from Diptera. Table I summarizes the host range of these viruses and some of their properties.

111. STRUCTURAL FEATURES

As in the case of the other occluded insect viruses, the most prominent feature of mature virus particles is their occlusion in a proteinaceous matrix termed spheroid (Goodwin and Filshie, 1969,1975)which accumulate in the cytoplasm at the end of the viral replicative cycle. Spindle-shaped proteinaceous bodies (Spindles), free of virus, are also synthesized in the infected cell, and in some instances they are also occluded in the spheroid.

A . The Virion The entomopox virions have many structural similarities to the orthopoxviruses. The virion is brick-shaped or oval with sizes ranging from 150 to 470 nm long, and from 165 to 300 nm wide. Negatively stained virus particles exhibit a folded outer membrane to give the appearance of a mulberry-like surface (Fig. 1A). These are similar to the M-form of vertebrate poxviruses (Westwood et al., 1964). These spherical folds vary in size depending on virus species and measure approximately 40 nm for Anascata moorei EPV (AmEPV) and 22 nm for Melolontha melolontha EPV (MmEPV) (Granados and Roberts, 1970; Bergoin et al., 1971). In cross-section, or when the negative strain penetrates the particle, the virion is seen to contain an electron-dense core surrounded by a multilayer membrane. EPVs from Orthoptera and Lepidoptera generally contain cyclindrical core and two lateral bodies (Fig lB), while those infecting Diptera contain a biconcave core and two well-developed lateral bodies.

THE ENTOMOPOXVIRUSES

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entomopoxvirus. (A) Intact virions isolated FIG.1. Structure of Choristoneura biennis from spheroids by dissolution with alkali. Note the mulberry-like surface structure. (B) Virus particle damaged by alkali. Note the rectangular core (C),core outer membrane (CM), lateral bodies (LB), and viral outer membrane (OM).

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BASIL M. ARIF

EPVs from Coleoptera contain a unilaterally concave core and one lateral body located in the cavity of the core. For excellent electron microscopic analyses of EPV, refer to Bergoin and Dales (1971), Granados and Roberts (1970), Stoltz and Summers (1972), Granados (1973a), and Kurstak and Garzon (1977). Some EPVs have an outer diffused layer termed “halo” (Bergoin etal., 1968),the nature of which is still not at all clear, but it is probably proteinaceous in nature since it is susceptible to trypsin (McCarthy etal., 1974). These authors also reported that removal of the “halo” by proteolytic digestion results in particles with higher relative infectivity and exhibit RNA polymerase activity. Isopycnic centrifugation of AmEPV in CsCl showed that virus particles with “halo” have a density 1.282 g/ml and without “halos” a density of 1.262 g/ml (McCarthy etal., 1974). However, virus particles with “halos” did not band sharply in CsCl indicating heterogeneity in such preparations. Such diffused outer layers do not appear to be associated with EPVs from the Choristoneura spp. (Arif, 1976).

B. Spindles Cells infected with EPVs from Lepidoptera and Coleoptera, but not from Orthoptera or Diptera, produce a large number of paracrystalline proteinaceous spindles. The molecular lattice of the spindle is approximately 58 A (Bird, 1974). The spindles vary in size, and even within the same species their range could be from 0.5 to 12 pm (Vago and Bergoin, 1968), and spindles up to 25pm have been described (Kurstak and Garzon, 1977). The function and significance of the spindles is still obscure, but their protein appears to be antigenically and chemically different from the 1970; Croizier and Veyrnues, 1971; Berspheroid protein (Bergoin etal., goin and Dales, 1971). The spindles are occasionally not dissolved when the spheroids are treated with carbonate solution, and as a result they band on a sucrose density gradient with virus particles that have been damaged by the alkali treatment (Fig. 2) (B. M. Arif, unpublished results). In such preparations the lattice structure of the spindles becomes quite visible. IV. VIRALCOMPONENTS The current knowledge on the viral structural proteins, enzymes, and nucleic acids will be reviewed in this section. It will be seen that biochemical data on certain EPVs confirm electron microscopic observations in that the structure of the virions is highly complex.

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FIG.2. Spindels of C. biennis entomopoxvirus. Occasionally spindles do not dissolve in alkali and band on a sucrose gradient with alkali-damaged virus particles. Note the paracrystalline structure of the spindles (S). Virion (V) and viral core (C).

A. Structural Proteins 1 . The Virion

Spheroids are usually dissolved in a solution of sodium carbonate containing a disulfide reducing agent such as sodium thioglycollate, and the released virions are then purified on either sucrose or CsCl gradients. Subsequent analysis on polyacrylamide gels show that purified virus particles contain a large number of structural proteins. Virions from Euxoa auxiliaris (EaEPV), AmEPV, and from Melanoplus sanguinipes (MsEPV)

202

BASIL M. ARIF

contained 24, 36, and 39 polypeptides, respectively (Langridge and Roberts, 1982). They compared the EPVs polypeptide profiles to that of vaccinia and found that there was little or no homology, in as far as the molecular size is concerned. It was also suggested that the similarity in size of DNA between vaccinia andEPVs (Section IV,C,l) is reflected in the number of structural proteins, and that also indicated that approximately the same percentage of the virus genome is expressed in vaccinia and in EPV (Langridge and Roberts, 1982). This is an interesting observation but remains to be documented. Virions from Choristoneurubiennis (CbEPV) revealed the presence of at least 40 polypeptides ranging in molecular weight from 12K to 250K (Bilimoria and Arif, 1980). The molecular weight of these proteins is 2.9 X lo6 which is approximately 38% of the total coding capacity of the genome (137 X lo6,Section IV,C,l). Densitometric measurement of the viral proteins showed that 12 were major polypeptides constituting approximately 95% of the total viral proteins (VPs), and of these major proteins, V12, VP59, VP89, and VP46 have a relative abundance of 24,15,12, and 9%, respectively. Viral cores, isolated by treatment with nonionic detergent NP-40, contained only one of the major protein, VP59, plus other minor proteins. Major proteins such as VP102 and VP89 were present, but in a much reduced amount when compared to the profile of total virions. The latter two proteins are probably surface polypeptides or associated with the lateral bodies, while VP59 is a core protein, probably internal and associated with the viral DNA. 2. Inclusion Body Protein -Spheroidin

Following the accepted convention to call the inclusion body protein of NPV as polyhedin, and of GV as granulin (Summers, 1975), it was, therefore, decided to call the equivalent protein of EPVs as spheroidin (Bilimoria and Arif, 1979). In order to determine the size of spheroidin of CbEPV, spheroids were dissolved in SDS, 2-mercaptoethanol, and 8 M urea in a 100°C water bath and analyzed on polyacrylamide gels. Spheroids were found to be composed of a single polypeptide with a molecular weight of 102K (MP102, Bilimoria and Arif, 1979). This is similar to the results found with NPVs and GVs (Summers and Smith, 1978),except that spheroidin is 2 - 3 times larger than polyhedrin or granulin. The matrix protein of AmEPV was also shown to be composed of single polypeptide with a molecular weight of llOK (Langridge and Roberts, 1982). Amino acid analyses of the spheroidins of AmEPV, EaEPV, and MsEPV showed that the acidic amino acids, aspartic and glutamic acids, and the basic amino acids, lysine and arginine, were present in approximately equimolar amounts (Langridge and Roberts, 1982). The authors also

THE ENTOMOPOXVIRUSES

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noted that the sulfur containing amino acids, cysteine and methionine in the spheroidins of AmEPV, EaEPV, and MsEPV constituted 9,8.1, and 3.7% of the total amino acids, respectively. They made the point that the need for a disulfide bond reducing agent, such as sodium thioglycollate or 2-mercaptoethanol, during alkali dissolution of spheroids is likely due to the high content of these two amino acids. Bergoin et al. (1970) were the first to report the need for a disulfide reducing agent along with alkali to liberate virions from spheroids.

B. Enzymes of Virion and Inclusion Body Four enzymatic activities are associated with the virion particles: a nucleotide phosphohydrolase, acidic and neutral DNases, and a DNA-dependent RNA polymerase. It is likely that in the future other virion enzymatic activities will be detected. An endogenous alkaline proteolytic activity is also reported to be associated with spheroidin. I . Nucleotide Phosphohydrolase (NPH)

Approximately 50- 100 pg of virus (E.acrea EPV) were incubated with [ Y - ~ ~ P ] Ain T Pa buffer composition similar to that used with vaccinia virus NPH assay (Gold and Dales, 1968). After 60 minutes of incubation, 3048 nmol of ATP were hydrolyzed per milligram of viral protein (Pogo et al., 1971). This level of NPH activity is comparable to that observed with vaccinia virus. Treatment with a nonionic detergent, NP-40, and 2-mercaptoethanol did not enhance the enzymatic activity, probably because the viral membrane is permeable to ATP. The activity of NPH was not affected by ouabain and rutamycin, known inhibitors of cellular ATPases (Pogo et al., 1971). 2.Acidic Deoxyribonuclease

Purified EPV was incubated with %'-labeled L cells DNA in acidic buffer. After 60 minutes approximately 34 pug of denatured DNA was degraded per milligram of viral protein (Pogo et al.,1971). The optimum pH was 5.0. The acidic DNase activity was considerably lower than that observed with vaccinia virus. One probable explanation is a partial inactivation of the enyzme when the virions were in high pH environment during alkali dissolution of the spheroids. 3. Neutral Deoxyribonuclease

Approximately 46 pg of denatured L cells DNA was degraded by 1mg of viral protein in 60 minutes in assay conditions similar to that used for

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BASIL M. ARIF

vaccinia virus neutral DNase (Pogo etal., 1971). The pH optimum for this enzyme is 7.8. 4.DNA-Dependent RNA Polymerase

The reaction mixture is similar to that used in the assay of vaccinia virus RNA polymerase, except in this case (AmEPV) the reaction is carried out at 26 instead of 37°C. In 60 minutes 5 - 25 pm of UMP were incorporated into acid precipitable product per 1 mg of viral protein when all four ribonucleotides triphosphates were present (McCarthy et al., 1974). Other investigators were not as successful as these authors in detecting such activity in EPV, and the reason for the success of McCarthy and coinvestigators is likely due to the fact that once the spheroids had dissolved, the pH of the reaction was lowered to 8.5-9.0 and subsequently treated the virions with trypsin. The RNA product labeled with [3H]ATP in the presence of the other three ribonucleotides triphosphates had an apparent sedimentation coefficient of 8-23 S which was reduced to 3 - 5 S after treatment with RNases 1975). It is probable that this RNA contained poly(McCarthy etal., adenylic acid sequences since it was not completely RNase sensitive. In addition, when [3H]ATPwas used as the sole enzyme substrate, the reaction was not inhibited by actinomycin D, and the product was resistant to RNase hydrolysis (McCarthy etal., 1975). This indeed indicated the possible presence of another enzyme, a polyadenylic acid polymerase, in AmEPV. Of course, vaccinia is known to contain a polyadenylic acid polymerase whose activity is resistant to actinomycin D (Moss etal., 1973; Sheldon and Kates, 1974).

5.Alkaline Protease The inclusion body protein of baculoviruses contains an endogenous alkaline protease which degrades the matrix protein into smaller polypeptides (e.g., Epstein and Thoma, 1975; Tweeten etal., 1978). A similar enzymatic activity was observed in the case of entomopoxviruses (Bilimoria and Arif, 1979;Langridge and Roberts, 1982). As mentioned earlier the matrix protein of CbEPV has a molecular weight of 102K. This protein is very quickly degraded into the 52K polypeptides and eventually into smaller polypeptides when the spheroids are dissolved in alkali to release the virions (Bilimoria and Arif, 1979). Azocall assays (Levinsohn and Aronson, 1967) demonstrated a pH optimum of 10-11 for the CbEPV protease. The activity was inhibited by heating at 60°C for 10 minutes, soybean trypsin inhibitor, SDS (2.5%), and 1mMHgC1,. Tissue culturederived spheroids did not exhibit alkaline protease activity even when the

THE ENTOMOPOXVIRUSES

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matrix protein is incubated at 28°C for 2 hours (Langridge and Roberts, 1982). This phenomenon is similar to that observed earlier with tissue culture derived baculovirus inclusion bodies (Zummer and Faulkner, 1979). C. Nucleic Acid

The genome of EPVs is a double-stranded DNA molecule similar to that of orthopoxviruses and constitute approximately 5% of the virus particle (Gotz etal., 1969; Granados and Roberts, 1970; McCarthy etal., 1974). The double-stranded nature of the DNA was demonstrated by characteristic hyperchromicity observed during thermal melting analyses at 260 nm, by reaction of the DNA with formaldehyde, and from buoyant density measurement in cesium chloride gradients (Arif, 1976; Langridge etal., 1977). 1 . Size

The size of the genome isolated from EPVs of Lepidoptera appear to be smaller than those of Diptera of Coleoptera. Arif (1976) reported the size of the genome of CbEPV to be in the order of 137 X lo6daltons. Contour length measurements of the genomes of several EPVs in the electron microscope put the size in the range of 135-200 X lo6 daltons (Langridge and Roberts, 1977). Table I1 illustrates the properties of several EPV DNAs from different genera. Electron microscopic analysis also showed the DNA to be linear, similar to orthopoxviruses. However, the size of vaccinia DNA is somewhat smaller than that of EPVs, 122 X lo6 daltons (Geshelin and Berns, 1974). Restriction endonuclease analyses on Chospp. EPV showed that the size of the DNA is slightly larger ristoneura than reported earlier, in the order of 150 X lo6daltons (B. M. Arif, unpublished results). 2.BaseComposition

It appears that a fundamental difference between orthopoxviruses and EPVs is reflected in the low G C content of the latter group. Genomes of EPVs contain from 17 to 27% G C (Arif, 1976; Langridge etaL, 1977), whereas the G C content of orthopoxviruses range from 32.5 to 39% (Pfau and McCrea, 1962; Joklik, 1962; Szybalski etal., 1963; Yau and Rouhandeh, 1973). Since there has been no effort made to carry out detailed physical or transcriptional mapping of EPV genomes, it is difficult to speculate at this stage on what is the significance or the effect of such low G C content on the genome structure and function.

+

+

+

+

206

BASIL M. ARIF TABLE I1

PROPERTIES OF VIRALDNA" Size X average

Host Lepidoptera Amsacta moorei (Arctiidae) Choristoneura biennis (Tortricidae) Choristoneura fumiferana (Tortricidae) Euxoa auxiliaris (Noctuidae) Coleoptera Othnonius batesi (Scarabaeidae)

Diptera Chironomus near decorous (=attenuatus) (Chironomidae) Goeldichironomus holoprasinus ( Chironomidae) Orthoptera Melanoplus sanguinipes (Archididae)

T,,, Density

G+C average

(X)

Reference Langridge and Roberts (1977); Langridge et al. (1977) Arif (1976)

134.7

76.gb

1.6878

18.5

137

61.6'

1.6849

26

62.2"

1.6838

25.7

135.6

-

-

-

Langridge and Roberts (1977)

200.4

-

-

-

-

-

1.676

16.3

Langridge and Roberts (1976) Langridge et al. (1977)

-

77.8b

1.681

21.2

199.2

-

-

-

123.2

-

1.678

18.6

Arif (1976)

Langridge et al. (1977) Langridge and Roberts (1977) Langridge and Roberts (1976)

Modified from Harrap and Payne (1979). *InlXSSC. In 0.1 XSSC.

v. VIRUS INFECTION AND MULTIPLICATXON Detailed electron microscopic studies on the replication of EPVs in larvae have been carried out by several laboratories. Such studies in tissue culture cells have been much more limited. The following is a brief outline of the present knowledge of EPV replication in viuo and in uitro.

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207

A. I n Larvae 1. Adsorption and Penetration

As in the case of t.he other occluded insect viruses, infection begins when larvae ingest viral inclusion bodies and the virions are released in the alkalkine environment of gut. Granados (1973b) observed that AmEPV is first detected in the gut lumen 1- 2 hour after per 0s inoculation of E.acrea larvae. The virus adsorbs on to the cells then the viral membrane fuses with the plasma membrane of microvilli and subsequently the viral core with portions of the lateral bodies enter the cell. This appears to be the usual mechanism of entry into cells, andviropexis does not appear to play a significant role when larvae are infected by per 0s inoculation (Granados, 1973b). However, viropexis is the normal mechanism of entry into cells when larvae receive an intrahemocoelic injection of virus (Devauchelle et al., 1971). The virus appears in a vacuole in the cell cytoplasm and at a later stage the vacuole breaks down and the virions uncoat to release free cores. 2. Morphogenesis and Maturation Electron microscopic studies on the development of EPVs in larvae have been excellently presented by Devauchelle et al. (1971), Stoltz and Summers (1972), Granados (1973a), Bird (1974), Bergoin et al. (1976), and Kurstak and Garzon (1977). Basically these studies show that after uncoating and a certain period of latency, cytoplasmic foci consisting of either electron-dense amorphous material (type I viroplasm), or aggregates of granular material interspersed with spherical vesicles (type I1 viroplasm) begin to appear in infected cells. The first recognizable viral structures are incomplete crescent-like shells or membranes appearing at the periphery of the virogenic stroma. These membranes develop and eventually enclose fully a mass of dense material. It is difficult to define or say at what point of the infectious cycle an immature virion develops. Stoltz and Summers (1972) defined the immature virion as a spherical structure containing a mass of amorphous material. Electron micrographs show that these immature particles consist of an inner trilaminar structure of unit membrane and a spicule coat (Stoltz and summer, 1972). In type I1 viroplasm, crescent or arch-like envelopes are present in association with fibrilar material of low to moderate density containing a large number of vesicles. Bergoin et al. (1969) suggested that these vesicles may well play an important role in the formation of immature viral envelopes. The incomplete viral envelopes progressively close, and in the process

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BASIL M. ARIF

appear to engulf the granulated material which eventually condense to give the appearance of immature particles found in type I viroplasm. The material inside these particles begins to differentiate and a viral nucleoid forms as highly condensed mass. A t this stage the nucleoid has no obvious shape or a particular structure. As the nucleoid structure differentiates further into a mature core surrounded by three-layered membrane, the particle begins to assume a more rectangular shape with a concomitant loss of the outer layer of spicules. The lateral bodies also assume a more recognizable structural form. Later the outer membrane is modified by folding to give the appearance of a beaded mulberry-like structure. 3. Occlusion

Mature virus particles are occluded within a proteinaceous mass that forms in the cytoplasm of infected cells. The occlusion of immature particles has also been noted (Stoltz and Summers, 1972),but this is obviously a rare phenomenon because thin sections of mature spheroids do not reveal the presence of immature particles. The occluded virion is a more compact structure than the nonoccluded form. There does not appear to be a control on the number of particles that become occluded. Some spheroids contain more particles than others and some appear to be devoid of occluded particles. Paracrystalline spindles are also formed in infected cells by crystallization of electron-dense material inside unit membranes. The spindles are also occluded together with their membranes (Bird, 1974). It is not certain that the membrane is a prerequisite to spindle occlusion. Occasionally only spindles and no virions become occluded. 4.Nonoccluded Particles

Nonoccluded particles are released at the cell surface by a budding mechanism during which process they are enveloped by a modified cell membrane (Granados, 1973b). Presumably the function of the nonoccluded virus particles is to initiate infection in other larval tissues similar to that observed for baculoviruses (Volkman etal., 1976).

B. In Tissue Culture Cells AmEPV was successfully shown to infect primary ovarian and hemocyte cultures from E.acrea(Granados and Naughton, 1975). These cells were initially infected with virus liberated from purified spheroids by alkali, and later with nonoccluded virus present in the supernatant medium of infected cultures. Viral inclusion bodies usually appeared in infected hemocytes 3 - 4 days postinfection (pi), and in ovarian cells 5 -6 days pi. By 10

THE ENTOMOPOXVIRUSES

209

days pi, 100% of the cells were infected, as judged by production of viral inclusion bodies. Spindles were not observed in these infected cultures. Also, the number of occluded virions appeared to be very low averaging 2 - 10 particles per plane of section and occasionally no virion was observed in the spheroids (Granados and Naughton, 1975). It appears that the development of EPV in tissue culture cells is similar to that in the host larva. Quiot etal. (1975) observed, however, that in Lymantria dispar cell culture infected with AmEPV, structures similar to immature virions appeared in the cell nucleus. This seems to implicate the nucleus in the replication of EPVs, but further studies on his virus-cell system are obviously needed to document this observation. Two early passage hemocyte cell lines for E.acreu, designated EA 1174A and EA 1174H, were also shown to support the replication of AmEPV (Granados, 1976). By 36-40 hours pi, the first cytopathic effects were seen by phase contrast microscopy. The cells were considerably enlarged and contained many refractile granules. Typical spheroids were observed intracellularly 2 days pi. The rate O f ViNS multiplication was higher in EA 1174A cell line. In this line most of the cells (90-95%) were infected by 48 hours pi compared to 20-25’ of the cells in EA 1174H line. Total cell infection (100%) was achieved at 72 hours pi for EA 1174A line, and at 96-120 hours pi for EA 1174H line. Other cell lines that support EPV replication have also been established (Granados, 1981). The viral replicative events were not studies at the molecular level in infected cells. Recently however, Langridge and Greenberg (1981) used the ELISA method to detect AmEPV proteins in E. acrea cell line BTIEAA at 12-50 hours pi. Very low concentrations of viral proteins in Trichoplusia ni cells (Tn. 368) were also detected from 1 to 96 hours pi.

VI. Is THERE A POTENTIAL FOR THESE VIRUSES IN PESTCONTROL? In evaluating the potential of EPVs as possible biological control agents one has to consider the efficacy of these viruses, safety to nontarget organism, and economical viability. These are very much the same parameters that are considered when a baculovirus is thought of as possible biological control agents. The advantages of baculoviruses over other insect viruses is that they do not resemble any known vertebrate or plant virus group, and are generally host specific. In considering EPVs one has to remember the fact that they have close morphological similarities to orthopoxviruses even though the two groups appear to be different biochemically. There are some examples in the literature indicating that natural EPV infection was responsible for reduction in certain insect populations. For example,

210

BASIL M. ARIF

studies of field populations of Oncopera alboguttata in Australia between 1971 and 1974 showed that a dual infection of EPV and a microsporidian parasite were responsible for a drastic reduction of the density of this insect in 1973 (Milner, 1977). An EPV and an NPV of Wiseana spp (Lepidoptera: Hepialidae) were shown to control populations of these soildwelling pasture pests in New Zealand (Crawford and Kalmakoff, 1977). Similar findings on the effectiveness of an EPV against larval midges, Chironomus decorus complex in California was reported by Harkrider and Hall (1975,1978). These authors also found that under controlled conditions the effect of EPV is dependent on virus concentration and inversely proportional to the age of the larval population at the time of treatment (Harkrider and Hall, 1979). On the other hand an EPV of Othnonius batesi has such a low pathogenicity against its host and the disease takes such a long time to develop that it is considered an ineffective control agent (Milner and Lutton, 1975). An EPV of the eastern spruce budworm was tested experimentally in the field to determine its effectiveness as a control agent. It was sprayed from helicpoters against infested forest plots (Bird et al., 1972). These authors found some measure of control against the budworm, but the EPV preparations were heavily contaminated with NPV and CPV, which makes it difficult to evaluate their results. In summary, if EPVs are to be considered in biological control of insects, in depth studies will have to be carried out on host specificity, efficacy, safety to nontarget organism, and on basic biochemical characterizations with emphasis on comparative studies to orthopoxviruses.

VII. CONCLUSION

A great deal of basic biochemical and biological characterization is still to be carried out on entomopoxviruses in order to better understand their nature and function. To date there has been little published REN analyses on the genome of these viruses, and studies on viral proteins were mostly confined to the identification of structural proteins. There are now cell lines that support EPV replication. This should be of great help in understanding the replicative cycle at the molecular level and the effect of virus on the infected cell. Also, since there is a resemblance to orthopoxviruses, a study on the interaction with nontarget organisms should receive more attention.

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REFERENCES Arif, B. M. (1976). Virology 69, 626-634. Bergoin, M., and Dales, S. (1971). In “Comparative Virology” (K. Maramorosch and E. Kurstak, eds.), pp. 169-205. Academic Press, New York. Bergoin, M., Davauchelle, G., Duthoit, J. L., andVago, C. (1968). C. R. Hebd. SeancesAcad. Sci., Ser. D 2 6 6 , 2126-2128. Bergoin, M., Devauchelle, G., and Vago, C. (1969). Arch. Gesamte Virusforsch. 28,285-302. Bergoin, M., Veyrunes, J. C., and Scalla, R. (1970). Virology 40,760-763. Bergoin, M., Devauchelle, G., and Vago, C. (1971). Virology 43,453-457. Bergoin, M., Devauchelle, G., and Vago, C. (1976). J . Ultrastruct. Res.55, 17-30. Bilimoria, S. L., and Arif, B. M. (1979). Virology 96, 596-603. Bilimoria, S. L., and Arif, B. M. (1980). Virology 104,253-257. Bird, F. T. (1974). J. Inuertebr. Pathol. 1 8 , 159-161. Bird, F. T., Sanders, C. J. andBurke, J. M. (1971). J . Inuertebr. Pathol. 18, 159-161. Bird, F. T., Cunningham, J. C., and Howse, G. W. (1972). Proc. Entomol. SOC.Ont. 102, 69-75. Buchatskyi, L. P. (1974). Mikrobiol. Zh. 36, 797-798. Crawford, A. M., and Kalmakoff, J. (1977). J. Inuertebr. Pathol. 29, 81-87. Croizier, G., and Veyrunes, J. C. (1971). Ann. Inst. Pasteur, Paris 120, 709-715. Cunningham, J. C., Burke, J. M., and Arif, B. M. (1973). Can. Entomol. 105, 767-773. Devauchelle, G., Bergoin, M., and Vago, C. (1971). J . Ultrastruct. Res.37,301-321. Epstein, D. A., and Thoma, J. A. (1975). Biochem. Biophys. Res.Commun. 62,478-484. Federici, B. A,, Granados, R. R., Anthony, D. W., and Hazard, E. I. (1974). J. Inuertehr. Pathol. 23, 117-120. Geshelin, P., and Berns, K. I. (1974). J . Mol. Biol. 88,785-796. Gold, P., and Dales, S. (1968). Proc. Natl. Acad. Sci. U.S.A. 60,845-852. Goodwin, R. H., and Filshie, B. K. (1969). J . Inuertebr. Pathol. 13,317-329. Goodwin, R. H., and Filshie, B. K. (1975). J.Inuertebr. Pathol. 25,35-46. Goodwin, R. H., and Roberts, R. J. (1975). J. Inuertebr. Pathol. 25, 47-57. Gotz, P., Huger, A. M., and Krieg, A. (1969). Naturwksenschaften 56, 145. Am. 9,73-94. Granados, R. R. (1973a). Mkc. Publ. Entomol. SOC. Granados, R. R. (1973b). Virology 5 , 305-309. Granados, R. R. (1976). Adu. Virus Res. 20,189-236. Granados, R. R. (1978). In “Viral Pesticides: Present Knowledge and Potential Effects on Public and Environmental Health” (M. D. Summers and C. Y. Kawanishi, eds.), pp. 89- 102. U.S. Environmental Protection Agency, Research Triangle Park, North Carolina. W. DavidGranados, R. R. (1981). I n “Pathogenesis of Invertebrate Microbial Disease” (E. son, ed.), pp. 101-126. Alanhead, Osmun. Granados, R. R., and Roberts, W. D. (1970). Virology 40, 230-243. Granados, R. R., and Naughton, M. (1975). Interuirology 5, 62-68. Harkrider, J. R., and Hall, I. M. (1975). Proc. Calif. Mosq. Control Assoc. 4 3 , 103-106. Harkrider, J. R., and Hall, I. M. (1978). Enuiron. Entomol. 7 , 858-862. Harkrider, J. R., and Hall, I. M. (1979). Enuiron. Entomol. 8, 631-635. Harrap, K. A., andPayne, C. C. (1979). Adu. Virus. Res.25, 273-355. Henry, J. E., Nelson, B. P., and Jutila, J. W. (1969). J. Virol. 3,605-610. Huger, A. M., Krieg A,, Einschermann, P., and Gotz, P. (1970). J. Inuertebr. Pathol. 15, 253- 261.

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Hurpin, B., and Robert, P. (1967). Entomophugu12,175-180. Hurpin, B., and Vago, C. (1963). Reu.Puthol. Veg. Entomol. Agric. Fr. 42,115-117. Joklik, W. K. (1962). J. Mol.Biol. 6, 265-274. Katagiri, K. (1973). J. Znuertebr. Puthol. 2 2 , 300-302. Katagiri, K., Kushida, T., Kasuga, S., and Ohba, M. (1975). Jpn.J. Appl. 2001.1 9 , 243. Kurstak, E., and Garzon, S. (1977). In“Atlas of Insect and Plant Viruses” (K. Maramorosch, ed.), pp. 29-66. Academic Press, New York. Langridge, W. H. R., and Greenberg, J. F. (1981). J. Gen.Virol. 67,215-219. Langridge, W. H. R., and Roberts, D. W. (1976). Proc. Znt. Colloq. Inuertebr. Puthol., Ist, 1976 p. 327. Langridge, W. H. R., and Roberts, D. W. (1977). J. Virol. 2 1 , 301-308. Langridge, W. H. R., and Roberts, D. W. (1982). J. Znuertebr. Puthol. 39,346-353. Langridge, W. H. R., Bozarth, R. F., and Roberts, D. W. (1977). Virology 76,616-620. Levinsohn, S., and Aronson, A. I. (1967). J.Bmteriol. 93,1023-1030. Lipa, J. J., and Bartkowski, J. (1972). J.Invertebr. Puthol. 2 0 , 218-219. McCarthy, W. J., Granados, R. R., and Roberts, D. W. (1974). Virology 69,59-69. McCarthy, W. J., Neser, C. F., and Roberts, D. W. (1975). Interuirology 6,69-75. Matthews, R. E. F. (1979). Znteruirology 1 2 , 132-296. Meynadier, G., Fosset, J., Vago, D., Duthoit, J. L., and Bers, N. (1968). Ann.Epiphyt. 1 9 , 703- 706.

Milner, R. J., (1977). J. Aust. Entomol. 16, 21-26. Milner, R. J., and Lutton, G. G. (1975). Entomophugu20,213-220. Moss, R., Rosenblum, E. N., and Paolett, E. (1973). Nature (London),New Biol. 2 4 6 , 59-62.

Pfau, C. J., and McCrea, J. F. (1962). Nature (London) 1 9 4 , 894-895. Pogo, B. G. T., Dales, S., Bergoin, M., and Roberts, D. W. (1971). Virology 4 3 , 306-309. Quiot, J. M., Bergoin, M., and Vago, C. (1975). C.R. Hebd. Seances Acud. Sci., Ser. D 2 8 0 , 2273- 2275.

Roberts, D. W., and Granados, R. R. (1968). J.Znvertebr. Puthol. 12,141-143. Sheldon, R., and Kates, J. (1974). J. Virol. 1 4 , 214-224. Stoltz, D. B., and Summers, M. D. (1972). J. Ultrastruct. Res.40, 581-598. Summers, M. D. (1975). In “Baculoviruses for Insect Pest Control: Safety Considerations” (M. D. Summers, R. Engler, L. A. Falcon, and P. Vail, eds.), pp. 17-29. Am. SOC. Microbiol., Washington, D.C. Summers, M. D., and Smith, G. E. (1978). Virology 8 4 , 390-402. Sutter, G. R. (1972). J. Znuertebr. Puthol. 19,375-382. Szybalski, W., Erikson, R. L., Gentry, G. A., Gafford, L. G., and Randall, C. C. (1963). Virology 19, 586-589. Tweeten, K. A., Bulla, L. A., Jr., and ConFigii, R. A. (1978). J. Virol. 26, 702-711. Vago, C. (1963). J. Insect Puthol. 6, 275-276. Vago, C., and Bergoin, M. (1968). Adu. Virus Res.1 3 , 247-303. Vago, C., Amargier, A., Hurpin, B., Meynadier, C., and Duthoit, J. L. (1968a). Entomophugu 13,373-376.

Vago, C., Monsarrat, P., Duthoit, J. L., Amargier, A., Meynadier, G., and Van Waerebeke, D. (1968b). C. R. Hebd. Seances Acud. Sci. 2 6 6 , 1621-1623. Vago, C., Robert P., Armagier, A., and Duthoit, J. L. (1969). Mikroskopie 26,378-386. Volkman, L. E., Summers, M. D., and Hsieh, C. (1976). J.Virol. 19,820-832. Weiser, J. (1969). Actu Virol. 13, 549-553. Weiser, J., and Vago, C. (1966). J. Inuertebr. Pathol. 8, 314-319.

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Weiser, J., Tchubianishvili, C., and Zizka, 2. (197). ActaVirol. 14, 314-317. Westwood, J. C. N., Harris, W. J., Zwartouw, H. T., Titmuss, D. H. J., and Appleyard, G. 34, 67-78. (1964). J. Gen.Microbiol. Yau, T., and Rouhandeh, H. (1973). Biochirn. Biophys. Acta299,210-217. Zummer, M., and Faulkner, P. (1979). J.Inuertebr. Pathol. 33,383-384.

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ADVANCES IN VIRUS RESEARCH. VOL . 29

USE OF PROTOPLASTS AND SEPARATE CELLS I N PLANT VIRUS RESEARCH Evamarie Sander and Gabriele Mertes

lnstitut fur Biologie II Universitat Tubingen Tubingen. Federal Republic of Germany

I. Introduction and Scope of the Review .

. . . . . . . . . . . . . . . .

I1. Isolation of Protoplasts from Leaves . . . . . . . . . . . . . . . . .

A . Culture of Plants . . . . . . . . . . . . . . . . . . . . . . . . B . Pretreatment of Leaves . . . . . . . . . . . . . . . . . . . . . C. Enzyme Solutions . . . . . . . . . . . . . . . . . . . . . . . D . Protoplast Isolation Procedures . . . . . . . . . . . . . . . . . I11. Isolation ofProtoplastsfrom Cell Suspension Cultures . . . . . . . . . IV. Inoculation of Protoplasts . . . . . . . . . . . . . . . . . . . . . . A . Inoculation with Virus Particles . . . . . . . . . . . . . . . . . B . Inoculation with Viral Nucleic Acids and Viroids . . . . . . . . . . C. Culture of Infected Protoplasts . . . . . . . . . . . . . . . . . . V. Determination of Virus Replication . . . . . . . . . . . . . . . . . . A . Bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Serological Virus Detection . . . . . . . . . . . . . . . . . . . C. OtherMethods . . . . . . . . . . . . . . . . . . . . . . . . . VI . Infection of Cells from Callus Tissue and Cell Suspension Cultures . . . . A. Infection with Virus Particles . . . . . . . . . . . . . . . . . . B . Infection with Viroids . . . . . . . . . . . . . . . . . . . . . . VII . Resistance and Antiviral Substances . . . . . . . . . . . . . . . . . VIII . Concluding Remarks and Perspectives . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 217 217 218 219 221 223 226 226 235 242 243 244 244 247 248 248 250 251 255 258

I. INTRODUCTION AND SCOPE OF THE REVIEW Plant protoplasts have become an indispensable part in plant virus research since Cocking (1960) first presented a method for their isolation from tomato fruit with strong evidence of successful infection with TMVl (Cocking and Pojnar. 1969) and since Takebe and co-workers (1968) overAbbreviations of virus names used in the review are explained in Table I; SDS. sodium dodecyl sulfate; EDTA. naphthaleneacetic acid . 215

Copyright 0 1984 by Academic Press. Inc . All rights of reproduction in any form reserved . ISBN 0-12-039829-X

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came the difficulties in preparing protoplasts from leaf mesophyll and showed substantial virus replication resulting from infection with TMV RNA (Aoki and Takebe, 1969) and TMV (Takebe and Otsuki, 1969). These discoveries allowed for the first time plant virological studies to proceed in separated cells under controlled and synchronized conditions instead of multicellular, nonsynchronous plant tissue or calli, a fact that had so far hampered advancement and also comparison of results obtained in plant virology with those in monolayer of bacteria and animal cells infected with bacteriophage or zoo-pathological virus. Many plant virological problems had to remain shelved for the lack of separate cells from plants, and the rather explosive increase in scientific data reflects the impact of the introduction of the protoplast/virus system into this field of investigation. The papers are now legion and have been reviewed under the aspect of protoplast preparation, virus uptake and multiplication by Cocking (1970, 1977), Zaitlin and Beachy (1974), Takebe (1975, 1980, 1983), and Sarkar (1977). Before the advent of protoplasts, tissue cultures were widely used in in uitro systems to investigate plant virus on a cellular level (reviewed by Kassanis, 1967), but since they consist of cell groups rather than separate cells and therefore could not be inoculated synchronously with virus, they were fast replaced by protoplasts. The considerable progress made in elucidating successive steps in the replication of virus, viral nucleic acid, andviroids in protoplasts and tissue cultures and the accompanying induction of proteins has been recently reviewed by Muhlbach (1982). Consequently, this aspect will not be enlarged upon in this review. Emphasis is placed on the advancement in knowledge with regard to the type and role of parameters that improve protoplast isolation, yield, and viability and efficiency of infection with viria and viral nucleic acids. Based on this knowledge and assisted by technical development in methods for separation and quantitative detection of substances and reactions on a molecular level, protoplasts are used increasingly to understand the mechanism of resistance against virus infection, which is reflected in this review. Equally promising is the employment of plant cell suspension cultures and calli in this respect and in order to study factors controlling the movement of virus from cell to cell. Lately, however, plant tissue cultures have come into their own again primarily for the reason that protoplasts can be isolated from in uitro cells thus circumventing the seasonal influence known to affect protoplasts from leaves. The references cited are by no means comprehensive for each topic reviewed, but rather exemplary.

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11. ISOLATION OF PROTOPLASTS FROM LEAVES A . Culture of Plants Of great influence on the stability of the protoplasts and their suitability for virus replication are the growth conditions of the plants (Takebe et al., 1968;Takebe, 1975;Kubo etal., 1975). In general, fast and uninterrupted growth is recommended for the plants, beginning with the early separation of the young seedlings. Among the growth conditions light intensity and relative humidity are the most critical factors for successful protoplast isolation. For tobacco plants the light intensity should not deviate significantly from 10,000 lux. Even though intensities higher than that for 1and 2 hours per day, which cannot always be prevented in a greenhouse, are not quite deleterious, best results are obtained by growing the plants in a growth chamber under controlled conditions. For the isolation of tobacco protoplasts intended for later infection with virus Kubo etal. (1975) recommends 10,800lux for 10-12 hours per day as optimum. A higher light intensity or a longer photoperiod may sometimes result in increased yield of protoplasts, but the faculty of these protoplasts to support virus replication appears reduced. For the isolation of mesophyll cell protoplasts from potato (Shepard and Totten, 1977) even a reduction of the light intensity from 15,000 to 7,000 lux and shortening the photoperiod from 12to 6 hours several days before protoplasts isolation is recommended. For the isolation of tomato leaf protoplasts, leaves of tomato plants grown at low light intensity which causes soft leaves should be used (Cassells and Barlass, 1976). Increasing light intensity leads to thicker cell walls as well as to an increase in calcium pectate which results in a certain resistance of the tomato leaf tissue to protoplast isolation. Cowpea plants should preferably be grown at 10,000 lux for 14 hours a day (Hibi et al., 1975). The relative humidity should be within 40-70% at the temperature appropriate for each plant species. The leaves of plants grown at relative humidity higher than 70% are less suited for the isolation of intact protoplasts as the leaves may collapse already in the solutions for surface sterilizing. Therefore, also the watering of the plants should be avoided about 24 hours previous to harvesting (Takebe et al., 1968). Feedings with a complete nutrient solution at weekly intervals and 1day before protoplast isolation is generally thought to be sufficient. For the isolation of protoplasts, leaves should be harvested for instance from Nicotiana tabacum at an age of 5-6 weeks from planting the seed which usually corresponds to the 4-6 leaf stage. The upper, almost fully expanded leaves are usually preferred. Most suited for the isolation of

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cowpea protoplasts are fully expanded primary leaves from plants only 9 - 11days old. As compared to tobacco the cowpea protoplast system has the great advantage of a much briefer period of plant culture during which culture conditions need to be kept constant. For plants grown in greenhouses during the winter season in geographical zones with temperate climate, the photoperiod should be intensified by additional light about 3000 lux for about 10 hours a day. However, it is common experience among protoplast workers that as a consequence of the influence of the seasons on the physiological condition of the source plant, variation in yield, stability, and biological properties of isolated protoplasts can hardly be avoided.

B. Pretreatment ofLeaves All steps for the isolation of protoplasts must be carried out under sterile conditions. Contaminations by microorganisms in the solutions used during the isolation procedure, which usually takes 6-8 hours, would greatly impair the viability of protoplasts. The first step in the procedure of protoplast isolation is the sterilization of the surface of the selected leaves. They are usually submersed in 70% ethanol for 15-30 seconds followed by another submersion of about 20 minutes in a 0.5-0.7% sodium hypochlorite solution. Commercial hypochlorite solutions such as “Clorix” or “Domestos” might be used instead, but ought to be diluted. Subsequently, the leaves must be washed for 15 minutes in 3 changes of distilled water. Leaves showing extended dark green areas or appearing even collapsed and limp should be discarded because a high yield of viable protoplasts cannot be expected. To improve the penetration of the enzyme solutions into the intercellular spaces, the cutting of the leaves into small pieces of about 1to 0.2 cm2 (Jensen etaL, 1971) improves contact between leaf tissue and enzyme solution, but removal of the epidermis usually results in a higher protoplast yield. Some kinds of leaves, especially fully expanded tobacco, tomato, and petunia leaves, permit peeling of the epidermis with forceps (Sarkar, 1977). Yet, when using leaves from other plants or not fully expanded leaves, the peeling is often impossible. In these cases the epidermis can be removed by treating the leaves with a soft nylon brush until they appear shiny green as reported by Shepard (1975; Shepard and Totten, 1977) for tobacco and potato leaves. Beier and Bruening (1975) removed the lower epidermis of cowpea leaves by adding Carborundum powder (320 mesh) during the brushing. This method also leads to a high yield of stable protoplasts from tobacco leaves. The sterilized Carborundum powder is spread onto the lower epidermis and removed by gentle brushing with a l-cm-wide paint brush until the leaf looks shiny green. Thereafter, the

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leaves must be washed by dipping them into sterile distilled water, changing the water at least twice. Problems with residual Carborundum do not occur during subsequent isolation steps, even when the protoplasts are used for electron microscopic studies (Mertes, using Carborundum 200 mesh). As an alternative to these mechanical methods for epidermis removal Schilde-Rentschler (1972) employed a solution of the enzyme pectin-glucosidase from Aspergillus.

C. Enzyme Solutions Since Takebe et al. (1968) isolated tobacco mesophyll protoplasts with “Macerocyme,” a crude pectinase preparation from Rhizopus and “Onozuka” cellulase, prepared from Trichoderma viride (both enzymes: Kinki Yakult Biochemicals Co., Nishinomiya, Japan), these enzymes have been almost exclusively used for the successful isolation of protoplasts from many different plant species. Yet, from some plant species no protoplasts can be obtained with this enzyme combination. This is attributed t o the varying constituents of hemicelluloses or the pectic material contained in the primary cell wall which do not yield to the specific activity of the enzymes derived from these fungi. Several other enzyme preparations are available, varying in their cell wall degrading specificity. “Macerocyme” might be replaced by “Macerase” (Calbiochem, La Jolla, Calif.), e.g., for the isolation of lettuce mesophyll protoplasts (Engler and Grogan, 1983) and cowpea protoplasts (Beier and Bruening, 1975) or by “Pectinase” (Serva Feinbiochemical GmbH, Heidelberg, Germany, or Sigma Chemical Co., St. Louis, Mo.), for the isolation of protoplasts from leaves of tomato (Cassells and Barlass, 1976) and spinach (Rose, 1980). “Pectolyase-Y 23” (Seishin Pharmaceutical Co., Nagareyama, Japan), a macerating enzyme derived from Aspergillus japonicus, was found to be well suited for the isolation of mesophyll protoplasts from soybean (Schwenk et al., 1981), tobacco, rice, and oat (Nagata and Ishii, 1979). With this enzyme the period of incubation for mesophyll protoplast isolation from tobacco could be reduced from the usual 2 hours and more to 25 minutes. Suitable cellulases are “Driselase” (Kyowa Hakko Kogyo Co. Ltd., Japan), “Meicelase P” (Meiji Seika Kaisha Ltd., Tokyo, Japan) for protoplast isolation from different cultivars of Nicotiana tabacum (Watts et al., 1974),“Cellulysin” (Calbiochem, La Jolla, Calif.) for the isolation of cowpea protoplasts (Beier and Bruening, 1975), or “Enzeco AP 650” (Enzyme Development Corp., New York, N.Y.) for protoplasts from Pisum satiuum (Bogers, 1973). Patnaik and Cocking (1982) used an enzyme mixture of “Rhozyme H P

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150” (Rhozyme, Rhom and Haas Co., U.S.) and “Macerocyme” as pectolytic enzymes in combination with “Mayvill” cellulase (Mayvil Chemicals Ltd., Sandbach, Cheshire, U.K.) for the isolation of petunia and tobacco protoplasts. This resulted in high yield and viability, comparable to that of protoplasts isolated with Japanese enzymes. By varying the “Rhozyme” concentration the seasonal influence on protoplast quality could be diminished. Desalting of the enzymes on BioGel P6 (Kao etal., 1971) or using the desalted preparations “Macerocyme R-10” and “Onozuka R-10” is discussed as to reduce toxic effects on the protoplasts, but some enzymes may become less active without improving the viability as reported for tomato leaf protoplasts (Cassells and Barlass, 1976). When preparing the enzyme solution it must be kept in mind that protoplasts are an osmotically active system which takes up water from a surrounding medium of low osmotic potential. To prevent rupturing the plasmalemma, the osmolality of the medium must be balanced by the addition of osmotic stabilizers. The one most commonly used is mannitol, because it is not metabolized by the protoplasts and can be successfully combined with sorbitol. Sorbitol, even though less inert, seems t o impair protoplast quality only in some cases (Schwenk etal., 1981; Gamborg et aL, 1973; Beier and Bruening, 1975). Depending on species and growth conditions of the plants the optimum osmolality ranges from 0.35 to 0.8 M. With protoplasts from Nicotiana tabacum cv. Samsun, mannitol concentrations above 0.75 M reduced virus replication (Shabtai etal., 1982)probably due to decreased RNA and protein synthesis under such high osmotic stress (Premecsz etal., 1978). The addition of salts as osmotic stabilizers such as potassium chloride and magnesium sulfate was reported to give good results despite their strong tendency to penetrate into the protoplasts (Meyer, 1974). Since it is sometimes difficult to dissolve the enzymes in the osmoticum it is recommended that the solute be adjusted first to pH 8 and then to pH 5.4 - 5.8 suitable for the physiological condition of the cells. Alternatively, turbid enzyme solutions can be clarified by centrifugation at about 5000 g. Sterilization of the solution should be achieved by filtration (Millipore filter, pore size about 0.4 pm). The addition of several substances such as potassium dextran sulfate (MW about 560, sulfur content 17.1%, Meito Sangyo Co., Nagoya, Japan), bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.), CaCl,, MES (2-N-morpholino-ethanesulfonic acid, Sigma Chemical Co., St. Louis, Mo.), or sodium citrate was found to be beneficial for protoplast release. Potassium dextran sulfate, probably exerting a stabilizing effect on the plasma membrane, should not exceed 0.1 -0.5% because concentrations of 1%or more are liable to decrease protoplast

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viability (Beier and Bruening, 1975). Bovine serum albumin was used for the stabilization of protoplasts from Betulu and Rhododendron (Smith and McCown, 1983). The quality of soybean mesophyll protoplasts (Schwenk et ul., 1981) was improved more by the addition of CaC1,.2H20 (0.12%) than by MES (0.01 M ) , which is successfully used for the isolation of mesophyll protoplasts from potato (Shepard and Totten, 1977)and lettuce (Engler and Grogan, 1983). The effect of calcium is believed to be related t n increased membrane stability (von Arnold and Eriksson, 1977). The possibility to substitute Ca2+with Mg2+indicates that many divalent cations may react with the negatively charged membrane groups. Sodium citrate (1.9 mM) markedly improves protoplast yield from tomato leaves (Cassells and Barlass, 1976), even when “hard leaves” from plants grown a t high light intensity were used. Citrate seems to weaken calcium pectate, the amount of which increases in tomato plants with increasing light intensity and is rather resistant to fungal polygalacturonases.

D. Protoplast Isolation Procedures The isolation of protoplasts from leaves can be approached by a sequential application of enzymes (two-step procedure) established by Takebe et al. (1968) or by mixed enzymes (one-step procedure) established by Power and Cocking (1970). Both procedures are described below with Nicotiana tabacum as an example; common to both is the sterilization of the leaf surface previous to subsequent steps. In the two-step procedure the lower epidermis is removed from the surface sterilized leaves which are then cut into pieces of about 1cm2. For the release of free cells these are incubated in a maceration solution consisting of 0.5- 1.0% Macerocyme (“Macerocyme R-10”: 0.03-0.05%, 1982), 0.5-0.7 M mannitol, and 0.5% potassium dextrane Shabtai et al., sulfate at a proportion of 10-20 ml/g tissue. To improve the enzyme action, the solution can be infiltrated into the intracellular spaces by installing a low vacuum 3 times for 20 seconds. During the incubation period the leaf fragments are agitated vigorously in the solution on a reciprocal shaker at 25-30°C up to 120 excursions per minute. After the first 20 minutes and after an additional 20 minutes the supernatant is discarded and the maceration solution renewed. By this exchange, broken cells and the cells released from spongy mesophyll are discarded so that protoplasts finally obtained were derived only from palisade cells. After further incubation for 120 minutes the maceration solution is removed by centrifugation (100- 300 g, 3 minutes). The removal can be improved by washing the cells in mannitol. The separate cells and remnants of semidigested tissue are suspended in a cellulase solution (20 ml/g original tissue)

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containing 1-2% enzyme and 0.5-0.7 M mannitol. This mixture is incubated for 60- 120 minutes while gently shaking (about 40 excursions/min) at 30-36°C. Although cellulase is more active near 37”C,the protoplasts are in better condition after incubation at 30°C. In the one-step procedure the lower epidermis is removed from the surface sterilized leaves which are then floated on 0.5-0.7 M mannitol for about 45 minutes. This preplasmolysis prevents spontaneous protoplast fusion during the following period of protoplast release. It also reduces toxic effects of various components contained in most enzyme preparations, by detaching the protoplast from the surrounding cell wall and thereby minimizing the contact with the surrounding medium. Spontaneous fusion of protoplasts was often mentioned as a disadvantage of the one-step procedure. The preplasmolyzed leaves are then floated on an enzyme mixture containing 0.5% “Macerocyme,” 1.5% cellulase, and 0.5 0.7 M mannitol. While gently shaking for 2-3 hours at 30”C,preferably on a rotatory shaker, the protoplasts are released and of the leaves only the cuticula is left undigested on the surface of the solution. Reciprocal shakers are less efficient in furthering the release of protoplasts. A common objection against the mixed enzyme procedure is that spongy and palisade mesophyll cells remain unseparated resulting in a heterogeneous protoplast preparation. But also in the mixed enzyme solution the spongy mesophyll cells are released first and can be discarded after about the first hour of incubation (Cassells and Barlass, 1976). The protoplasts obtained by either one of these methods are separated from residues by filtration through nylon cloth (pore size 100 ,urn), and freed of the enzymes by low speed centrifugation at 30 g and 3 washings in mannitol. The protoplasts should be handled most carefully, preferably with special so-called “Kommagome” pipets which have an opening of 2 mm diameter. Sterility is achieved by stuffing the pipets with cotton and/or by using an electric pipet aid (“Pipetman P” :Gilson France, Villeiers Le Bel; “Hirschmann pipetus” :Hirschmann Laborglas, Eberstadt, Germany). If separation of broken protoplasts from intact protoplasts is required, the protoplast suspension can be centrifuged with a commercial density buffer called “Lymphoprep” (Nyegaard A/S, Oslo, Norway). As established by Larkin (1976) on the top of 1 volume “Lymphoprep” up to 3 volumes of the protoplast suspension are layered. Centrifugation at 50100g for 10 minutes collects the intact protoplasts at the interface and the broken cells at the bottom of the centrifuge tube. Usually the mixed enzyme method leads to higher yields of viable protoplasts. Huber (1983) compared TMV replication in one-step- and twostep-isolated tobacco protoplasts and found the one-step-isolated proto-

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plasts (leaves pretreated with Carborundum) to support virus replication better and the protoplast quality to vary less throughout the year. A shorter exposure to the enzyme solutions, less mechanical stress through vigorous shaking, and centrifugation during isolation of protoplasts are thought to be the reason for this effect.

111. ISOLATION OF PROTOPLASTS FROM CELLSUSPENSION CULTURES The use of cells from suspension cultures for the isolation of protoplasts has several advantages as compared to protoplasts from leaves. In virus studies with protoplasts from leaves reproducability of experiments frequently suffers from the variation in yield and viability of the protoplasts, reflecting the season-dependent influence on the physiological state of the initial plant. This difficulty can be overcome by employing plant cells cultured in uitro under controlled conditions throughout the year. Already the cultivation of plants for the isolation of protoplasts from leaves requires considerable space and expense and together with the pretreatment of the leaves also considerable time and labor. In comparison, cells in suspension culture need less space, they have the great advantage to grow already under sterile conditions, and allow good contact with the enzyme solution without pretreatment because the cells are growing in small, loose clumps or even separately. Also, protoplasts derived from an in vitro system can be considered as better adapted to being cultured in vitro for further studies than protoplasts isolated from fresh leaves. A precondition for the isolation of protoplasts well suited for virus experiments are fast dividing, finely growing cell suspension, forming small cell groups. Cell cultures with a maximum doubling time of 1.5 days, consisting of spherical cells in clumps up to 100cells, are considered to be a good source for protoplasts. The callus tissue intended for the initiation of a cell suspension culture should be grown on solidified nutrient medium without kinetin to obtain a friable callus. According to Negrutiu et al. (1975) the solidity of callus tissue is positively correlated with the kinetin concentration in the nutrient medium, and not with auxins. Kinetin is necessary, however, for the first initiation of callus tissue from germinated hypocotyls or from protoplasts. The resulting callus tissue can be used for the initiation of a crude cell suspension in kinetin-free nutrient medium. After 5- 10days of culture this cell material is placed on kinetin-free medium solidified with agar and the more friable callus tissue thus developed will provide a better source for initiation of cell suspensions which should be grown in nutrient

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medium also without kinetin. The selection of a finely dissociating cell suspension culture for the isolation of protoplasts is often a result of a series of passages over solidified medium (Wilson and Street, 1975). To achieve a culture of cells with the desirable spherical shape the reaction to various auxins must be tested as auxin requirements vary for each cell line. Regarding such lines of which protoplasts were isolated for virus replication, for cells of Glycine max 1mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) proved sufficient (Jarvis and Murakishi, 1980; Mertes and Sander, 1981). Cells from Nicotiana tabacum cv. Bright Yellow-:! (BY-2) and from Vinca rosea (Fukunaga etal., 1981; Kikkawa et al., 1982) require 0.5 mg/liter 2,4-D. Mertes (1983) found 1mg/liter 2,4-D in addition to 1mg/liter NAA best suited for the cell line established from Nicotiana tabacum cv. Xanthi nc.; within 31 days of culture more than 90% of the cells showed spherical form. Protoplasts should be isolated from cell suspension cultures before the culture reaches the stationary growth phase, usually 4-5 days after subculturing (Uchimiya and Murashige, 1974; Mertes and Sander, 1981). Jarvis and Murakishi (1980) described cells at the first doubling time as best suited for highly reproducible virus infection experiments. Some cell cultures, however, show a considerable degree of resistance to cell wall degrading enzymes and even cell suspension cultures giving a high percentage of protoplasts sometimes show culture periods with reduced protoplast release. Wallin et al. (1977) could markedly improve the protoplast yield from Haplopappus gracilis, cells quite recalcitrant to wall degradation (protoplast release only 14%)by the addition of 0.25 d L - c y s t e i n e or 0.25 mM L-methionine to the nutrient medium about 2 days prior to protoplast isolation. The protoplast release could be raised up to 50%. From cell suspension cultures of Nicotiana sylvestris and Daucus carota, the yield is usually close to 100% free protoplasts and can be maintained during a culture period of decreasing yield by supplementation of the nutrient medium with the mentioned amino acids. From cell suspension cultures protoplasts can be isolated only by the mixed enzyme method. A mixture of 1-376 cellulase “Onozuka” (SS or R-10) and 0.5- 1%“Macerocyme” was used for the isolation of protoplasts from cell suspensions of Nicotiana tabacum (Uchimiya and Murashige, 1974; Mertes, 1983), Glycine max (Mertes and Sander, 1981), and Arabidopsis thaliana (Negrutiu et al., 1975). Protoplasts from the last two cell lines can be isolated with higher yield and in a shorter time when cellulase “Onozuka” is replaced by 1.5- 2% “Driselase.” In both cases, the incubation time could be reduced from 6 to 2 -4 hours and protoplast yields of 80% were achieved with “Driselase” and cells of Glycine max even when isolation with “Onozuka” indicated culture periods with decreased protoplast

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yield. Jarvis and Murakishi (1980) used a mixture of 2% “Driselase,” 1% “Macerase” (Calbiochem, La Jolla, Calif.), and 2% “Cellulysin” for successful protoplast isolation from Glycine max cells within 2.5 hours. A mixture of 2% Cellulase “Onozuka R-10” and 1%“Pectolyase-Y 23” was found equally beneficial for the isolation of Vinca rosea protoplasts in 1 hour with a yield of nearly 100% (Fukunaga et al., 1981) as for Nicotiana tabacum BY protoplasts (Kikkawa et al., 1982). A mixture of 2% cellulase “Onozuka-SS” and 1%“Pectinase” was used for the isolation of protoplasts from Glycine rnax(Fowke et al., 1974) and for the isolation of Daucus carota and Nicotiana sylvestris protoplasts (Wallin et al., 1977). The supplementation of the enzyme mixture by an anorganic salt solution (Negrutiu et al., 1975; 0.2 mM KH,PO,, 1 mM KNOB, 1 mM CaCl,.2H,O, 1 mM MgSO,. 7H,O; 0.001 mM KJ, diluted 1:10) was employed in protoplast isolation from cells of Glycine max (Mertes and Sander, 1981) and Nicotiana tabacum cv. Xanthi nc. (Mertes, 1983) for optimizing ionic strength for the enzymes. The addition of 0.5- 1%potassium dextrane sulfate improved the release of intact protoplasts from tobacco cell suspension culture (Mertes, 1983) to double the yield. Plasmolytica suited for protoplasts from cell suspension cultures are the same as described for leaf protoplasts. Either mannitol (Fukanaga et al., 1981) or sorbitol (Jarvis and Murakishi, 1980) or a mixture of both (Mertes and Sander, 1981)can be used. The enzyme mixtures will be prepared and sterilized as described for leaf protoplasts. The isolation procedure of protoplasts from cell cultures involves, in general, the following steps (Mertes and Sander, 1981). Four days after subculturing, the cell suspension is allowed to settle down in graduated tubes. The supernatant is removed and of the enzyme mixture twice the amount is added. Of the cell - enzyme mixture, 20 - 50 ml are incubated in a 100- 200 ml conical flask at 28”C by gently shaking at 40 rotations per minute. When protoplast release is completed (3-6 hours) the protoplasts are filtered twice through nylon cloth, first of a pore size of 100pm and then of 20- 30 pm to separate undigested cell material. The protoplasts are low speed centrifuged a t 30 - 50 g and washed three times in mannitol. Recently a nonenzymatic method for the isolation of protoplasts from callus of Saintpaulia ionantha was published (Bilkey and Cocking, 1981). Preincubation of the callus tissue on highly auxin-enriched media produced callus cells with thin cell walls which could be removed mechanically. These protoplasts offer the possibility of detailed physiological studies on the effects of cell wall degrading enzymes on plant protoplasts and perhaps the penetration process of virions and viroids.

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IV. INOCULATION OF PROTOPLASTS A . Inoculation with Virus Particles Since plant protoplasts provide an experimental system which allows synchronous infection with and replication of viruses and viroids, protoplasts have been isolated from various plant species for the study of such processes (Table I). For a long time protoplasts from cell suspension cultures were considered to be less suited for plant virus replication than those from leaf cells. The idea was that protoplasts derived from undifferentiated only meristem like cells support virus replication insufficiently in comparison to protoplasts isolated from truly differentiated cells of leaves. Recently, this difficulty has been overcome by several laboratories that have developed infection systems with protoplasts from cell suspension cultures that reached the same efficiency of infection and virus synthesis as in leaf protoplasts. Also one-step growth curves were established comparable to those with mesophyll protoplasts (Jarvis and Murakishi, 1980; Lesney and Murakishi, 1981; Kikkawa et aL, 1982). The infection procedure involves basically the same steps regardless of which of the two sources the protoplasts are derived. Infection of protoplasts is effected while they are suspended in an inoculation medium; the role of the constituents will be discussed successively. Most workers prefer to expose the protoplasts to the inoculation medium by the “indirect method,” i.e., they are first suspended in one volume of an osmolyticum to which an equal volume of the inoculation medium is added (Takebe and Otsuki, 1969). With the “direct method” the pelleted protoplasts are suspended directly in the inoculation medium (Siege1 et al., 1978). Comparing the two procedures, Morris-Krsinich et al. (1979)found a third variation. They suspended the protoplasts first in a small volume of mannitol and then transferred them to the inoculation medium. The difference in the efficiency of the three methods was slight. 1. Poly-L-ornithine (PLO)

The addition of PLO, a macromolecular polycation, is reported by most authors as being necessary to improve the virus infection of protoplasts. Usually, an optimum effect is achieved when virus and PLO are incubated before addition to the protoplasts and not when preincubation is performed with the protoplasts and the polycation. Different findings were reported for BMV at low-pH infection (Okuno and Furusawa, 1978a), PEMV (Motoyoshi and Hull, 1974), and BPMV (Lesney and Murakishi, 1981). For these viruses PLO was not essential; it only stimulated infection when added to the inoculation medium. How-

227

PROTOPLASTS IN PLANT VIRUS RESEARCH TABLE I INFECTION SYSTEMS OF PROTOPLASTS AND PLANT VIRUSES ~

_ _ _ _ _ _ ~

~~

Plant species Leaf protoplasts Avena sativa

Brassica rapa

Brassica sinensis Chenopodium hybridum Cucumis sativus

Hordeum vulgare

Lycopersicon esculentum Nicotiana benthamiana

Nicotiana rustica Nicotiana tabacum

Reference BMV (brome mosaic virus) BYDV (barley yellow dwarf virus) CYMV (cauliflower mosaic virus) TRosV (turnip rosette virus) TYMV (turnip yellow mosaic virus) TYMV (turnip yellow mosaic virus)

Furusawa and Okuno (1978) Barnett et al. (1982)

BMV (brome mosaic virus) CMV (cucumber mosaic virus) TMV (tobacco mosaic virus) BMV (brome mosaic virus) BSMV (barley stripe mosaic virus) BYDV (barley yellow dwarf virus)

Okuno and Furusawa (1979) Maule etal. (1980)

TMV (tobacco mosaic virus)

Motoyoshi and Oshima (1975)

RRV (raspberry ringspot Virus) TRV (tobacco rattle virus)

Harrison et a2. (1977)

PYDV (potato yellow dwarf virus) AMV (alfalfa mosaic virus) CCMV (cowpea chlorotic mottle virus) CGMMV (cucumber green mottle mosaic virus)

Riesterer and Adam (1981) Motoyoshi et al. (1975) Watts and Dawson (1980)

Howell and Hull (1978) Morris-Krsinich et al. (1979) Renaudin et al. (1975) Renaudin et al. (1975)

Coutts and Wood (1976) Loesch-Fries and Hall (1980) Chiu Ben-Sin and Tien Po (1982) Barnett et al. (1982)

Harrison et al. (1977)

Sugimura and Ushiyama (1975) -

(continued)

228

EVAMARIE SANDER AND GABRIELE MERTES TABLE I (continued) Plant species

Petunia sp. Phaseolus vulgaris Raphanus satiuus Triticum aestivum Vigna sesquepedalis Vigna sinensis

Vigna unguiculata

virus CMV (cucumber mosaic virus) CPMV (cowpea mosaic virus) PEMV (pea enation mosaic virus) PVX (potato virus X) RRV (raspberry ringspot virus) TBRV (tomato black ring virus) TNDV (tobacco necrotic dwarf virus) TMV (tobacco mosaic virus) TRV (tobacco rattle virus) TMV (tobacco mosaic virus)

Reference Otsuki and Takebe (1973)

Huber et al. (1977) Motoyoshi and Hull (1974)

Otsuki et al. (1974) Barker and Harrison (1978)

Fritsch etal. (1978)

Kubo and Takanami (1979)

Takebe and Otsuki (1969)

Mayo (1982) Hibi et al. (1968)

BGMV (bean golden mosaic virus)

Bajet and Goodman

BMV (brome mosaic virus)

Furusawa and Okuno

BMV (brome mosaic virus)

Furusawa and Okuno

CMV (cucumber mosaic virus) CPMV (cowpea mosaic virus) TMV (tobacco mosaic virus)

Koike et al. (1977)

AMV (alfalfa mosaic virus) CMV (cucumber mosaic virus) CPMV (cowpea mosaic virus) TMV (tobacco mosaic virus)

(1981)

(1978)

(1978)

Beier and Bruening (1975)

Koiki et al. (1976)

Nassuth et al. (1981)

Gonda and Symons (1979)

Hibi et al. (1975) Huber et al.(1981)

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PROTOPLASTS IN PLANT VIRUS RESEARCH TABLE I (continued) Plant species

Virus

Reference ~~~~

Zea mays Cell culture protoplasts Glycine max

Nico tiana tabacum

BMV (brome mosaic virus)

Furusawa and Okuno (1978)

BPMV (bean pod mottle virus) CPMV (cowpea mosaic virus) SBMV (southern bean mosaic virus) TMV (tobacco mosaic virus)

Lesney and Murakishi (1981) Jarvis and Murakishi (1980) Jarvis and Murakishi (1980)

TMV (tobacco mosaic virus)

Kikkawa et al. (1982)

Mertes and Sander (1981)

ever, the stimulatory effect was not obtained when PLO and virus were preincubated. Viruses for which PLO is not essential to effect infection have higher isoelectric points, whereas polycation-requiring viruses are of negative charge in inoculation media with acidic pH. Therefore, the main effect of PLO is apparently based on complexing with the negatively charged virus and thus facilitating ingress into the protoplasts. Mayo and Roberts (1979) demonstrated by electron microscopy the formation of specific virus/PLO aggregates with TMV, TRV, and RRV. The effect of charge balancing was substantiated by infection experiments with BMV (Okuno and Furusawa, 1978a). A t infection a t low pH, when the net charge of BMV particles is positive, the infection of barley protoplasts does not depend upon PLO, whereas at high pH and negative net charge of the virus, PLO is essential for infection. Infection of protoplasts with BMV particles at low pH required also a higher amount of inoculum than with PLO complexed virions, which net positive charge is probably higher. For PLO independent infection the concentration of BMV could be raised to 50 ,ug/ml being still connected with increasing infection efficiency, whereas at PLO-mediated infection and at high pH, the infection rate declined with BMV concentrations above 1pg/ml. This, however, may partly be due to an unfavorable BMV/PLO ratio. Lesney and Murakishi (1981) reported a PLO independent, stimulated infection of protoplasts from soybean suspension culture with BPMV, a virus with a higher isoelectric point, whereas, for infection with CPMV, the PLO was essen-

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tial. According to Hibi etal. (1975) the infection with CPMV of tobacco mesophyll protoplasts depends on PLO whereas up to 90% of leguminose protoplasts could be infected without any PLO. This indicates that action on the net charge of virions is not the only feature of this cation. The optimum preincubation time of virus and PLO is within 5 to 10 minutes as reported by most workers, e.g., Morris-Krsinich etal. (1979) working with TRosV. The effect was correlated with an increase of turbidity in the virus/PLO mixture as measured at 320 nm, indicating that aggregate formation mediates infection. That PLO can enhance the infection rate also when infection does not depend on the cation indicates an effect on the plasma membrane of the protoplasts. This is postulated from the observation of enhanced pinocytosis of virus particles (Otsuki etal., 1972) and by virus uptake through specific lesions in the membrane caused by the virus/PLO aggregates (Burgess etal., 1973; Kassanis etal., 1977). When the cation acts on the plasma membrane, preincubation of virus and PLO is not necessary (Lesney and Murakishi, 1981)or even detrimental for the infection process (Okuno and Furusawa, 1978a). Should PLO create lesions in the membrane thus facilitating virus ingress, there are also cases known where virus enters the protoplast in absence of the cattion. Here, perhaps, small amounts of viria are taken up through wounds in the membrane possibly caused by the very procedure of protoplast isolation. Also the hypothesis that viria find ingress by pinocytosis is not generally supported, since in many protoplast systems inoculation is effected at 0" C when an energy requiring process such as pinocytosis should not take place. Further studies are necessary to elucidate the interaction between PLO and the plasma membrane as well as the modes of ingress of viria into the protoplast. The optimum concentration of PLO is 1,ug/ml in most of the protoplast infection systems. Concentrations of PLO higher than 1-2 pg/ml are reported by most workers to reduce the viability of protoplasts from a wide range of plant species. 2. OtherPolycations

PLO can be replaced by other polycations. The infection of suspension culture tobacco protoplasts with TMV (Kikkawa etal., 1982) was stimulated even more by polyethyleneimine (Polymin P, MW 30,000-40,000; Bethesda Research Laboratories) than by PLO. The limiting Polymin P concentration was 0.8pg/ml because of protoplast aggregation and damage a t higher concentrations. For infection of tobacco mesophyll protoplasts with CCMV the PLO could be replaced by poly-D-lysine (Motoyoshi etal.,

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1974a). When cowpea protoplasts were infected with TMV by usingpolyD-lysine instead of PLO the efficiency of infection was halved, but the virus amount produced per infected protoplast remained the same (Huber etal., 1981). 3.Buffers

In the beginning of protoplast infection with virus particles citrate was the most commonly used buffer based on the first reproducible protoplast infection system established by Takebe and Otsuki (1969). Later, phosphate buffer was found to be more stimulatory in comparison with citrate for many virus/protoplast systems, e.g., for tobacco protoplasts infected with TMV (Takebe, 1975;Mertes and Sander, 1981) and RRV (Barker and Harrison, 1977), for cowpea protoplasts infected with TMV (Huber etal., 1981), CPMV (Beier and Bruening, 1976), and AMV (Alblas and Bol, 1977),for tomato protoplasts infected with TMV (Motoyoshi and Oshima, 1975), and for soybean suspension culture protoplasts infected with TMV (Mertes and Sander, 1981) and CPMV (Jarvis and Murakishi, 1980). As with PLO, preincubation of the virus in the buffer seems to be necessary for a stimulatory effect on the efficiency of infection (Lesney and Murakishi, 1981). Tris -chloride buffer was found to be much more effective than citrate or phosphate when turnip leaf protoplasts were infected with TRV (Morris-Krsinich etal., 1979). It was also used successfully for the infection of barley protoplasts with BMV (Okuno and Furusawa, 1978a), tobacco protoplasts with TRV (Mayo, 1978), and soybean protoplasts from suspension culture with SBMV (Jarvis and Murakishi, 1980). Mayo and Roberts (1979) made an approach to explain the influence on the buffer on the infectivity of inocula. They found that in phosphate and Trischloride buffer the efficiency of infection was superior to that in citrate buffer when the optimum concentration of PLO for the inoculation system was dissolved in the buffer for virus preincubation, usually 1pg/ml. Citrate buffer in turn is superior when suboptimal concentrations of this cation (up to 0.6 pg/ml) are dissolved. Electron microscopic analysis of the virus/PLO aggregates showed that the aggregate size is not correlated to the infectivity. In contrast, Kubo etal. (1976) suggested that smaller, but more numerous aggregates of virus and PLO in phosphate buffer are responsible for an infectivity superior to that of the aggregates in citrate buffer. Mayo and Roberts (1979) found that the aggregates of a certain virus and PLO were of different, but typical types in different buffers and that in a certain buffer the aggregates of different viruses and PLO were also of distinct types, but no attribute of the aggregate types could be related to their infectivity.

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EVAMARIE SANDER AND GABRIELE MERTES

4.p H

The optimum pH for protoplast infection depends on the net charge of the virus and the ionization pattern of the amino acids of its coat protein as much as on the pH tolerance of the protoplasts. Below pH 5.2 the success of infection decreases in protoplasts of many plant species as their viability is affected by this degree of acidity (Kubo and Takanami, 1979; Huber et al., 1981; Kikkawa etal., 1982). In phosphate and citrate buffered systems infection occurs mostly within the range of pH 5.2 -5.6. For BPMV the protonization/deprotonization requirement of the virus was interpreted by Lesney and Murakishi (1981) as due to histidine involved in the virus specific buffer effects. For CYMV, a virus with an isoelectric point at p H 5.3, maximum infection occurs at pH 6.0 whereas the infection rate was low at pH 5.4 when the net charge of the viria is about nil (Rao and Hiruki, 1978). When inocula are prepared in Tris-chloride buffer, in general maximum infection occurs at pH 7.1-8.9, the effective buffering range of Tris; in the case of TRosV at pH 7.0- 8.6 (Morris-Krsinich et al., 1979), TMV at pH 7.2-8.7 (Motoyoshi and Oshima, 1976), BMV at pH 8.0-8.8 (Okuno and Furusawa, 1978a), and for SBMV at pH 7.1 -8.6 (Jarvis and Murakishi, 1980). 5.Polyethylene Glycol (PEG)

Recently, a procedure for inoculation of protoplasts has been developed which employs fusion-promoting agents, such as PEG, in lieu of polycations. In the presence of 1.3 - 7.8% (w/v) PEG, protoplasts from Nicotiana tabacum leaves became infected with TMV (Cassells and Cocker, 1980), TMV, and CCMV (Dawson et al., 1978), tomato protoplasts with TMV (Cassells and Barlass, 1978), and cucumber protoplasts with CMV (Maule etal., 1980). The procedure of Cassells and Cocker (1980), who combined PEG with protoplast aggregation conditions in high pH/calcium solutions, involves several basic steps. To 1X lo6protoplasts in 0.4 m10.66 Mmannitol, pH 5.8, were added 120 pg TMV in 0.5 ml phosphate (0.005 M ) buffered mannitol and 0.1 ml8% PEG. Incubation for 20 minutes at 4°C resulted in precipitation of the virus onto the protoplast surface without visible clumping of the protoplasts. Then 0.3 ml8% PEG in mannitol and 0.7 ml phosphate (3.5 mM) buffered mannitol containing 11 mM CaC1, were added to initiate protoplast fusion at 25°C for 1 hour. Thereafter, the protoplasts were washed with 50 mMCaC1, in mannitol, pH 10.5, and then with minimal salt medium. More than 70% of the protoplasts became infected. Without precipitation of TMV previous to protoplast aggrega-

PROTOPLASTS IN PLANT VIRUS RESEARCH

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tion, the percentage of protoplasts infected reached only 20%. When protoplasts were aggregated before virus addition, no infection occurred. The process of virus uptake is not yet fully understood. The authors (Cassells and Cocker, 1980) suggest that the virus particles are incorporated into localized areas of membrane fusion or that virus uptake occurs a t destabilized areas between adjacent protoplasts. Vesicle formation, as reported for PLO-mediated infection (Otsuki et al., 1972), could not be observed. As for PLO, also with PEG, still further studies are necessary before the process of virus uptake can be explained. 6. Protoplast Concentration.

When protoplast infection experiments were first carried out, citratebuffered inocula were mainly used and the protoplast concentration in the inoculum varied from 5 X lo6 (Otsuki and Takebe, 1972)to 2 X lo6protoplasts/ml (Coutts and Cocking, 1972). Since phosphate and recently Tris -chloride-buffered inocula are used, several authors report that concentrations higher than 1- 2 X lo6 protoplasts/ml yield only a very low percentage of infected protoplasts (Morris-Krsinich et al., 1979; Lesney and Murakishi, 1981). Maximum infection is usually reached with 0.21 X lo6 protoplasts/ml. In general, 1 X lo6 protoplasts/ml is the preferred concentration, an amount that can also be handled technically with more ease than a lesser number. Mertes and Sander (1981) confirm this concentration for the infection of tobacco leaf protoplasts; however, for infection of the much smaller protoplasts from soybean cell suspension culture the optimum amount was 5 X lo6 protoplasts/ml. Mayo (1978) compared the influence of the protoplast concentration in different buffers on the efficiency of infection in the TRV/tobacco protoplast system and found the concentration of 0.5 - 5 X lo6 protoplasts/ml to be inversely related to the percentage of infected tobacco protoplasts when phosphate or Tris - chloride-prepared inocula were used. In citrate-buffered inocula no effect of the protoplast concentration was observed. Because the decrease of infection efficiency in phosphate buffer was counteracted by increased PLO concentration, Mayo (1978) discusses the ratio of this concentration/number of protoplasts as influencing the success of infection. As already described (Section IV,A,3) citrate buffer is more suited to effect satisfactory infection with suboptimum PLO concentration. With increasing amount of protoplasts the concentration of PLO can be raised only within limits because of its toxicity. 7. Concentration of Inocula When infection is mediated by PLO, increase of inoculum effects also an increase in the efficiency of infection, approximately 1,ug virus/ml being

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the optimum amount. Further increase in concentration results in a decreased percentage of infected protoplasts. The reason for this phenomenon is probably that higher virus concentrations require higher PLO concentrations which, in turn, are not applicable because of their toxic effect on protoplasts (Alblas and Bol, 1977). The amount of virus produced per infected protoplasts remains the same regardless of the concentration of inoculum used (Huber etal., 1981). When infection of protoplasts takes place in the absence of PLO, the efficiency of infection reaches a plateau, e.g., for BPMV at 4.5pg vimslml (tested up to 9 pg/ml) and for BMV at 20 pg virus/ml (tested up to 50 pglml). This phenomenon may be caused either by saturation of receptor sites (BPMV; Lesney and Murakishi, 1981) or because the capacity of the host cell to replicate virus is limited. When BMV infection is performed with PLO, the inocuium requirement changes and an optimum virus concentration is reached at 1pg/ml (Okuno and Furusawa, 1978a). 8. Osmotic Stabilizer

An osmotic shock immediately before or during inoculation was reported to increase the percentage of barley protoplasts infected with BMV (Okuno and Furusawa, 1978b). Raising the mannitol concentration from 0.5 to 0.7 M or even to 0.85 M increased the infection efficiency whereas a decrease in the osmolality prior to or during inoculation inhibited infection. The authors discuss that shrinkage of the protoplasts renders membrane structure and properties more favorable for virus adsorption and uptake. Also Sarkar (1977) recommended the use of inoculation media with a slightly higher osmolality than the medium employed for the protoplast washing previous to inoculation of tobacco protoplasts with TMV. In contrast with Okuno and Furusawa (197813) no enhanced infection efficiency by osmotic shock was observed when protoplasts from Brassica rupa were inoculated with TRosV (Morris-Krsinich et ul., 1979) or cowpea protoplasts with TMV (Huber et al., 1981). In the latter system an increase of mannitol concentration in the inoculation medium from 0.6 to 0.8 M even led to a decreased amount of virus synthesized by the infected cowpea protoplasts. Further investigations will be necessary to understand the effect of osmotic shock on the protoplasts and to discern whether it is generally useful for infection of protoplasts or only for certain protoplast/virus systems. 9. Temperature during Inoculation

Most workers use a temperature of about 25°C during the inoculation period. However, when closely investigated, the uptake of most viruses by protoplasts seems to be independent of the temperature within a range from 0 to 30 C,as is the case of CCMV (Motoyoshi etal., 1974a), TRosV

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(Morris-Krsinich etal., 1979), TMV (Huber etal., 1981), and BPMV (Lesney and Murakishi, 1981). There are also exceptions, e.g., in the same protoplast system used for inoculation with BPMV and CPMV, the infection efficiency of CPMV suffered below 13°C (Jarvis and Murakishi, 1980). For another virus, AMV, even the optimum inoculation occurs at 0°C (Alblas and Bol, 1977), a fact that might be explained by the high sensitivity of AMV against ribonucleases and proteolytic enzymes. 10. CaC1,

Preincubation of virus with CaCl, in addition to PLO was found to be very stimulatory to the infection of soybean cell suspension culture protoplasts with CPMV, SBMV (Jarvis and Murakishi, 1980), and BPMV (Lesney and Murakishi, 1981). With the latter virus the presence of CaC1, even caused a synergistic effect at low virus concentration. The efficiency of infection increased with CaC1, concentrations from 0.25 to 1 mM, then reaching a plateau. The stimulating effect was markedly reduced when protoplasts were washed before inoculation with medium containing CaC1,. The mechanism of CaC1, action is not yet understood. The effect of the salt is not common to protoplasts from suspension cultures of all cell lines, since no stimulation by CaC1, was observed when suspension culture protoplasts from tobacco (Kikkawa etal., 1982) and from soybean (E. Sander and J. Gras, unpublished) were inoculated with TMV. The necessity to preincubate the virus with the salt to achieve stimulation indicates an interaction with the virion itself. Particle stabilization by the salt is reported for such viruses as SBMV and TRosV (Hull, 1978) which contain Ca2+as a major stabilizing cation, but this effect is unlikely to occur with BPMV (Lesney and Murakishi, 1981). It is generally accepted that washing of protoplasts after inoculation in an osmoticum containing 0.1 mM CaCl, has a stabilizing effect on the membrane. Concentrations of 10 mM CaC1, are sometimes reported to induce clumping of or even injury to protoplasts, e.g., for those derived from turnip (Morris-Krsinich etal., 1979), cowpea (Alblas and Bol, 1977), and pea (von Arnold and Eriksson, 1977). Yet, Okuno and Furusawa (1978a) report 10 mM CaCl, in the postwashing medium to yield a higher infection efficiency with barley protoplasts than concentrations lower than that. For tobacco protoplasts (Kassanis etal., 1975) and soybean cell suspension protoplasts (Jarvis and Murakishi, 1980) the omission of CaC1, in the postwashing medium has no effect on the success of infection.

B. Inoculation withViral Nucleic Acidsand Viroids RNA. Until now, only protoplasts from plant leaves have been successfully inoculated with viral RNA by one of the standard procedures. With

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protoplasts from cell suspension cultures, RNA infection has to be mediated by liposomes. Mertes (1983) inoculated tobacco leaf protoplasts with TMV-RNA employing several modifications of standard procedures, but did not yet succeed in TMV-RNA infection of protoplasts from soybean cell suspension culture, although they can be inoculated with viria with a high degree of efficiency. Further studies will discern whether this is a common feature of cell culture protoplasts. The reason for this pheare more nomenon may be that conditions for growth of plant cells inuitro stressed than in the very leaf and that the stress enhances increased production of nucleases. This hypothesis was supported by Suzuki and Takebe (1978) who found that heterologous DNA becomes degraded more easily in protoplasts from cell suspension cultures than in those from leaves. For infection of leaf protoplasts with plant virus RNA, most authors emphasize the necessity of RNA concentrations as high as 10- 100 pg/ml of inoculation medium, e.g., for TMV-RNA (Sarkar etal., 1974; Dawson et al., 1978; Motoyoshi and Oshima, 1979). In general, the efficiencies of infection reported are considerably lower than those achieved in inoculation systems with complete virus particles. So, only up to 7% of the protoplasts could be inoculated with RNA of TMV (Aoki and Takebe, 1969),CCMV, andBMV (Motoyoshi etal., 1973,1974b). Modifications of inoculation methods raised the percentage of infected protoplasts to 25% for CCMV-RNA (Watts etal., 1975) and to 41% for TMV-RNA (Motoyoshi and Oshima, 1979). Infection efficiency comparable to that of infections with virus particles was only reported by Sarkar etal. (1974) for TMV-RNA and by Okuno and Furusawa (1978~)for BMV-RNA. The difficulties in inoculation of plant protoplasts with free viral nucleic acid are attributed to an enhanced production of nucleases in protoplasts which are delivered into the medium (Heyn and Schilperoort, 1973; Lurquin and Hotta, 1975). Apparently, in protoplasts the denouo synthesis of ribonucleases is stimulated by the hypertonic medium, since Premecz etal. (1977) found a positive correlation between the osmolality of the surrounding medium and the amount of RNase activity in the protoplasts. Therefore, successful inoculation of plant protoplasts with viral nucleic acid requires sufficient reduction of content or activity of nucleases in the inoculation system. 1 . Osmotic Stabilizer

As mentioned above, the osmolality of the mannitol solution plays an important role in the success of infection. Mannitol concentrations above 0.5 M were reported to cause a sharp decrease in the infection efficiency of barley protoplasts with BMV-RNA (Okuno and Furusawa, 1978~).Most other authors reporting lower infection efficiencies (Aoki and Takebe,

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1969; Motoyoshi etal., 1973, 1974b, 1979; Watts etal., 1975) used media with 0.7 or 0.8 M mannitol. Beier and Bruening (1976) who inoculated cowpea protoplasts with CPMV-RNA with an efficiency of 64% used a medium with as little as 0.45 M mannitol. According to Sarkar etal. (1974) the activity of RNase at highly alkaline pH and high salt concentrations is almost completely inhibited. Under these conditions he infected tobacco protoplasts with 4-40 pg RNA/ml reaching an infection efficiency of 70- 100%. Several authors, however, reported this method to be difficult to repeat with their infection systems (Dawson etal., 1978;Okuno and Furusawa, 1978c; Motoyoshi and Oshima, 1979; Maule etal., 1980). This is probably due to the tendency of protoplasts to deteriorate under conditions of high salt concentration. Also Mertes (1983) did not succeed in infecting tobacco leaf protoplasts with TMV-RNA under these conditions but achieved infection, even though of low efficiency, in 0.7 M mannitol solution of pH 9.0. 2.Zinc Salts

Zinc salts are reported to inhibit the activity of plant nucleases (FrischNiggemeyer and Reddi, 1957). The addition of 5 mM ZnC1, or ZnSO, enhanced the infection of tobacco protoplasts with CCMV-RNA (Dawson etal., 1978) and TMV-RNA (Mertes, 1983) as well as with heterologous DNS (Suzuki and Takebe, 1976). However, zinc salts may not be suitable for all protoplast systems. Maule etal. (1980) observed 5 mM ZnC1, to be slightly toxic to cucumber protoplasts and did not enhance infection. ZnC1, must not be employed with citrate or phosphate buffers, because it will precipitate. In the presence of Zn2+the inoculation should be carried out in mannitol solution and pH 5.2 (Mertes, 1983) or in potassium succinate buffer (Dawson etal., 1978). 3.CaC1,

Infection of protoplasts with 1pg viral RNA/ml in the presence of 1mM CaC1, and PLO at 0°C was carried out successfully with TMV-RNA and tobacco protoplasts (Mertes, 1983) and with BMV-RNA and barley protoplasts (Okuno and Furusawa, 1978c) yielding up to 90% infected protoplasts. The salt is thought to stabilize the protoplast membrane and to effect changes in the configuration of the RNA thus protecting it from the action of nucleases. 4. Polycations

The infection of protoplasts with viral nucleic acid does not dependuponthe presence of polycations, but several authors report PLO as stimulating the process (Aoki and Takebe, 1969; Suzuki and Takebe, 1976;Motoyoshi etal., 1973; Beier and Bruening, 1976; Okuno and Furusawa, 1978~).

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Sarkar etal.(1974) discuss the stimulating effect of PLO as due to a protection from nucleases by direct interaction between RNA and PLO, because no enhancing effect of the cation was observed at high pH and salt conditions, which inhibit RNase activity. It is unlikely that the cation enhances pinocytosis. This is postulated from experiments by Okuno and Furusawa (1978~) who observed infection to be stimulated by PLO at 0°C when pinocytosis, as an energy-requiring process, must have ceased. When Zn2+ salts are added during preincubation of nucleic acid with PLO, there is no stimulatory effect of PLO on infection (Suzuki and Takebe, 1978). Therefore, Zn2+salts should only be added to the protoplast suspensions. The salts probably hinder the interaction of PLO with the RNA, so protection of RNA from nucleases appears, indeed, to be the main effect of PLO. PLO can be replaced by poly-D-lysine without change in effect as reported for tomato protoplasts and TMV-RNA (Motoyoshi and Oshima, 1979). 5.Cycloheximide

The increase of RNase activity in hypertonic mannitol solutions can be inhibited by adding cycloheximide (Premecz etal., 1977). Also, a reduced nuclease activity in protoplasts was observed when the source leaves for oat protoplasts were treated with cycloheximide or L-lysine (Sawhney et 1977). al., When barley protoplasts were isolated in an enzyme solution containing either one of these substances, the infection with BMV-RNA was considerably stimulated (Okuno and Furusawa, 1978a). The same effect was observed when tobacco protoplasts were isolated in the presence of 0.1 pg/ml cycloheximide and infected with TMV-RNA (O'C, PLO, CaC1,; Mertes, 1983). The TMV yield was increased 4- to 7-fold as determined by biotest. Cycloheximide is thought to reduce protein synthesis in protoplasts during the incubation period in enzyme solution in which they are set free. In turn, the nuclease content becomes reduced, thus facilitating infection of the protoplasts with free viral RNA. 6. Buffers

In contrast to recent observations with the infection of protoplasts with virions, citrate buffer seems to be more suitable for RNA inoculation systems than phosphate or Tris buffer (Okuno and Furusawa, 1978c; Motoyoshi and Oshima, 1979). 7. Temperature

In nearly all RNA infection systems a temperature as low as 0 to 10°C was much more efficient than higher temperatures, most probably because

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of reduced enzyme activity to low temperatures. Different findings are, therefore, only reported in those inoculation systems in which nuclease activity was inhibited by high pH/high salt conditions (Sarkar etal., 1974) or by Zn2+salts (Dawson etal., 1978; Mertes, 1983). In both cases infection was effected at inoculation temperatures of 25 - 30"C. 8.Polyethylene Glycol (PEG)

Recently, PEG-mediated infection has also been extended to viral RNA. Dawson etal. (1978) inoculated tobacco protoplasts with RNA of CCMV, TMV, and BMV by this procedure reporting that CaC1, was also essential for infection. The increase of infection efficiency compared to that by other methods was up to one order of magnitude. M a d e etal. (1980) infected cucumber protoplasts with CMV- and TRSV-RNA in the presence of PEG with an efficiency up to 60%. The cucumber protoplasts were suspended in 0.2 ml mannitol at a concentration of 0.5-1.5 X 10' protoplasts/ml and 2 ml40% PEG with 3 mMCaCl, and 0.1 mg RNA were added. The PEG causes rapid aggregation of the protoplasts. After 10 seconds at 0°C the suspension was diluted with 20 ml mannitol containing 0.1 mM CaCl,, incubated further 30 minutes at 20°C, and washed with CaC1,-mannitol. The infection mechanism is suggested to be the same as for PEG-mediated infection with virus particles, i.e., entrapping and uptake of the viral nucleic acid at areas of membrane fusion.

9. Liposornes When amphipathic phospholipids are dispersed in aqueous solutions, vesicles with a bilayered membrane can be formed, encapsulating some of the aqueous solution. These lipid structures, called liposomes, can interact with protoplasts and deliver the encapsulated material into the cell. This ability makes them apromising tool for introducing nucleic acids into protoplasts with a higher efficiency than with conventional methods, since the nucleic acids are highly protected from nucleases when entrapped in liposomes. The preparation of liposomes, possible by at least 8 different methods, their interaction with protoplasts, and some examples of specific liposome/protoplast interactions have recently been reviewed in detail by Makins (1983). While so far cell suspension culture protoplasts could not be infected with free viral nucleic acid by the conventional methods, several authors now report successful liposome-mediated infection of such protoplasts with viral nucleic acid. Fukunaga etal. (1981) inoculatedprotoplasts from Vinca roseacell suspension culture with TMV-RNA, encapsulated in phosphatidylserine liposomes. The efficiency was up to 80% infected protoplasts. The inoculation was performed in the presence of PEG or

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polyvinyl alcohol (PVA), both substances yielding comparable results. The liposomes were prepared by the Ca2+-EDTA chelation method, developed by Papahadjopoulos and co-workers (Wilson et al., 1979). Phosphatidylserine in chloroform-methanol, vortex mixed with buffer, is ultrasonicated to form small liposomes. Addition of calcium induces the liposomes to form cochleate cylinders by aggregation and fusion. These cylinders are collected by centrifugation and resuspended in a TMV-RNA containing aqueous solution. Addition of EDTA induces the cochleate cylinders to change into large liposomes which encapsulate 2 - 10%of the available RNA (Fukunaga et al., 1981). Nagata et al. (1981) inoculated suspension culture protoplasts from Nicotiana tabacum with liposomes, prepared by the reverse phase evaporation method, with TMV-RNA. This method gave rise to a high efficiency of encapsulation (40-60% of the available RNA); thus, the amount of RMV-RNA, necessary for protoplast inoculation, could be significantly reduced. The reverse phase evaporation method was developed by Szoka and Paphadjopoulos (1980). Here, the phospholipid containing ether phase and the RNA containing aqueous phase are briefly sonicated t o give small droplets of aqueous phase, surrounded by a monolayer of phospholipid molecules. Removing the ether in a rotary evaporator under reduced pressure increases the concentration of the phospholipid vesicles up to a point where some of these monolayer vesicles become unstable and rupture by forming bilayered liposomes with neighboring vesicles. With similarly prepared liposomes Fraley et al. (1982) inoculated suspension culture protoplasts from Nicotiana tabacum with TMV-RNA in the presence of PEG or PVA, of which he found PVA to be more than twice as efficient as concluded from virus multiplication. Comparing the toxicity of different liposome preparations toward protoplasts, the authors found liposomes carrying positive charge to be more toxic than negatively charged and neutral liposomes. As most efficient RNA carriers to protoplasts they found negatively charged phosphatidylserine/cholesterol liposomes. Cholesterol is reported to reduce the permeability of the liposome membranes to hydrophilic solutes (Makins, 1983) and thus reduces leakiness of the liposomes resulting in a higher TMV-RNA amount to be available for inoculation. Liposome-mediated infection was reported by Rollo and Hull (1982) for Brassica rupumesophyll protoplasts and TRosV-RNA. The liposomes were prepared by the reverse evaporation method. The mechanism of liposome-mediated infection cannot be explained yet, but hypotheses are advanced. As inoculation is established during a rather short period in the presence of fusion-promoting agents such as PEG and PVA, and is enhanced by postinoculation washing with a high

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pH/high Ca2+buffer (Fukunaga etal., 1981), it is supposed that liposomes and protoplasts fuse, thereby shifting the nucleic acid from the liposomes to the host cell (Makins, 1983). An alternative hypothesis, based on endocytosis, was substantiated by an ultrastructural study of Fukunaga et al. (1983). A new fixation procedure allowed the fragile liposomes and their interaction with protoplasts to be examined by transmission electron microscopy. No evidence whatsoever could be found for fusion of liposomes and protoplasts. The liposomes adhered to the protoplasts surface and, after invagination of the plasmalemma, they were observed within vesicles of cellular origin. Direct entry by lesions in a few cases was also discussed by the authors. The further fate of the vesicles in the cell was not investigated. These observations correspond to the fact that the PEG concentration, necessary for liposome-mediated infection, is suboptimal for protoplast fusion. The main effects of PEG a t this concentration may therefore be precipitation by dehydration and enhancing of pinocytosis. Calcium in the postwashing medium may stabilize the protoplast/liposome contact by “calcium bridges” (Makins, 1983). DNA. Infection of protoplasts with plant virus DNA is described by Yamaoka et al. (1982) who infected turnip protoplasts with 1 pg/ml CaMV-DNA at conditions described for RNA inoculation systems. A t room temperature in citrate-buffered mannitol, pH 5.0, and in the presence of 1pg/ml PLO protoplasts were infected with an efficiency of 16%. Infection of bean mesophyll protoplasts with BGMV-DNA is reported by (1981), yet no information is given about the methods. Haber etal. Viroids. Viroids are plant pathogens that exist as uncoated circular single-stranded RNA (Sanger, 1982; Muhlbach, 1983). Infection of protoplasts with viroids was achieved by Muhlbach and Sanger (1977), who inoculated tomato leaf protoplasts with CPFV (cucumber pale fruit viroid), PSTV (potato spindle tuber viroid), and CEV (citrus exocortis viroid). Significant viroid replication was observed only for CPFV. The inoculation procedure was derived from the high pH/high salt method, developed by Sarkar etal. (1974) for the TMV-RNA/tobacco protoplast system. Because the tomato protoplasts were seriously damaged in the salt medium, viroid inoculation was performed in glycine - KOH-buffered mannitol a t pH 9.0 to which 0.2 pg/ml PLO was added. For successful infection of 1X lo6protoplasts/ml a t least 10 pg/ml viroid was required. The rate of viroid synthesis, as assessed by incorporation of [3H]uridine into RNA and extractable viroid infectivity, was much lower than reported for plant viruses; the first newly synthesizedviroid RNA was detected at 36 hours after inoculation. The addition of a-amanitin (Muhlbach and Sanger, 1979) inhibited viroid synthesis in protoplasts. On the basis of the calculated intracellular concentrations of a-amanitin it was suggested that

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the DNA-dependent RNA-polymerase I1 is involved in viroid replication. Further understanding of viroid replication is expected from a potato cell suspension culture, continuously replicating PSTV; this culture is also a source for the isolation of protoplasts (Muhlbach and Sanger, 1981).

C. Culture ofInfected Protoplasts For virus synthesis the inoculated protoplasts are usually incubated in liquid media from which aliquots are taken in succession for virus assay. Virus synthesis is often significantly influenced by growth substances present in the medium. In general, kinetin is not required for the survival of protoplasts and virus replication improves in its absence, e.g., in the tobacco protoplast/TMV system (G. Mertes, unpublished) and the cowpea protoplast/TMV system. In the latter the addition of kinetin reduced virus production about 80% (Huber etal., 1981). Virus synthesis can also take place in the absence of auxins. However, when the presence of 2,4dichlorophenoxyacetic acid (2,4-D) is advised, its concentration is critical. In protoplasts from leaves of a local lesion tobacco host, virus replication is enhanced by addition of 2,4-D to the postinoculation medium whereas, in protoplasts from a systematic tobacco host, replication is decreased in its presence as compared to the replication in auxin-free media (Loebenstein etal., 1980). Because 2,4-D is a commonly accepted auxin in protoplast culture media, these results can explain why frequently the virus synthesis was not observed to differ in protoplasts from either host plant, quite contrary to the synthesis of the virus in the intact plants. However, when protoplasts from either host plant were cultured after virus inoculation in a medium without growth substances then virus synthesis was 4- to 10-fold higher in protoplasts from systemic hosts than from local lesion hosts. This corresponds with the results obtained with the intact plants. The effect of increasing mannitol concentrations on virus multiplication was quantified by Shabtai etat. (1982). At molarities above 0.75 he found virus synthesis considerably decreased in protoplasts from a systemic tobacco host, the effect being somewhat less in protoplasts from local lesion hosts. The light intensity required by protoplasts is determined by the source tissue from which they are derived. In protoplasts from cell suspension cultures cultivated in the absence of light (Mertes and Sander, 1981) and nongreen hypocotyl protoplasts from Chinese cabbage (Renaudin etaL, 1981)virus multiplication proceeds satisfactorily when the infected protoplasts are cultured in darkness; the application of light had no effect. Also, chlorophyll-containing protoplasts from leaves allow virus multiplication

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in the dark, but the infection efficiency is frequently improved by constant light of low intensities (Hibi etal., 1975; Renaudin etal., 1981). Light intensities above 5000 lux are harmful to many protoplasts (Sarkar, 1977). For the tobacco leaf protoplasts/TMV system, culture at constant light of 1000 lux raised the virus synthesis up to 3.6-fold over that at 2000 lux for 16 hours in change with 8 hours of darkness. Regeneration of infected protoplasts to callus clones was recently reported for CaMV-infected turnip protoplasts (Paszkowski etal., 1983). The plating efficiency was markedly reduced in the infected protoplast system. The hypothesis of competition between cell proliferation and virus multiplication was possibly further substantiated by a reduction of growth in infected calli derived from infected protoplasts after 5 months in contrast to healthy clones. The authors discuss that initial low numbers of viria permit cell proliferation which is gradually inhibited by increased virus multiplication. If protoplasts withstand regeneration, the callus formation can be markedly improved by using filter paper as a carrier for protoplast cultures. aquilinum (Partanen, 1981) Established for protoplasts from Pteridium the method was also found very beneficial for the regeneration of tobacco leaf and soybean cell suspension culture protoplasts (Mertes, 1983) as well as for Medicagomesophyll protoplasts (Arcioni etal., 1982). The filter paper is placed upon nutrient medium solidified with 0.6-0.7% agar and the protoplast suspension is applied to the filter surface. The renewal of nutrient medium for the developing clones is best effected by the transfer of the clone-carrying filter paper to fresh agar medium. Inhibitors to cell proliferation released by dead cells or such cells that accumulate metabolic contaminating compounds such as phenolic substances can be removed by adsorption to activated charcoal (Carlberg etal., 1983), thus improving the protoplast regeneration frequency.

V. DETERMINATION OF VIRUSREPLICATION It must be clearly kept in mind that the bioassay is the only means by which both infectivity of virus and increase and decrease of infective particles can be determined. All other methods assess presence and quantity of viria, complete or defect and also virus constituents like coat protein subunits or nucleic acid, but do not supply information as to the very criterion of completeness of a virion, the infectivity, i.e., its biological activity. Consequently, for the study of steps in biosynthesis or degradation of virus, bioassay and serological and/or staining methods should be em-

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ployed in combination, as the bioassay, in turn, does not recognize virus constituents except infective nucleic acid.

A. Bioassay The amount of virus produced per protoplast can be calculated by determination of the number of infected protoplasts in a sample (Section V,B,l) and measuring the infectivity of aliquots of these protoplasts on a local lesion host for the virus. To release viria, protoplasts must be disrupted either by mortar and pestle or by homogenization on a ground-glass tissue grinder (Potter mini-homogenizer). Preferably, the homogenate is assayed on half leaves and compared to a standard concentration of a purified virus suspension on the opposite half of each leaf, thus minimizing the effect of the variation in sensitivity to the virus between test plants and leaves in different positions on the plant. The virus concentration in the protoplasts is determined by that dilution of the protoplast homogenate which induces lesions in the same order of magnitude as does the standard solution of known virus content (Huber et al., 1981). To estimate by which degree the protoplast homogenate should be diluted, the protoplast extracts can be examined in an electron microscope following negative staining with uranyl acetate to determine the density of virus particles in the sample (Kassanis et al., 1975; Cassells and Cocker, 1980). If no local lesion host for the virus is known, the relative infectivity can be determined on a systemic host by determining the dilution end point which is generally defined as that dilution of the virus to which 50% of the inoculated test plants react with symptoms in several separate assays (Matthews, 1981).

3.Serological Virus Detection 1, Staining Methods

The proportion of infected and uninfected protoplasts in a sample can be discerned by staining with antibodies directed against the virus which are conjugated with a dye as marker. For this purpose, the protoplasts are usually fixed either in suspension (Cassells and Cocker, 1980) or on slides (Otsuki and Takebe, 1969)with a fixative, like acetone or 1 to 3% glutaraldehyde, before the actual staining. a. Fluorescent Antibody Technique. The most commonly employed procedure for determination of the amount of infected protoplasts is the staining of accumulated virus antigen in the protoplasts by antibodies

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conjugated with a fluorescent dye. Of two fluorochromes, the more convenient and widely used is fluorescein isothiocyanate (FITC) (Otsuki and Takebe, 1969); the other, lissamine rhodamine B was reported to be more sensitive (Cassells and Gatenby, 1975),but found no general application Yet, the availability of two different fluorescing dyes could be useful for the detection of antigen in protoplasts infected with different viruses. Fluorescein isothiocyanate can be directly conjugated to the virus-specific antibody (Otsuki and Takebe, 1969)or to an antibody directed against the virus-specific antibody (Mertes and Sander, 1981). According to the latter procedure the fixed and washed (phosphate-buffered saline, pH 7.2) protoplasts are incubated with antivirus immunoglobulins from rabbit for 1 hour at 37°C and after subsequent washing covered with FITC-conjugated anti-rabbit immunoglobulin from goat (Behring AG, Marburg, Germany) for 1hour a t 37°C. After final washing and embedding in glycerol the protoplasts are examined under a microscope with high pressure mercury lamp and specific filter combinations. The virus-containing fluorescing protoplasts can be distinguished from the noninfected protoplasts which show no or slight unspecific fluorescence of another shade. Since the intensity of the fluorescence fades with time, the slides have to be examined in quick succession. b. Peroxiduse Staining. To overcome the problems of interpretation of unspecific fluorescence in FITC-stained protoplasts and to become independent from microscopes with fluorescence tops, recently, an immunoperoxidase technique was developed to determine the efficiency of infection in barley protoplasts infected with BSMV (Chiu Ben-Sin and Tien Po, 1983). 1982) and in tobacco protoplasts infected with TMV (Sander etul., The protoplasts are spread on slides, dried fast in a stream of warm air, and fixed for 30 minutes in a 96% methanol containing 0.074% HC1 to inhibit action of endogenous peroxidase. After washing the fixed protoplasts in phosphate-buffered saline, they are incubated with virus-specific antibodies from rabbit (0.5 mg/ml) for 1 hour at 37°C. Antibodies not adsorbed are removed by washing, followed by incubation of the protoplast preparations with antibodies against rabbit antibodies from sheep or goat (Behring AG, Marburg, Germany). These antibody preparations may be used a t a dilution of 1:10 or 1: 50. After 30 minutes at 37°C antibodies not adsorbed are removed by washing and the protoplast preparations are covered with antibodies from rabbit against horseradish peroxidase complexed with horseradish peroxidase (PAP, peroxidase - antiperoxidase complex; Paesel GmbH & Co., Frankfurt, Germany). The preparation of the soluble PAP complex is described by Chiu Ben-Sin and Tien Po (1982). After 30 minutes incubation at 37°C the protoplasts are again

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washed and stained for 10 minutes with 0.05% 3,3’-diaminobenzidine (DAP) in 0.05 M Tris-HC1 buffer, pH 7.6, containing 0.01% hydrogen peroxide. After final washing in Tris- HC1 buffer the protoplasts can be examined under a light microscope. Brown staining of the protoplasts indicates virus antigen, whereas uninfected protoplasts remain faint yellow. In contrast to Chiu Ben-Sin and Tien Po who reported no nonspecific staining and thus higher specificity of the PAP-staining procedures than that with fluorescent dyes, nonspecific staining, especially of broken protoplasts was observed by Sander et al. (1983). This effect, however, could be completely excluded by treating the protoplast preparations previous to application of virus-specific antibodies with normal rabbit serum (Flow Laboratory GmbH., Bonn, Germany) by which specific staining was not impaired. The advantage of the PAP method is the possibility of storing the stained preparations for several days and examining them several times without the danger of the stain fading. 2. Quantitative Determination of Virus Antigen in Protoplasts

a. Enzyme-Linked Immunosorbent Assay (ELISA). In this method the virus contained in the test sample is selectively trapped by virus-specific antibodies, adsorbed to the surface of the wells of immulone microtiter plates (Dynatech GmbH, Denkendorf, Germany). The trapped virus, in turn, attaches to antibodies labeled with an enzyme which reacts with an adequate substrate. The enzyme/substrate reaction results in a yellow color in the case of alkaline phosphatase measurable photometrically at 405 nm. Because of its specificity and the possibility to assay protoplast samples for virus content within 2 days, the method finds increasing application (Loebenstein and Gera, 1981;Shabtai et aL, 1982;Chiu Ben-Sin and Po, 1982; Sander et al., 1983)based on the work of Clark and Adams (1977) who developed the ELISA for detection of plant viruses. For the detection of TMV in both, protoplasts from tobacco leaves and soybean cell suspension culture the ELISA and the bioassay were employed (E. Sander and J. Gras, unpublished). The results obtained with the serological assay correspond strongly with those obtained by bioassay; virus quantities as low as 10 ng were still measurable. b. Radioimmunassay (RIA). The assay resembles that for detection of virus by enzyme-labeled antibodies (ELISA), only that here radioactive iodine (1251) is coupled to the virus-specific antibodies instead. To each well 125 pl labeled antibody (about 2 X lo6 cpm) is added (Fraley et al., 1982). Instead of color intensity the amount of radioactivity, determined in scintillation vials, is measured indicating the amount of virus in the protoplast sample. Virus quantities as low as 1 ng could still be detected. c. Dilution End Point. From homogenized virus-infected protoplasts a

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series of dilutions is made to each of which a constant amount of virus-specific antibodies is added in order to precipitate the virus (Coutts and Cocking, 1972). The dilution endpoint in the sample is compared with the dilution end point in a dilution series with purified virus of known concentration which precipitated with the same constant amount of virus-specific antibodies. Thus, the virus content in the protoplast sample is calculated. The least amount of virus detectable by this method is 1 pg/ml.

C. Other Methods 1. Sucrose Gradient Assay

The amount of virus produced in infectedprotoplasts can be assayed by a sucrose gradient procedure (Motoyoshi et al., 1973; Morris-Krsinich et al., 1979). The protoplast sample is homogenized and clarified by centrifugation. Aliquots are layered onto 10 to 40% sucrose in acetate buffer gradients and high-speed centrifuged for 2 hours. The virus yield can be determined photometrically by comparison with virus samples of known concentrations. The gradients are scanned with an ISCO density gradient analyzer. The lowest amount of virus detectable is 0.3 p g . 2. Electron Microscopic Determination

The concentration of virus particles in a homogenate of infected protoplasts can be estimated by mixing the samples with a known concentration of latex particles (Coutts and Cocking, 1972). Droplets of known volume of the mixture are observed in the electron microscope and the number of virus particles is estimated in relation to the number of the latex particles. 3.Dot Molecular Hybridization

Virus RNA in protoplasts can be detected by using a 32P-labeledcDNA, prepared by reverse transcription of virus RNA (Sela and Weissbach, 1983). From TMV-infected protoplasts aliquots of 4 droplets (lo6protoplasts) were diluted 1 :3 in 0.1%SDS and incubated at 42°C for 10 minutes to lyse the protoplasts and disrupt the virus. In the lysates the RNA was denaturated at 65’C with formaldehyde and subsequently hybridized with cDNA on nitrocellulose. The lowest amount of virus RNA detectable is 2.5 pg. The procedure was very specific. The reverse transcription of the RNA to the cDNA can be prepared rapidly with a commercially available enzyme. In contrast to the serological tests no production of specific antisera is necessary; the rate of virus RNA synthesis in the protoplasts can be determined and virus concentration is measured in terms of its RNA rather than its coat protein.

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VI. INFECTION OF CELLSFROM CALLUS TISSUE AND CELL

SUSPENSION CULTURES

A. Infection withVirus Particles Constant effort has been invested to establish tissue culture systems that support virus multiplication. Their advantages over protoplasts are (1)the stability of the cells and their resistance to rupture and disintegration, (2) the availability of the system throughout the year without the season-dependent physiological variation known in leaves, (3) the absence of isolation procedures, and (4) the ease with which they can be handled experimentally. Tissue culture offers the possibility to culture infected cells for virus production over long periods of time whereas infected protoplasts are a transient system unless tissue cultures are established from such protoplasts. A decided disadvantage of tissue cultures for the investigation of virus replication is that, contrary to the singly occurring protoplasts, even in suspension cultures the cells grow in small aggregates and are connected by plasmodesmata (Spencer and Kimmins, 1969; Pelcher etal., 1972). This growth pattern and the presence of a rigid cell wall render synchronous and sufficient infection of a large number of cells difficult. Early attempts to inoculate cells of plant tissue cultures involved injury of cells with needles or a glass rod stirrer in the presence of virus (Motoyoshi and Oshima, 1968), resulting in low levels of infection. A more synchronous infection resulting in over 90% infected cells was introduced by Murakishi etal. (1970) who inoculated suspension culture calli by treating the cell/virus mixture (100- 200pg virus/ml) with a vortex mixer for 20 seconds at about 800 rpm. This simple procedure was found to be very efficient with several cell culture systems besides tobacco (Beachy and Murakishi, 1971;Mertes, 1983),especially for cell suspension cultures from soybean (Wu and Murakishi, 1979) and several Trifolium species (Jones etal., 1981). Yet, the disadvantage of these virus multiplication systems is still the lack of synchrony in virus replication, since the virus can spread from cell to cell by plasmodesmata (Pelcher etal., 1972). The entry of virus into the vortexed cells was found to occur at exposed ends of ruptured plasmodesmata (Murakishi etal., 1971). The virus uptake is not influenced by polycations. Intracellular vesicles, indicating pinocytosis, are not described. A promising step to achieve synchronous virus replication in plant cell cultures was the “different temperature” treatment, established by Dawson and Schlegel(l976) to synchronize TMV replication in tobacco plants, applied to soybean cell suspension cultures (White etal., 1977; Wu and

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Murakishi, 1979). First, the cells are inoculated by vortex mixingat 25"C, followed by incubation at 6°C for 4 days and a shift back to 25°C. Wu and Murakishi (1979) found a putative replicative form (RF) accumulated in the cells during the incubation period at 6"C, from which virus replication started synchronously after cell transfer to 25"C, the permissive temperature. The synthesis of the RF of virus RNA and complete virions could be studied in detail in this synchronized cell culture system, although slight problems in the pulse-chase experiments occurred, since in the cell aggregates the influxlefflux conditions could not be standardized entirely. Callus or cells cultured in suspension may be valuable for further understanding of the process of necrotic response of plants to virus infection. In infected protoplasts derived from such hypersensitive plants no necrotic symptoms become manifest. Apparently, cell to cell contact is essential for the visible expression of the genetic factor responsible for the necrosis. Different results have been obtained with cell cultures derived from tobacco plants hypersensitive to TMV when such cells were vortex inoculated with the virus and subsequently spread onto agar for further cultivation (Beachy and Murakishi, 1971). Here, a hypersensitive response was expressed; about 44 hours after inoculation the formation of local lesions started on the calli. Another indication of differences in the reaction to virus infection between cells from hypersensitive and systemic host plants is received from the observation that in suspension cultures of the former, virus multiplication occurs in two cycles reaching a maximum about 72 hours and a second maximum about 100 hours after inoculation (Beachy and Murakishi, 1973), whereas in suspension cultures of cells from a systemic host plant virus multiplication proceeded in one cycle with a maximum at 5 - 8 days after inoculation (Motoyoshi and Oshima, 1968;Murakishi et al., 1971;Wu and Murakishi, 1978). At least two maxima were observed when suspension culture cells of Nicotiana tabacum cv. Xanthi nc. were inoculated with TMV by vortexing (Mertes, 1983);the first maximum was reached within 48- 72 hours after inoculation followed by a marked decrease of infectivity in the cells as detected by bioassay (up to 75%). When the cell suspension culture was supplied with fresh medium about 120 hours after inoculation, a second maximum of infectivity developed. Aliquots of the samples were subjected to a serological detection of virus antigen in the cells (ELISA); here, the TMV decrease appeared not as drastic as that determined by the bioassay, but the pattern of virus multiplication detected serologically was essentially the same. Probably, the first maximum of infection is the result of primary infected cells in which the first replication cycles of virus take place. By spread of the virus to noninfected neighboring cells through the plasmo-

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desmata a second cycle becomes initiated distinguishable from that in the primary infected cells by onset of necrosis in the latter. Hirth and Durr (1971) showed that healthy cells in TMV-inoculated tissue cultures are nevertheless susceptible to the virus. Further approaches will show whether cell culture systems are suited for studies to elucidate the mechanism of the hypersensitive reaction of plants to virus infection. The occurrence of 4 additional proteins, characteristic for local lesion host plants (Van Loon and Van Kammen, 1970),could not be detected by Beachy and Murakishi (1976) in the callus system from hypersensitive tobacco, indicating marked differences in the reaction of cell cultures and whole plants.

B. Infection withViroids PSTV replicates continuously in a potato cell suspension culture at a rate much higher than in other described inuitro systems, such as callus cultures or protoplasts, and in the tomato plants (Muhlbach and Sanger, 1981). The conditions for rapid growth of the suspension culture and for extensive viroid biosynthesis are described in detail by Muhlbach etal. (1983). The optimum conditions for PSTV accumulation paralleled those for the optimum growth rate of the cell suspension culture. This inuitro system allows detailed analyses of intermediates occurring in PSTV replication in intact living cells and from these results a model for the mechanism of viroid replication was developed. The potato cells contain besides circular and linear ( )PSTV other single-stranded RNA molecules complementary to ( )PSTV, with a greater chain length [“longer-than-unitlength (-)PSTV”] (Miihlbach etal., 1983). In highly purified nuclei, isolated from the PSTV-infected potato cell suspension culture, 2 oligomeric forms of (+)PSTV, were accumulated (Spiesmacher etal., 1983). The authors propose the following model for PSTV replication: (1)The circular ( )PSTV molecule is transcribed within the nucleus into “longer-than-unit-length ( - )PSTV” oligomers, possibly by a rolling circle-like mechanism, the transcription being catalyzed by the nuclear DNA-dependent RNA-polymerase 11. (2) The ( - )PSTV oligomers serve as a template for transcription into ( )PSTV oligomers, the precursors of (+ )PSTV monomers. (3) The ( )PSTV oligomers are spliced into monomers by specific endonucleolytic cleavage and are ligated to covalently closed circles after having become modified at their termini. The cleavage and ligating steps can be carried out by normal enzymes of the host cell. An RNA ligating enzyme, also capable of forming circular viroid molecules, is already found in tissue of higher plants (Branch etal., 1982; Kikuchi etal., 1982).

+

+

+

+

+

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Further studies on viroid-infected cell suspension cultures will contribute to the understanding of viroid replication and the host cell/viroid interaction. The establishment of another cell suspension culture, replicating PSTV continuously, was reported by Zelcer etal. (1982),the culture being derived from tomato. VII. RESISTANCE AND ANTIVIRAL SUBSTANCES In whole plants resistance against virus infections can be attributed to various mechanism: (1)virus entry is rendered more difficult, e.g., by a thicker epidermis; (2) cell-to-cell movement of the virus is inhibited; (3) cell-to-cell movement of the virus is reduced (necrotic response); (4) spreading of the virus in the vascular bundles is inhibited; (5) virus replication is inhibited on the cellular level. Except for the first aspect, explanations for the four others can be attempted by experiments with in vitro systems such as plant cell cultures and protoplasts. Inhibited cell-to-cell movement of the virus was demonstrated by Sultzinski and Zaitlin (1982) who investigated the difference in susceptibility of cowpea (Vigna sinensis) plants and protoplasts to TMV. Cowpea plants did not show any symptoms after TMV inoculation andonly minute amounts of TMV could be recovered from infected tissue whereas in cowpea protoplasts when inoculated with TMV, the virus multiplies well. The authors inoculated cowpea plants with TMV, isolated protoplasts from the infected leaves at various times after inoculation, and stained the protoplasts with fluorescent antibodies. Thus, 1protoplast in 50,000- 150,000 proved to be infected and no increase in the number of infectedprotoplasts could be detected within 11 days after inoculation. However, a heavy increase of infected cells was observed in control experiments with Nicotiana tabacurn cv. Samsun, a host plant susceptible to TMV. From the results that there was a low number of well infected cells in the leaf tissue of cowpea and that virus multiplied in protoplasts, it can be postulated that cowpea plants build up a resistance against TMV infection by inhibiting cell-to-cell movement of the virus, whereas TMV replication at the level of the infected cell proceeds unaffected. Further evidence for this hypothesis was put forward by Leonhard and Zaitlin (1982) who found a temperaturesensitive strain of TMV, defective in cell-to-cell movement in Nicotiana tabacum, to differ from the type strain only by a small 30,000 M , polypeptide. Since this alteration is correlated with the inhibited cell-to-cell movement of TMV at a restrictive temperature, it is plausible to consider the small polypeptide as a “translocation protein.” This deduction is further substantiated by the observation of a reduced number of plasmo-

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desmata in plants infected at the permissive temperature with either the temperature-sensitive or the type strain. Apparently, alteration of the plasmodesmata is a function of the “translocation protein.” In nonhost plants for TMV as cowpea, this protein is perhaps destroyedand resistance thus established by restricting the virus entirely to the very cell infected. It would be interesting to know whether similar observations can be made with other nonhost/virus systems in which protoplasts support virus synthesis although the whole plant does not. An indication for virus-induced spreading exists for CPMV, a virus with a split genome encapsidated in two (bottom and middle) nucleoprotein particles. On infection of cowpea protoplasts with the bottom component alone, the bottom component RNA is replicated and expressed independently (Goldbach etal., 1980) and induces cytopathic structures in the protoplasts. However, when cowpea leaves are inoculated with the bottom component alone, symptoms are not developed as the infection is localized, obviously because the bottom component is incapable of spreading (Rezelman etal., 1982). Perhaps one of the polypeptides translated by the middle-component RNA (Franssen etaL, 1982) will have the function of a “translocation protein.” Reduced cell-to-cell movement of the virus by necrotic response of the infected plant cannot be assayed with protoplast systems since this type of necrotic response is not expressed in protoplasts; however, infected protoplasts isolated from infected necrotic hosts replicate virus heavily and show a high degree of viability. Yet, differences between protoplasts from necrotic hosts and those isolated from systemic hosts were observed by Loebenstein and Gera (1981). They isolated a substance from TMV-infected protoplasts derived from Nicotiana tubacum cv. Samsun NN which inhibits virus replication (IVR). This tobacco variety is well known for its necrotic response to infection with TMV. Protoplasts from a systemic host did not produce any IVR when infected with virus. Treatment of inoculated protoplasts with IVR inhibited virus replication in protoplasts from the necrotic host as well as in protoplasts from the systemic host. Characterization of the chemical and biological properties of IVR can perhaps also contribute to the understanding of the basic steps preceding the necrotic response. It would be interesting to find out whether a metabolic connection to the key enzymes of necrotization, e.g., phenylalanine ammoniumlyase (Sela, 1981) is evident. Also, callus and cell suspension cultures from local lesion hosts of a virus may be suited for investigation of the necrotic response, since callus tissue, derived from tobacco varieties reacting necrotically, is reported to develop lesions after inoculation with TMV (Beachy and Murakishi, 1971). In a cell suspension culture from Nicotiana tubacum cv. Xanthi nc. Mertes

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(1983) observed TMV replication occurring in cycles which, perhaps, is due to dying of cells primarily infected, and the establishment of sequential infection cycles in healthy cells within the small cell aggregates by passage of TMV through the plasmodesmata. Beachy and Murakishi (1976) assayed the calli reacting hypersensitively to the virus. They could not detect the additional proteins which are found in the protein pattern of leaves from hypersensitive tobacco plants (Van Loon and Van Kammen, 1970) and which are absent in leaves of systemic virus hosts. Further investigations are desired with regard to the spread of virus through plasmodesmata and the influence of IVR systems with cell to cell contact. With regard to inhibition of virus transport in vascular bundles some basic information can be gained from experiments with protoplasts, e.g., the influence of special cell types on virus replication. Since TNDV, a virus restricted to the phloem of its host plant, can multiply in mesophyll protoplasts (Kubo and Takanami, 1979) it would be interesting to know if virus restricted primarily to the mesophyll of leaves can be propagated in protoplasts or cell cultures from vascular tissue. No such test system has been established so far. The inhibition of virus replication by infectedplants on a cellular level is well demonstrated by the isolation of antiviral substances as reviewed by Sela (1981). Such substances are produced in infected cells and can be translocated to neighboring noninfected cells rendering them resistant. Apparently, it is virus replication on which these substances act rather than on penetration or uncoating of the virus, since replication is also reduced or inhibited when infection was incited by viral RNA instead of the virion. The inhibitory effect can be considered as dosage dependent. This could be confirmed with a substance isolated from spinach and assayed with TMV in tobacco protoplasts, where the same degree of inhibition was effected as in tobacco leaves only by about 1/800 the amount of antiviral substance (Junga, 1978). The molecular mechanism of antiviral activity is not yet fully understood (Sela, 1981), but contributions to the elucidation have been put forward by several authors. Mozes (1980) found the earliest virus-specific protein in virus replication, a molecule of high molecular weight, to be blocked by treatment with an antiviral factor. This corresponds with the findings of Junga (1978) that a treatment of protoplasts with the antiviral substance before inoculation with TMV resulted in strong inhibition of virus synthesis even at very low dosage; when the protoplasts were treated soon after inoculation, only high concentrations of the substances were still inhibitory. With regard to the fact that antiviral substances from plants are not species specific since they are capable of inciting resistance in protoplasts and in plants of species other than the one from which they were isolated, they are a promising tool

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against plant virus infections, especially once their mode of action on a cellular level has become known. The hypothesis that virus replication in nonhosts is restricted by an active response of the plant to infection has also been substantiated by Maekawa etal. (1981). They compared the influence of actinomycin D on BMV-infected protoplasts isolated from the natural host, barley and from nonhosts, radish and turnip. Actinomycin D inhibits the DNA-dependent RNA-polymerase and, thus, eventually the protein synthesis of the host cell. An effect of actinomycin D on the replication of RNA viruses can only be explained by a host cell specific step in virus replication. Actinomycin D is known to inhibit virus replication in many virus/natural host protoplast systems, when it is added during or soon after inoculation, as for PVX (Otsuki etal., 1974),PEMV (Motoyoshi and Hull, 1974), CMV (Takanami etal., 1977), TRosV (Morris-Krsinich etal., 1979), CPMV 1982),AMV (Alblas and Bol, (Rottier etal., 1979),TYMV (Renaudin etal., 1977; Nassuth etal., 1983), and TMV (Huber, 1983; Mayo and Barker, 1983). The results of the experiments about the influence of actinomycin D on BMV infection of barley protoplasts are in agreement with these data. In addition, Maekawa etal. (1981) found that in nonhost protoplasts actinomycin D had a significantly enhancing effect on the replication of BMV. This indicates that nonhost protoplasts may produce an antiviral substance. A reduced host cell-specific protein synthesis could mean reduced antiviral activity and, thus, virus replication would be enhanced. Comparable results were obtained by Sander (1969) when assaying the influence of actinomycin C on TMV multiplication in tobacco leaf disks. Similar to experiments with tobacco protoplasts and TMV (Huber, 1983) actinomycin C reduced TMV multiplication in the leaf disks; however, at low concentrations even in this natural host an enhancing effect on TMV multiplication occurred. In tobacco protoplasts these low concentrations were still inhibitory to virus replication (Huber, 1983). Yet, this single cell system reacts much more sensitively than whole plants as was observed by Junga (1978) in comparative experiments with antiviral substance and tobacco leaves and protoplasts, respectively. The observation that actinomycin D or C inhibits a very early step(s) in virus replication in protoplasts derived from a natural virus host plant led to the hypothesis that an “essential factor” necessary for first reactions after virus inoculation is produced in the cell. Recently, Mayo and Barker (1983) suggested that the uncoating of virus might be this host-dependent step, based on experiments in which the antimetabolite inhibited virus multiplication when tobacco protoplasts were inoculated with viria of TMV or TRV, but not when the nucleic acid of the two viruses was used as inoculum. So far, the results submitted t o explain the mechanism of

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actinomycin D action are still controversial, since for CPMV (Rottier et al., 1979) and AMV (Nassuth, 1982) virus RNA synthesis was inhibited, whereas virus coat protein continued to accumulate in the presence of the antimetabolite. In any case, actinomycin D is likely to prove a key substance in the attempt to elucidate an important step or steps in the specific virus/host cell relationship that may reveal whether and how resistance to virus infection is induced in plants. The fact that protoplast/virus systems are sensitive to the action of antiviral substances isolated from plants and to antimetabolites such as actinomycin D or C recommends them as tools for the screening of potential antiviral substances, e.g., well known compounds such as acetylsalicylic acid (White, 1979). Only a few efforts in this direction are known, e.g., with virazole, a nucleoside analog (Streeter et aL, 1973) which inhibits virus replication specifically and almost completely (Renaudin etal., 1982; Huber, 1983). Experiments should be conducted cautiously, because virazole is thought to be carcinogenic and teratogenic to man. The establishment of such screening systems is further facilitated by the possibility of isolating large amounts of protoplasts of comparable quality throughout the year from cell suspension cultures less laboriously and more rapidly than from leaves. Also, the possibility to assess the effect of antiviral substances on virus synthesis by serological assays contributes to such screening schemes. The decisive experiment for the evaluation of an antiviral substance, however, is the bioassay on plants. Knowledge of the mechanism of resistance of plants against virus infection and the activation of this mechanism may also assist in the explanation of the increased resistance observed in infected plants against further infection by other pathogens. McIntyre et al. (1981) reported that a hypersensitive tobacco variety, first inoculated with TMV, did not only develop resistance against further infection with other viruses, but also against fungi (Phytophthora parasitica, Peronospora tabacina), bacteria (Pseudomonas tabaci), and the reproduction of aphids (Myzuspersicae). The authors discuss a single agent to induce resistance, both systemic and persistent, in tobacco plants against various pathogens and they point out the potential of induced resistance as a means of pathogen control in the field.

VIII. CONCLUDING REMARKS AND PERSPECTIVES The advancement of knowledge has made protoplasts and plant tissue cultures a multipurpose system and a most workable tool in plant virologi-

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cal investigations, ranging from intricate molecular aspects like the role of a messenger RNA for TMV coat protein (e.g., Ogawa etal., 1983) to the adaptation of protoplasts and tissue cultures for the purpose of screening 1983), antiviral substances (e.g., Mertes and Sander, 1981; Sander etal., from tissue culture as a means for the propagation of viroids (e.g., Muhlbach and Sanger, 1981)to the study of virus resistance (Foxe and Prakash, 1983). From the original source of protoplasts, tomato fruit and tobacco leaves, modified isolation methods and new enzymes (Davey, 1983) have since extended the experimental scope to protoplasts from lower plants such as algae (e.g., Berliner etal., 1978), fungi (e.g., Hashib and Yamada, 1982), mosses (e.g., Jenkins and Cove, 1983), ferns (e.g., Partanen, 1981), and many species of higher plants including gramineae (Dale, 1983), recalcitrant to protoplast isolation for a long time, including their culture and regeneration. The improvement of the procedure for inoculation of protoplasts with viria, viral nucleic acid, and viroids and the improvement of conditions that promote their replication, as well as the increased knowledge of steps involved in both processes, seem to open an intriguing avenue of investigation in gentechnology: the use of virus as vehicle for genetic information heterologous to the cell. No such experiments have yet been reported, but efforts in this direction were made with another plant pathogen, the plastumefaciens (Davey etal., 1980). With virus the mid of Agrobacterium transfer of genetic information could be achieved by encapsulating foreign nucleic acid in the virus coat protein as suggested earlier (Zaitlin and Beachy, 1974) or by introducing foreign DNA segments into the genome of a DNA plant virus by cloning and its subsequent encapsulation in virus coat protein or liposomes. A possible advantage of a plant virus as vehicle for genetic material would be the virus’ adaptation to this plant and also the plants’, and respectively the protoplasts’ faculty to support the replication of the virus in principle. In this light the regeneration of a plant from such treated protoplasts would gain importance with regard to improvement of crop plants. Protoplast culture and plant regeneration of cereals, legumes, and other recalcitrant crops have been reviewed by Dale (1983) and application of protoplast technology in agriculture by Cocking (1983). Another interesting application of protoplast technology would be the investigation of the influence of virus infection on the production of antibiotics by fungi. Of Penicillium chrysogenum strains are known that carry virus-like particles (VLP). Aberle (1975) found that such a strain showed up to 10-fold higher antibiotic activity against Bacillus subtilis than the virus-free strain. Grabski (1980), working with the same strains, traced this effect to an additional antibiotic in the VLP-carrying strain. Proto-

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plasts were produced from the virus-free strain but proved recalcitrant to inoculation with VLPs. However, the advancement in techniques will facilitate investigating whether virus infection enhances production of secondary metabolites and/or leads to new antibiotics. Protoplasts of filamentous fungi in genetics and metabolite production (Ann6, 1983), recent advances in protoplast methodology for antibiotic-producing Streptomyces (Baltz, 1983),as well as current questions of gene transfer via protoplast fusion (Ferenczy, 1983) have been recently reviewed, but not under strictly virological aspects. Of the monolayer cultures, plant protoplasts compare well with animal and insect cells and bacteria in their usefulness to study biochemical steps in virus and viral nucleic acid replication. As a matter of fact, they are the only means available for doing so. But contrary to animal cells that are surrounded by a cell wall regardless of their existence as tissue or in single cell culture, protoplasts are plant cells without a cell wall. Such processes as ingress of virus into cells in plant tissue, or adsorption of virus to yet hypothetical receptors on or closely connected with the cell wall cannot be studied in protoplasts. It should also be considered that the stressing culture conditions may cause an altered physiological state in protoplasts compared to that of cells in plants. Here, suspension cultures have gained importance, even more so since the synchronizing effect of temperature shifts on virus inoculation and replication has been reported (e.g., Wu and Murakishi, 1979). With regard to synchronous virus replication in protoplasts, consideration should be given when one-step growth curves are established by the criterion of protoplasts stained with fluorescent antibodies. If efficiency of infection increases up to 60 hours after inoculation, this could indicate that virus replication and accumulation of coat protein is not absolutely synchronized. In describing infection systems, instead of the term “pg” of virus or nucleic acid/number of protoplasts, usually employed in virus/protoplast work, the term “multiplicity of infection” (number of viria or nucleic acid molecules/number of protoplasts) is recommended whenever possible, as is the habit in work with animal viruses and bacteriophage, for better comparison of results. Since protoplasts opened the possibility to investigate on similar experimental lines, as with monolayer cultures of zoological and bacterial cells, comparison of plant and animal virus and bacteriophage in efficiency of infection with regard to multiplicity of infection, rate of virus synthesis, appearance of proteins, and other quantitative and biochemical aspects would be interesting, in order to observe differences and similarities. On this ground new hypotheses could evolve.

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ACKNOWLEDGMENTS We wish to thank Drs. Dale, Davey, Loebenstein, Makins, Mayo, Muhlbach, Sarkar, and Takebe for providing us with information in press or prior to publication.

REFERENCES Aberle, H. (1975). Diploma Thesis, Universitiit Tiibingen (Biology), Federal Republic of Germany. Alblas, F., and Bol, J. F. (1977). J. Gen. Virol. 3 6 , 175-185. AnnB, J. (1983). In “Protoplasts 1983,” Proc. Int. Protoplast Symp., 6th, 1983,Birkhaeuser, Basel. Aoki, S., and Takebe, I. (1969). Virology 39,439-448. Arcioni, S., Davey, M. R., Dos Santos, A. V. P.,and Cocking, E. C. (1982). Z. PfEanzenphysiol. 106,105 - 110.

Bajet, N. B., and Goodman, R. M. (1981). Phytopathology 71,201. Baltz, H. R. (1983). I n “Protoplasts 1983,” Proc. Int. Protoplast Symp., 6th, 1983, Birkhaeuser, Basel. Barker, H., and Harrison, B. D. (1977). J. Gen. Virol. 3 5 , 125-133. Barker, H., and Harrison, B. D. (1978). J. Gen.Virol. 40,647-658. Barnett, A., Hammond, J., and Lister, R. M. (1982). J. Gen. Virol. 5 7 , 397-401. Beachy, R. N., and Murakishi, H. H. (1971). Phytopathology 61,877-878. Beachy, R. N., and Murakishi, H. H. (1973). Virology 65,320-328. Beachy, R. N., and Murakishi, H. H. (1976). I n Vitro 12,517-520. Beier, H., and Bruening, G. (1975). Virology 64,272-276. Beier, H., and Bruening, G .(1976). Virology 7 2 , 363-369. Berliner, M., Wood, N. L., and Damico, J. (1978). Protoplasma 96,39-46. Bilkey, P. C., and Cocking, E. C. (1981). Z. Pfinzenphysiol. 105,285-288. Bogers, R. J. (1973). Colloq. Int. C. N. R. S. 212,397-401. Branch, A., Robertson, H. D., Greer, C. H., Gegenheimer, P., Peebles, C., and Abelson, J. (1982). Science 2 1 7 , 1147-1149. Burgess, J., Motoyoshi, F., and Fleming, E. N. (1973). Plantu 112,323-332. Carlberg, I., Glimelius, K., and Eriksson, T. (1983). I n “Protoplast 1983,” Proc. Int. Protop l a t Symp., 6th, 1983, Birkhaeuser, Basel. Cassells, A. C., and Barlass, M. (1976). Physiol. Plant. 3 7 , 239-246. Cassells, A. C., and Barlass, M. (1978). Virology 87, 459-462. Cassells, A. C., and Cocker, F. M. (1980). 2.Naturforsch. C 35,1075-1061. Cassells, A. C., and Gatenby, A. A. (1975). Z. Naturforsch. C 30, 696-697. Chiu Ben-Sin, and Tien Po (1982). J. Gen. Virol. 68, 323-327. Clark, M. F., and Adams, A. N. (1977). J. Gen. Virol. 34,475-483. Cocking, E. C. (1960). Nature (London) 187,962-963. Cocking, E. C. (1970). Int. Rev. Cytol. 28,89-124. Cocking, E. C. (1977). Znt.Rev. Cytol. 48,323-343. Cocking, E. C. (1983). In “Protoplasts 1983,” Proc. Int. Protoplast Symp., 6th, 1983, Birkhaeuser, Basel. Cocking, E. C., and Pojnar, E. (1969). J. Gen. Virol. 4, 305-312. Coutts, R. H. A., and Cocking, E. C. (1972). J. Gen.Virol. 1 7 , 289-294. Coutts, R. H. A., and Wood, K. R. (1976). Arch. Virol. 5 2 , 59-69.

PROTOPLASTS IN PLANT VIRUS RESEARCH

259

Dale, P. (1983). In “Protoplasts 1983,” Proc. Int. Protoplast Symp., 6th, 1983, Birkhaeuser, Basel. Davey, M. R. (1983). In “Protoplasts 1983,” Proc. Int. Protoplast Symp., 6th, 1983, Birkhaeuser, Basel. Davey, M. R., Cocking, E. C., Freeman, J., Pearce, N., andTudor, I. (1980). PZantSci. Lett. 18,307-313.

Dawson, J. R. O., Dickerson, P. E., King, J. M., Sakai, F., Trim, A. R. H., and Watts, J. W. (1978). 2. Naturforsch. C 33, 548-551. Dawson, W. O., and Schlegel, D. E. (1976). Phytopathology 66,177-181. Engler, D. E., and Grogan, R. G. (1983). PlantSci. Lett. 22, 223-229. Ferenczy, L. (1983). In “Protoplasts 1983,” Proc. Int. Protoplast Symp., 6th, 1983, Birkhaeuser, Basel. Fowke, L. C., Bech-Hansen, C. W., Constabel, F., and Gamborg, 0. L. (1974). Protoplasma 81,189-203.

Foxe, M. J., and Prakash, J. (1983). In “Protoplast 1983,” Poster Proc. 6th Int. Protoplast Symp. Birkhaeuser, Basel. Fraley, R. T., Dellaporta, S. L., and Papahadjopoulos, D. (1982). Proc. Natl. Acad. Sci. U.S.A. 7 9 , 1859-1863. Franssen, H., Goldbach, R., Broekhuijsen, M., Moerman, M., and Van Kammen, A. (1982). J . Virol. 4 1 , 8-17. Frisch-Niggemeyer, W., and Reddi, K. K. (1957). Biochim. Biophys. Acta 26,40-46. Fritsch, C., Mayo, M. A., and Murant, A. F. (1978). J.Gen.Virol. 4 0 , 587-594. Fukunaga, Y., Nagata, N., and Takebe, I. (1981). Virology 113,752-760. Fukunaga, Y., Nagata, N., Takebe, I., Kakehi, T., andMatsui, C. (1983). Exp.Cell Res.1 4 4 , 181- 189.

Furusawa, I., and Okuno, T. (1978). J. Gen.Virol. 4 0 , 489-492. Gamborg, 0.L., Kao, K. N., Miller, R. A., Fowke, L. C., and Constabel, F. (1973). Colloq. Int. C.N.R.S. 2 1 2 , 155-161. Goldbach, R., Rezelman, G., and Van Kammen, A. (1980). Nature(London) 286,297-300. Gonda, T. J., and Symons, R. H. (1979). J . Gen.Virol. 45,723-736. Grahski, C. (1980). Diploma Thesis, Universitat Tubingen (Biology), Federal Republic of Germany. Haber, S., Ikegami, M., Bajet, N. B., and Goodman, R. M. (1981). Nature(London)2 8 9 , 324- 326.

Harrison, B. D., Hutcheson, A. M., and Barker, H. (1977). J. Gen. Virol. 36,535-539. Hashib, T., and Yamada, M. (1982). Phytopathology 72,849-853. Heyn, R. F., and Schilperoort, P. A. (1973). Colloq. Int. C.N . R. S. 212,385-395. Hibi, T., Yora, K., and Asuyama, H. (1968). Ann.Phytopathol. Soe. Jpn. 34,375. Hibi, T., Rezelman, G . ,and Van Kammen, A. (1975). Virology 6 4 , 308-318. Hirth, L., andDurr, A. (1971). Colloq. Int. C. N . R. S. 193,481-498. Howell, S. H., and Hull, R. (1978). Virology 8 6 , 468-481. Huher, 1. (1983). Diploma Thesis, Universitat Tubingen, (Biology), Federal Republic of Germany. Huher, R., Rezelman, G., Hibi, T., andVan Kammen, A. (1977). J. Gen.Virol. 34,315-323. Huber, R., Hontelez, J., and Van Kammen, A. (1981). J. Gen.Virol. 5 5 , 241-245. Hull, R. (1978). Virology 89, 418-422. Jarvis, N. P., and Murakishi, H. H. (1980). J. Gen.Virol. 48,365-376. Jenkins, G. I., and Cove, D. J. (1983). Pluntu 1 5 7 , 39-45. Jensen, R. G., Francki, R. I. B., and Zaitlin, M. (1971). Plant Physiol. 4 8 , 9-13. Jones, R. A., Rupert, E. A., and Barnett, 0. W. (1981). Phytopathology 7 1 , 116-119.

260

EVAMARIE SANDER AND GABRIELE MERTES

Junga, U. (1978). Diploma Thesis, Universitat Tubingen (Biology), Federal Republic of Germany. Kao, K. N., Gamborg, 0. L., Miller, R. A., and Keller, W. A. (1971). Nature (London)232, 124.

Kassanis, B. (1967). Methods Virol. 1,537-566. Kassanis, B., White, R. F., and Woods, R. D. (1975). J. Gen. Virol. 28, 185-191. Kassanis, B., White, R. F., Turner, R. H., and Woods, R. D. (1977). Phytopathol. 2. 88, 215-228.

Kikkawa, H., Nagata, T., Matsui, C., and Takebe, I. (1982). J . Gen. Virol. 63, 451-456. Kikuchi, Y., Tyc, K., Filipowicz ,W., Sanger, H. L., andGross, H. J. (1982). Nucleic AcidRes. 10,7521-7529.

Koike, M., Hibi, T., and Yora, K. (1976). Ann. Phytopathol. SOC.Jpn. 4 2 , 105. Koike, M., Hibi, T., and Yora, K. (1977). Virology 83,413-416. Kubo, S., and Takanami, Y. (1979). J. Gen. Virol. 42, 387-398. Kubo, S., Harrison, B. D., and Barker, H. (1975). J. Gen. Virol. 2 8 , 255-257. Kubo, S.,Robinson, D. J., Harrison, B. D., and Hutcheson, A. M. (1976). J. Gen. Virol. 3 0 , 287-298.

Larkin, P. J. (1976). Phnta 1 2 8 , 213-216. Leonhard, D. A., and Zaitlin, M. (1982). Virology 117,416-424. Lesney, M. S., and Murakishi, H. H. (1981). J. Gen. Virol. 6 7 , 387-395. Loebenstein, G., and Gera, A. (1981). Virology 1 1 4 , 132-139. Loebenstein, G., Gera, A., Barnett, A., Shabtai, S., and Cohen, J. (1980). Virology 1 0 0 , 110-115.

Loesch-Fries, L. S., and Hall, T. C. (1980). J. Gen. Virol. 4 7 , 323-332. Lurquin, P. F., and Hotta, Y. (1975). Plant Sci. Lett. 5 , 103- 112. McIntyre, J. L., Dodds, J. A., and Hare, J. D. (1981). Phytopathology 71,297-301. Maekawa, K., Furusawa, I., and Okuno, T. (1981). J. Gen. Virol. 5 3 , 353-356. Makins, J. F. (1983). I n “Protoplasts 1983,” Proc. Znt. Protoplast Symp., 6 t h 1983, Birkhaeuser, Basel. Matthews, R. E. F. (1981). “Plant Virology,” 2nd ed. Academic Press, New York. Maule, A. J., Boulton, M. I., Edmunds, C., and Wood, K. R. (1980). J. Gen. Virol. 4 7 , 199-204.

Mayo, M. A. (1978). Znteruirology 9, 184-188. Mayo, M. A. (1982). Znteruirology 17, 240-246. Mayo, M. A., and Barker, H. (1983). J. Gen. Virol. 6 4 , 1775-1780. Mayo, M. A., and Roberts, I. M. (1979). J. Gen. Virol. 44,691-698. Mertes, G. (1983). Dissertation, Universitat Tiibingen (Biology), Federal Republic of Germany. Gent 46,1019Mertes, G., and Sander, E. (1981). Meded. Fac. Landbouwwet., Rijksuniu. 1029.

Meyer, Y. (1974). Protoplasma 81,363-372. Morris-Krsinich, B. A. M., Hull, R., and Russo, M. (1979). J. Gen. Virol. 4 3 , 339-347. Motoyoshi, F., and Hull, R. (1974). J. Gen. Virol. 24,89-99. Motoyoshi, F., and Oshima, N. (1968). Jpn. J. Microbiol. 12,317-320. Motoyoshi, F., and Oshima, N. (1975). J. Gen. Virol. 2 9 , 81-91. Motoyoshi, F., and Oshima, N. (1976). J. Gen. Virol. 32,311-314. Motoyoshi, F., and Oshima, N. (1979). J. Gen. Virol. 44, 801-806. Motoyoshi, F., Bancroft, J. B., Watts, J. W., and Burgess, J. (1973). J. Gen. Virol. 2 0 , 177- 193.

Motoyoshi, F., Watts, J. W., and Bancroft, J. B. (1974a). J. Gen. Virol. 2 5 , 245-256. Motoyoshi, F., Bancroft, J. B., and Watts, J. W. (197413). J. Gen. Virol. 25,31-36.

PROTOPLASTS IN PLANT VIRUS RESEARCH

261

Motoyoshi, F., Hull, R., and Flack, J. H. (1975). J. Gen.Virol. 27, 263-266. Mozes, R. (1980). Ph.D. Thesis, Hebrew University of Jerusalem. Miihlbach, H. P. (1982). Curr. Top. Microbiol. Immunol. 99, 81-129. Muhlbach, H. P . (1983). In “Protoplasts 1983,” Proc. Int. Protoplust Symp., 6th, 1983, Birkhaeuser, Basel. Muhlbach, H. P., and Sanger, H. L. (1977). J . Gen.Virol. 35, 377-386. Miihlbach, H. P., and Sanger, H. L. (1979). Nature (London)278, 185-188. Miihlbach, H. P., and Sanger, H. L. (1981). Biosci Rep.1, 79-87. Muhlbach, H. P., Faustmann, O., and Sanger, H. L. (1983). Plant Mol. Biol. 2,239-247. Murakishi, H. H., Hartmann, J. X., Beachy, R. N., and Pelcher, L. E. (1970). Virology 4 1 , 365- 367. Murakishi, H. H., Hartmann, J. X., Beachy, R. N., and Pelcher, L. E. (1971). Virology 43, 62-72. Nagata, T., andIshii, S. (1979). Can. J. Bot. 57, 1820-1823. Nagata, T., Okada, K., Takebe, I., and Matsui, C. (1981). Mol. Gen.Genet. 184, 161-165. Nassuth, A. (1982). Ph.D. Thesis, Rijksuniversiteit, Leiden. Nassuth, A., Alblas, F., and Bol, J. F. (1981). J . Gen.Virol. 53, 207-214. Nassuth,A.,Alblas,F.,VanderGeest, J. M.C.,andBol, J.F. (1983). Virology124,517-524. Negrutiu, J., Beetfink, F., and Jakobs, M. (1975). Plant Sci. Lett. 5,293-304. Ogawa, M., Sakai, F., and Takebe, I. (1983). Phytopathol. 2.107, 146- 158. Okuno, T., and Furusawa, I. (1978a). J. Gen.Virol. 38, 409-418. Okuno, T., and Furusawa, I. (1978b). J. Gen.Virol. 39, 187-190. Okuno, T., and Furusawa, J . (1978~).J . Gen.Virol. 41,63-75. Okuno, T., and Furusawa, I. (1979). Virology 99, 218-225. Otsuki, Y., and Takebe, I. (1969). Virology 38,497-499. Otsuki, Y., and Takebe, I. (1972). Virology 49, 188- 194. Otsuki, Y., and Takebe, I. (1973). Virology 53,433-438. Otsuki, Y., Takebe, I., Honda, Y., and Matsui, C. (1972). Virology 4 9 , 188-194. Otsuki, Y., Takebe, I., Honda, Y., Kajita, S., and Matsui, C. (1974). J. Gen.Virol. 22, 375-385. Partanen, C. R. (1981). In Vitro 17, 77-80. Paszkowski, J., Shinshi, H., Koenig, I., Lazar, G. B., Hohn, T., Mandak, V., and Potrykus, I. (1983). In “Protoplast 1983,”Poster Proc. 6th Int. Protoplast Symp. Birkhaeuser, Basel. Patnaik, G., and Cocking, E. C. (1982). Z. Pflantenphysiol. 107, 41-45. Pelcher, L. E., Murakishi, H. H., and Hartmann, J. X. (1972). Virology 47, 787-796. Power, J. B., and Cocking, E. C. (1970). J. Ezp.Bot. 21,64-70. Premecz, G., Olah, T., Gulyas, A., Nytrai, A., Palfi, G., and Parkas, G. L. (1977). Plant Sci. Lett. 9, 195-200. Premecz, G., Ruzicska, P., Olah, T., and Farkas, G. L. (1978). Planta 141,33-36. Rao, D. V., and Hiruki, C. (1978). J . Gen. Virol. 38,303-312. Renaudin, J., BovB, J . M., Otsuki, Y., and Takebe, I. (1975). Mol. Gen.Genet. 141,59-68. Renaudin, J., Mouches, C., Brochard, M., Gandar, J., and BovB, J . M. (1981). Proc. Int. Congr. Virol., 5th, 1981 Abstract P20/07. Renaudin, J., Mouches, C., and BovB, J. M. (1982). Ann.Virol. 1 3 3 , 295-314. Rezelman, G., Franssen, H. J., Goldbach, R. W., Ie, T. S., and Van Lammen, A. (1982). J. Gen.Virol. 60, 335-342. Riesterer, C., and Adam, G. (1981). Proc. Int. Congr.Virol., 5th, 1981 Abstract P20/08. Rollo, F., and Hull, R. (1982). J . Gen.Virol. 60, 359-363. Rose, R. J. (1980). Aust. J. Plant. Physiol. 7, 713-725. Rottier, P. J. M., Rezelman, G.,and Van Kammen, A. (1979). Virology 92, 299-309. Sander, E. (1969). J . Gen.Virol. 4, 235-244.

262

EVAMARIE SANDER AND GABRIELE MERTES

Sander, E., Gras, J., Mertes, G., and Huber, I. (1983). Zn “Protoplast 1983,” Poster Proc. 6th Int. Protoplast Symp., Birkhaeuser, Basel. Sanger, H. L. (1982). Encycl. Plant Physiol. New Ser. 14 B . Sarkar, S. (1977). Methods Virol. 6,435-456. Sarkar, S., Upadhya, M. D., and Melchers, G. (1974). Mol. Gen. Genet. 1 3 5 , l - 9 . Sawhney, R. K., Adams, W. R. J., Tsang, J., and Galston, A. W. (1977). Plant Cell Physwl. 18,1309-1317.

Schilde-Rentschler, L. (1972). 2.Naturforsch. B 2 7 , 208-209. Schwenk, F. W., Pearson, C. A., and Roth, M. R. (1981). Plant Sci.Lett. 2 3 , 153- 155. Sela, I. (1981). Adu. Virus Res. 26, 201-237. Sela, I., and Weissbach, A. (1983). Zn “Protoplast 1983,” Poster Proc. 6th Int. Protoplast Symp. Birkhaeuser, Basel. Shabtai, S., Gera, A., and Loebenstein G. (1982). Plant Sci. Lett. 2 4 , 157-161. Shepard, J. F. (1975). Virology 66, 492-501. Shepard, J. F., and Totten, R. E. (1977). Plant Physiol. 60,313-316. Siegel, A., Hari, V., and Kolacs, K. (1978). Virology 8 5 , 494-503. Smith, M., and McCown, B. (1983). Plant Sci. Lett. 28, 1 4 9 - 156. Spencer, D. F., and Kimmins, W. C. (1969). Can. J.Bot. 47,2049-2050. Spiesmacher, E., Muhlbach, H. P., Schnolzer, M., Haas, B., and Sanger, H. L. (1983). Biosci. Rep. 3,767-774. Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell, R. W., Bauer, R. J., Robins, R. K., and Simon, L. N. (1973). Proc. Natl. Acad. Sci. U.S.A. 70,1174-1177. Sugimura, Y., and Ushiyama. R. (1975). J. Gen. ViroI. 29,93-98. Sultzinski, M. A., and Zaitlin, M. (1982). Virology 1 2 1 , 12-19. Suzuki, M., and Takebe, I. (1976). 2.Pflanzenphysiol. 78,421-433. Suzuki, M., and Takebe, I. (1978). 2.Pfknzenphysiol. 89,297-311. Szoka, F., and Papahadjopoulos, D. (1980). Annu. Reu.Biophys. Bioeng. 9,467-508. Takanami, Y., Kubo, S., and Imaizumi, S. (1977). Virology80,376-389. Takebe, I. (1975). Annu. Reu. Phytopathol. 13,105-125. Takebe, I. (1980). Symp. Biol. Hung.22,431. Takebe, I. (1983). Znt. Reu. Cytol. 16,89-111. Takebe, I., and Otsuki, Y .(1969). Proc:Natl. Acad. Sci. U.S.A. 64,843-848. Takebe, I., Otsuki, Y., and Aoki, S. (1968). Plant Cell Physiol. 9, 115-124. Uchimiya, H., and Murashige, T. (1974). Plant Physwl. 54,936-944. Van Loon, L. C., and Van Kammen, A. (1970). Virology40, 199-211. von Arnold, S., and Eriksson, T. (1977). Physiol. Plant. 3 9 , 257-260. Wallin, A., Glimelius, K., and Eriksson, T. (1977). Physiol. Plant. 40,307-311. Watts, J. W., and Dawson, J. R. 0. (1980). Virology 105,501-507. Watts, J. W., Motoyoshi, F., and King, J. (1974). Ann. Bot. (London)[N. S.] 38,667-671. Watts, J. W., Cooper, D., and King, J. M. (1975). Modif. Znf. Content Plant Cells,John Znnes Symp. 2nd 1974 p. 119. White, J. L., Wu, F. S., and Murakishi, H. H. (1977). Phytopathology 67,60-63. White, R. F. (1979). Virology 99, 410-412. Wilson, H. M., and Street, H. E. (1975). Ann. Bot. (London) [N. S.] 39,671-682. Wilson, T., Papahadjopoulos, D., and Taber, R. (1979). Cell 1 7 , 77-84. Wu, F. S., and Murakishi, H. H. (1978). Phytopathology 68,1389-1392. Wu, F. S., andMurakishi, H. H. (1979). J. Gen. Virol. 4 5 , 149-160. Yamaoka, N., Furusawa, I., and Yamamoto, M. (1982). Virology 122,503-505. Zaitlin, M., and Beachy, R. N. (1974). Adu.Virus. Res. 1 9 , 1-35. Zelcer, A., Zaitlin, M., Robertson, H. D., and Dickson, E. (1982). J. Gen. Virol.59,139- 148.

ADVANCES IN VIRUS RESEARCH. VOL. 29

VIBRIOPHAGES AND VIBRIOCINS: PHYSICAL. CHEMICAL. AND BIOLOGICAL PROPERTIES

.

S . N Chatterjee Biophysics Division Saha Institute of Nuclear Physics Calcutta. India

.

M Maiti Indian Institute of Chemical Biology Calcutta. India

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction to the Host Bacteria. the Vibrios . . . . . . . . . . . B. Historical Background of Vibriophage Research . . . . . . . . . . I1. Biological Properties of Vibriophages . . . . . . . . . . . . . . . . . A . HostRange . . . . . . . . . . . . . . . . . . . . . . . . . . B . Plaque Morphology . . . . . . . . . . . . . . . . . . . . . . . C. Phage Adsorption . . . . . . . . . . . . . . . . . . . . . . . D. Intracellular Phage Multiplication . . . . . . . . . . . . . . . . E . Serological Properties . . . . . . . . . . . . . . . . . . . . . . F. Mode of Action of the Phage 4149 on the Classical and El Tor Biotypes . . . . . . . . . . . . . . . . . . . . . . . . I11. Sensitivity of Vibriophages to Physical and Chemical Agents. . . . . . . A . Heat Inactivation . . . . . . . . . . . . . . . . . . . . . . . . B. Ultraviolet Inactivation and Photoreactivation . . . . . . . . . . . C. Photodynamic Inactivation . . . . . . . . . . . . . . . . . . . D . Inactivation by Chemicals . . . . . . . . . . . . . . . . . . . . E . pH Inactivation . . . . . . . . . . . . . . . . . . . . . . . . IV . Morphology and Other Properties of Vibriophages . . . . . . . . . . . A . Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . B. Sedimentation Properties . . . . . . . . . . . . . . . . . . . . C. Properties of Phage Components . . . . . . . . . . . . . . . . . V. Classification of Vibriophages . . . . . . . . . . . . . . . . . . . . VI . Practical Uses of Vibriophages . . . . . . . . . . . . . . . . . . . . A. Phage Typing of Cholera Vibrios . . . . . . . . . . . . . . . . . B. Differentiationofthe ClassicalandEl Tor Biotypes . . . . . . . . C. Therapeutic and Prophylactic Use . . . . . . . . . . . . . . . . D. Epidemiological Use . . . . . . . . . . . . . . . . . . . . . . VII. Lysogenic Vibriophages . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . B . Biological and Other Properties . . . . . . . . . . . . . . . . . C. Practical Use . . . . . . . . . . . . . . . . . . . . . . . . . 263

264 264 265 266 266 267 267 272 216 277 278 278 279 281 282 283 283 283 287 281 290 292 292 293 293 294 294 294 295 297

Copyright 0 1984 by Academic Press. Inc . All rights of reproduction in any form reserved. ISBN 0-12-039829-X

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VIII. Vibriocins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . B. Production and Detection . . . . . . . . . . . . . . . . . . . . C. H o s t R a n g e . . . . . . . . . . . . . . . . . . . . . . . . . . D. Morphology.. . . . . . . . . . . . . . . . . . . . . . . . . E. Physical and Chemical Properties . . . . . . . . . . . . . . . F. Mode of Action. . . . . . . . . . . . . . . . . . . . . . . . . G. Practical Use . . . . . . . . . . . . . . . . . . . . . . . . . IX. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

298 298 299 301 301 302 303 304 306

307

I. INTRODUCTION A. Introduction to the Host Bacteria, the Vibrios The genus Vibrio includes many different species of which few are pathogenic. The classic example of a pathogenic species of this genus is Vibrio cholerae, others being V.parahaemolyticus (Sakazaki et al., 1963), V.alginolyticus (Sakazaki, 1968) and certain nonagglutinable vibrios (often termed NAG vibrios). Sakazaki (1970) made a significant contribution toward classification and characterization of vibrios and defined the practical basis for the differential diagnosis of the genus Vibrios and the related genera, viz. Aeromonas, Plesiomonus, and the Enterobacteriaceae. The differentiation depended upon several important tests, e.g., the fermentation of glucose and mannitol, the production of hydrogen sulfide, oxidase, and lysine-decarboxylase, etc. Earlier studies of the antigenic structure of vibrios revealed different serological types and demonstrated the presence of heat-labile H and heat-stable 0 antigens. Gardner and Venkatraman (1935) made a significant contribution toward serological classification of the vibrios. Different workers (Bhattacharya et aL, 1971; Sakazaki, 1968, 1970; Hugh and Feeley, 1972) claimed that the species belonging to the genus Vibrio can be differentiated in accordance with their H-antigens. The particular species, V. cholerae, was again divided into six subgroups or serotypes according to their heat-stable O-antigens. Subsequently more than 50 serotypes of V. cholerae were detected (Sakazaki et al., 1970; Sakazaki and Shimada, 1975). All major epidemics and pandemics of cholera have practically been caused by strains of the same O-group (designated 0 group 1or 0 :1). However, strains belonging to other 0-groups (e.g., NAG vibrios) are also being encountered in outbreaks of cholera-like diarrheae (Barua and Burrows, 1974).

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V .cholerae was further shown to include two biotypes: V. cholerae bioclassical) and V. cholerae type classical (to be termed hereafter V. cholerae biotype El Tor (to be termed hereafter as V.cholerae El Tor). The El Tor variant was discovered as the causative agent of the cholera-like disease during the Seventh Pandemic of the disease cholera (Gallut, 19711, but the properties of these organisms were not sufficiently distinctive as to claim their identity as a separate species (Hugh, 1965a,b). Both V. cholerae classical and V.cholerae El Tor belong to the serotype 0 : 1. The vibrios of the serotype 0 :1are further differentiated serologicallyaccording to their subsidiary 0-antigens into three subgroups: INABA, OGAWA, and HIKOJIMA, the names denoting their historical origins.

B. Historical Background of Vibriophage Research The story of cholera bacteriophages started practically with the discovery of the “lytic principle” in the cholera stools by d‘Herelle (1922,1923). Subsequent works (Flu, 1924, 1925; Nobechi, 1926; Jotten, 1922) confirmed and established the existence of the bacteriophages. Large scale investigation on the cholera bacteriophage started in India in 1927 under the leadership of d’Herelle (d’Herelle and Malone, 1927) and under the auspices of the Indian Research Fund Association, which was subsequently taken up by Asheshov and others. Thirteen types of cholera bacteriophage were recognized. Types A, B, and C were described by Asheshov etal. (1930), types D, E, and F by Pasricha etal. (1932a,b),types G, H, and J by Morrison (1933), type K by Pasricha (1933), type L by Anderson (1935), type M by Pasricha etal. (1936), and type N by Pasricha etal. (1941). A variant of the type L, termed LL, was isolated and studied by White (1936, 1937). These classifications were mainly based on their reciprocal actions on the phage-resistant secondary growth of vibrios. A detailed account of these and other works on cholera bacteriophages prior to 1959 was documented by Pollitzer (1959). Systematic studies on the physical, chemical, and biological properties of the vibriophages started apparently around 1960. The studies on the vibriocins started still later. Practically no review article appeared on the vibriophages and vibriocins during the period between 1960 and 1980, except the one by Mukejee and Takeya (1974) emphasizing the epidemiological use of these agents and another on vibriocin typing by Brandis (1978). A review of the basic properties of the vibriophages (restricting to and vibriocins those infecting the pathogenic species of the genus Vibrio) as reported to date by different authors has been long overdue. The present article has been written with this objective in view and also with a

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view to update the available and relevant literature in the context of the reports of the International Committee on the Taxonomy of Viruses (Matthews, 1982). TI. BIOLOGICAL PROPERTIES OF VIBRIOPHAGES A. HostRange

Mukejee etal.(1957) isolated 30 choleraphage strains from stool samples in Calcutta and studied their lytic activity on 200 V. cholerae classical strains. On the basis of the lytic patterns, the 30 phages could be differentiated into four distinct groups. Studies on the host range of these phages were extended further by including V.cholerae El Tor, NAG vibrios, and Escherichia coli strains (Mukerjee etal., 1957, 1959, 1960). The results could be summarized as follows: 1. All the NAG vibrios were insensitive to group I1 choleraphages. 2. Group IV phages were totally inactive against any of the V.choleraeE1 Tor strains while they were universally lytic for all V. cholerae classical strains. 3. None of the E.coli strains tested was susceptible to any choleraphage.

Subsequently, the phages lysing the V.cholerae El Tor strains were also differentiated into 5 groups in accordance with their host range (Basu and Mukerjee, 1968). Lee and Furniss (1981) made an extensive study of the sensitivity of over 1500 vibrio strains to 14 phage strains, including Mukejee’s cholera phages I, 11,111,and IV, the five El Tor phages of Basu and Mukerjee (1968), and a selection of host range variants and contaminants of them, a and /3, 25 newly isolated from sewage and water in Dacca, Bangladesh, and 11 temperate phages. This study enabled the authors (Lee and Furniss, 1981) to develop an improved phage-typing scheme. A new vibrio-infecting phage was isolated from sewage in Fukuoka by using the H218 Sm’ strain of V. cholerae classical as the propagating strain and was named FK phage (Takeya etal., 1981). This phage lysed all 25 strains of V. cholerae classical at a 10X routine test dilution (RTD) but failed to lyse all 56 strains of V.cholerae El Tor and 37 strains of NAG vibrios. V. parahaemolyticus (24 strains) and species of different bacteria, e.g., Salmonella (24 strains), Shigella (21 strains), Escherichia (8strains), Bacillus (4 strains), Micrococcus (1 strain), and Staphylococcus (21 strains) were insensitive to FK phage even at 100,000 X RTD. Koga etal. (1982) studied bacteriophages using 18strains the host ranges of 18 V. parahaemolyticus

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representing 8 0 and 10 K serotypes. These auof V. parahaemolyticus thors corroborated the finding of Baross etal.(1978) that there was no correlation between the serotypes of the vibrio and the phage lytic spectra. Not enough work has been reported to decide whether the cholera phages can cut across the lines of bacterial classification. Also the nature of host range modification arising due to the phage mutation has not been investigated adequately.

B. PlaqueMorphology Mainly three types of plaque produced by cholera bacteriophages on the lawn of the host vibrio strains were reported (1)clear plaques of diameters depending on the phage and host strains and also on other factors, (2) clear plaques with peripheral halo appearing due to the lytic action of phage induced enzymes, and (3) clear plaques with a central turbid zone produced mostly by the lysogenic choleraphages. As is widely known today, the plaque morphology is not invariantly a characteristic of the phage strain only and depends on various environmental factors (Adams, 1959; Stent, 1963; Ackermann, 1969, 1976, 1983; Ackermann etal., 1978). This was equally appreciated in cases of cholera bacteriophages by Asheshov etal. (1933) and accordingly Mukerjee (1961a) admitted failure in the classification of choleraphages from the plaque morphology alone. However, there is still no denying the fact that plaques do represent characteristics of phage and host strains and almost any mutation in a phage is likely to alter the appearance of its plaques (Adams, 1959). Mutations affecting plaque types have, however, not been studied adequately for vibriophages.

C. PhageAdsorption The first step in the interaction between a phage particle and its host cell is specific adsorption. Various aspects of the adsorption have been studied only for a few vibriophages (Table I). The adsorption kinetics was mostly found to be biphasic in nature (Fig. l),the first phase exhibiting a faster adsorption and was followed by a much slower one in the second phase. Similar biphasic adsorption kinetics was exhibited by many other phages, viz. phage 7V of Pseudomonasueruginosa (Feary etal., 19641, phage q5W of P. acidovorans (Kropinski and Warren, 1970), etc. The origin of such a biphasic adsorption process is not well understood. It may be that the phage population concerned are heterogeneous in respect of the rate and degree of their adsorption capacities. Alternatively, one might imagine a heterogeneity not in the phage population but in the host bacteria in respect of their sensitivities to the respective phages. Electron

TABLE I ADSORPTION CHARACTERISTICS OF VIBRIOPHAGES Adsorption kinetics" Rate

Constant

First phase (ml/min) X los

Second phase (ml/rnin) X 10'O

References

1.39 (31.5) 1.09 (90.0) 1.03 (50.0) 0.42 (95.0) 0.37 (60.0) 5.50

2.9

Chanda and Chatterjee (1975)

2.08

Maiti and Chaudhuri (1979)

0.95

Maiti and Chatterjee (1971)

Host

Phage strains

PL 163/10 42 4149 4149

Ph-1

VcAl a

V. cholerae strains OGAWA 154 (classical) OGAWA 154 (classical) OGAWA 154 (classical) MAK 757b (El Tor) MAK 757 (El Tor) RV 79 (El Tor)

Nature Biphasic Biphasic Biphasic Monophasic Biphasic NR

Maiti (1978a,b) 2.14

-

Figures within parentheses represent percentage adsorption; NR, not reported.

Maiti et al. (1973) Weston et al. (1973)

* Although MAK 757 does not act as host of 4149 (no phage multiplication takes place), it possesses receptors of

4149 on its surface.

VIBRIOPHAGES AND VIBRIOCINS (

I

l

l

5

10

I

269

I

100

50

-* 01

cn a

-c

B

g

10

i

3

5

15 20 Time (min)

25

FIG.1. Kinetics of adsorption of the choleraphage 4149 to untreated ( 0 )and furazolidone (0.5 ,ug/ml)-treated (0)V. cholerae OGAWA 154 cells in nutrient broth medium at 37°C (Chatterjee and Maiti, 1973b).

microscopy could not detect any heterogeneity in morphological type in the cholera phage population in any of the cases. The effects of ionic environment on the adsorption of vibriophages have not been studied adequately. Adsorption of the phages Ph-1 (Maiti etal., 1973) and PL163/10 (Chanda and Chatterjee, 1975) did not increase by any significant extent in the presence of different amounts of Ca2+or Mg2+ ions. The pH of the reaction media had a significant effect on phage adsorption. Adsorption of phage Ph-1 was significantly reduced at pH 6.4 or 9.0 (Maiti etal., 1973). For the choleraphage (6149, the overall adsorption at pH 7.4 was much greater than the corresponding values at pH 6.0 or 8.0 or 9.0 (Maiti and Chatterjee, 1970,1971). The adsorption of the phage PL163/10 remained unaffected in the pH range 7.0 to 8.0 but was greatly reduced a t pH 9.0 (Chanda and Chatterjee, 1975). There is practically no report on the requirement of any cofactor for adsorption of vibriophages. The adsorption of the phage $149 to the strain OGAWA 154 pretreated with the drug furazolidone (0.5 pg/ml, 3 hours at 37°C) was faster and greater than the control or untreated cells (Chatterjee and Maiti,

270

S. N. CHATTERJEE AND M. MAITI

1973a,b). The phage adsorption to drug-treated cells was also biphasic ml/minute for the first phase (Fig. 1). with a rate constant of 2.7 X An interesting application of the phage adsorption data was made in the determination of the length of furazolidone-treated V. cholerae cells. The drug (0.5pg/ml) was known to inhibit DNA synthesis and cell division and led to filamentation of the V. cholerae cells (Chatterjee and Raychaudhuri, 1971; Raychaudhuri et al., 1970). Since the multiplicity of infection was kept low ( 0.001), the kinetics of phage adsorption was described by the equation

-

-dp/dt = KBp (1) where K is the adsorption rate constant, B the bacterial concentration, and

p the phage concentration at time t. The adsorption rate constant was related to the diffusion constant (D) of the phage particles by Schlesinger (1932) as

K = -4nDR (2) where R is the radius of a sphere having the same surface area as that of the bacterium. Using Eqs. (1) and (2) and assuming that the rod-shaped bacteria increased only in length after drug treatment (and this was found true by electron microscopy), the length, Zd, of the drug treated cells could be obtained from the equation ld

= K$!,/KE

(3)

where K, and & are the adsorption rate constants for untreated and drug treated cells, respectively, and I, is the length of the untreated cell. Using the measured values of Kd, K,,and l,, the length (1,J of the furazolidonetreated cells was found as 21 pm, i.e., seven times the length of the untreated cells (Chatterjee and Maiti, 1973b). Direct electron microscopic examination revealed that the lengths of the furazolidone-treated V. cholerae cells ranged between 6 and 11times the length of the untreated ones (Raychaudhuri et al., 1970), which agreed satisfactorily with the phage adsorption data. The specific attachment of phage particles to host bacteria is mediated through certain macromolecules on the surface of the cells and which are known as receptors. Jesaitis and Goebel(l955) and Weidel (1958) extensively studied the nature of the phage receptors on the surface of different bacteria. The nature of the vibriophage receptors has not been extensively investigated. For the cholera phages 4149 and $2, the presence of phage receptors on the isolated cell walls and the purified lipopolysaccharides (LPS)was demonstrated (Maiti and Chatterjee, 1971;Sur et aL, 1974; Maiti et al., 1977; Maiti and Chaudhuri, 1979). Cell wall and LPS were

VIBRIOPHAGES AND VIBRIOCINS

271

isolated from the host cell followingthe methods of Keeler etal. (1966) and Westphal etal. (1952), respectively. The purity of cell wall and LPS preparations was checked by electron microscopy, ultraviolet (UV) and visible extinction (Sur and Chatterjee, 1970), and chemical analysis (Chatterjee etal., 1971; Adhikari, 1971). Adsorption of the phage 4149 to isolated cell wall and LPS followed first-order reaction kinetics resulting in 70% inactivation by cell wall within 30 minutes and 50% by LPS within 60 minutes. After treatment with 0.5%(w/v) sodium deoxycholate (SDC) for 1hour a t 37"C, cell wall as well as LPS largely lost their phage inactivating capacity. The 50%phage inactivating concentration (IC,,) of LPS increased from the normal value of 7 yg/ml to about 3.6 mg/ml after SDC treatment (Fig. 2). The loss of phage-inactivating capacity might be due to the fact that SDC dissociates LPS of gram-negative bacteria into very small units with subsequent loss of biological activity (Ribi etaL, 1966; Lindberg, 1967). The opalescence of LPS solutions disappeared almost immediately after addition of SDC. When SDC was removedby extensive dialysis, the phage-inactivating capacity of the LPS was restored significantly, the IC,, value being 570 pg LPS/ml (Maiti etal., 1977). The p g LPSirnl 10

5

2

3

15

4

5

rng LPS/rnl

FIG.2. Inactivation of the choleraphage 4149 by different amounts of lipopolysaccharides (LPS) isolated and purified from the V. chokrae cells. A, LPS from V. chokrae classical; 13,LPS from V. cholerae El Tor; A,LPS from V. chokrae classical pretreated with 0.5% (w/v) sodium deoxycholate (SDC) and nondialyzed; 0,LPS from V. chokrae classical pretreatedwith 0.5%(w/v) SDC and dialyzed. The upper scale in the abscissa refers only to the solid triangles (Maiti et al., 1977).

272

S. N. CHATTEWEE AND M. MAITI

receptors of the choleraphage $2 were also detected in the cell wall and LPS. Adsorption of this phage to isolated cell walls and isolated LPS followed first-order reaction kinetics resulting in 80 and 50%phage inactivation, respectively, within 30 minutes at 37°C (Maiti and Chaudhuri, 1979). Choleraphage $149 belongs to Mukerjee's group IV and possesses the unique property of differentiating the V. cholerae classical and V. cholerae El Tor strains. The basis of the resistance of the El Tor strains to phage 4149 has not been adequately investigated. While investigating the presence of phage 4149 receptors on V. cholerae El Tor strains, Maiti etal. (1977) reported that LPS preparations from these cells could not inactivate by any significant degree the phage 4149 within 60 minutes at 37°C. It was subsequently reported (Maiti, 1978a,b) that the phage 4149 was inactivated by El Tor vibrios. The time course of phage adsorption to the El Tor strain MAK 757 obeyed first-order reaction kinetics with a rate constant of 4.194 X lo-'' ml/minute. Also the phage 4149 was adsorbed and inactivated by the cell walls isolated from El Tor strain MAK 757 and obeyed first-order reaction kinetics with a rate constant of 3.42 X 10+ ml/minute. The nature of the phage $149 receptors on the cell wall of El Tor vibrios has not yet been identified clearly and requires further investigation.

D. Intracellular PhageMultiplication The one-step growth experiment as originally designed by Ellis and Delbruck (1939) is widely used as the basic procedure for studying phage multiplication. The characteristics of the intracellular multiplication of vibriophages as obtained by this technique are summarized in Table 11. The eclipse periods were determined by the premature lysis technique originally designed by Doermann (1951, 1952) and modified by Skhaud and Kellenberger (1956). The values of the burst size corresponding to different phage-host systems were determined by the one-step growth experiment and, in some cases, were also obtained by the single burst technique (Delbruck, 1945). Different vibriophages growing on the same bacterial strain (OGAWA 154, classical) exhibited widely different latent periods. The burst sizes corresponding to different vibriophage - host systems also varied widely. The phage VcAl exhibited an unusually large burst size as compared to the burst sizes of other vibriophages or of such well studied coliphages as T1, T4, or 1 which have average bursts of about 100 (Doermann, 1952; Lederberg and Lederberg, 1952). Both VcAl and A are temperate phages and the latent periods of both are about 40-50

273

VIBRIOPHAGES AND VIBRIOCINS TABLE I1 INTRACELLULAR GROWTH CHARACTERISTICS OF VIBRIOPHACES ~~~~~~~~~~~~~~~~~~~

~

Host Phage strains

V. cholerae

PL 163/10

OGAWA 154 (classical) OGAWA 154 (classical) OGAWA 154 (classical) MAK 757 (El Tor) OGAWA 154 (classical) Burma J. (El Tor) RV 79 (El Tor)

+2 $149

Ph-l 5

7

VcAl

strains

~

Latent period (min)

Eclipse period (min)

Rise period (min)

Average burst size (PFU/cell)

31.0

13.0

29.0

40.0

38.0

22.0

17.0

36.0

20.0

24.0

50.0

32.0

45.0

120.0 (124.0)" 58.0 (51.0)" 103.0

60.0

-

65.0

75.0

Rizvi and Monsur (1965)

60.0

-

45.0

36.0

Rizvi and Monsur ( 1965)

45.0

-

-

References Chanda and Chatterjee (1975)

600.00

Maiti and Chaudhuri (1979)

Maiti and Chatterjee (1971) Maiti etal.(1973)

Weston etal.(1973)

(470)"

Data of single burst experiment.

minutes. The reason for VcAl producing four to six times as many infectious particles per burst is not known. The intracellular multiplication of vibriophages was studied extensively only in case of the phage $149 (Maiti and Chatterjee, 1971; Maiti, 1972; Chatterjee and Maiti, 1973a,b) infecting V. cholerae classical strain OGAWA 154,both in the presence and absence of the drug furazolidone ( a synthetic nitrofuran having a wide spectrum of antimicrobial activity). The presence of the drug significantly affected the burst size (Fig. 3), phage yield (Fig. 4), and macromolecular synthesis (Fig. 5) in the phage-infected V. cholerae cells. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration, the 50%reduction being achieved by a drug concentration of 0.05pg/ml. The latent period of phage multiplication and also the eclipse period did not, however, change significantly from the control values. The phage yield of infected bacteria decreased progressively with increasing concentration of the drug. The phage yield also depended significantly on the time of addition or withdrawal of the drug (Fig. 6). If the drug was added any time within the first 25 minutes of the latent period of infection, inhibition in phage production was maximal and consistently the same irrespective of the time of addition. When the drug was added a t later stages of infection,

274

S. N. CHATTERJEE AND M. MAITI

Time (rnin)

FIG.3. One step growth experiments of the choleraphage 4149 in the host V. cholerae OGAWA 154 arranged in the presence (triangles) and absence (circles) of 0.05 fig furazolidone per ml of the growth medium at 37°C. Samples taken a t intervals were either plated immediately for PFU (0,A)or subjected to premature lysis before assay (0,A).The inset shows the effect of drug concentration on the burst size (Chatterjee and Maiti, 1973b;Maiti, 1972).

its effect on the inhibition of phage production by the infected bacteria was significantly reduced. The greater the delay in the addition of the drug, the greater was the phage yield. The study of the time courses of synthesis of DNA and RNA, as measured by the incorporation of 32Pinto the respective chemical fractions, and of protein in V. cholerae cells infected with phage (6149 (Chatterjee and Maiti, 1973a,b) and grown in the presence or absence of furazolidone presented interesting data on intracellular phage synthesis. A concentratin of 0.05pg of furazolidone per ml inhibited DNA synthesis by about 50% in phage infected cells and only by about 18%in noninfected ones, relative to the respective controls (Fig. 5). RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. The nature of inhibition of phage yield and DNA synthesis in infectedcells

275

VIBRIOPHAGES AND VIBRIOCINS

Drug concn. (vgirnl)

FIG.4. Effects of mitomycin C (e)and furazolidone (0)on the yield of the choleraphage $149 in nutrient broth medium a t 37°C by the V. cholerae OGAWA 154cells. Effects on the free phages are shown by A,mitomycin C; A,furazolidone (Maiti and Chatterjee, 1971). 9 8 7

3 2 1

0

15

45

30

60

75

Time (rnin)

FIG.5. Kinetics of 32Pincorporation in DNA of noninfected and phage $149-infected V. cholerae OGAWA 154 cells in the presence and absence of 0.5 pg furazolidone per ml of growth medium at 37°C. 0, Noninfected cells; e, noninfected cells furazolidone; A, phage-infected cells; A,phage-infected cells furazolidone (Chatterjee and Maiti, 1973b).

+

+

276

S. N. CHATTERJEE AND M. MAITI

a,

n

3

Y

250 -

-

-

-

150 -

-

200

. 0

100 -

-

-

-

3

-n LL

50

I

I

I

I

30 40 50 60 Time of addition or removal of furazolidone ( m i n ) 10

.

20

FIG.6. Effects of time of addition ( 0 )or withdrawal (0)of furazolidone on the phage yield of infected bacteria as determined by the one-step growth experiment. For addition of furazolidone within 36 minutes of infection (latent period), samples from the first growth tube were diluted at the required time into second growth tubes containing 0.05 pg furazolidone per ml. For exposure at later periods of time, several second growth tubes were prepared (beforeonset of lysis) and furazolidone was addeddirectly at the appropriate times. Removal of furazolidone was made by dilutions into furazolidone-free medium a t appropriate times. Samples (0.1 ml) from the second growth tubes were taken after 100 minutes incubation in growth medium at 37°C for assay of PFU/O.l ml of second growth tube (Chatterjee and Maiti, 1973b).

led to the conjecture that the drug was interfering with a late stage in the reproduction of infective phage particles (i.e., phage assembly).

E. Serological Properties Asheshov etal.(1933) and Pasricha etal.(1936) were among the early workers who studied the antigenic properties of choleraphages. Mukejee (1962) made a more systematic study of the choleraphages which he previously classified into four distinct groups. All the antiphage sera were found to neutralize the corresponding phages. Also, each of the sera inhibited plaque formation by phage strains belonging to the corresponding group roughly to an equal extent. Cross neutralization studies revealed that the four groups of phages were antigenically unrelated. The virulent phages (BPVcA, BPVcD, PVcl20, and ATCC2) investigated by Guice and Newman (1969) were found antigenically related. Also, the phages PVcll7 and PVc301, temperate phages from a lysogenic V.cholerae and a lysogenic El Tor vibrio, respectively, were found serologically re-

VIBRIOPHAGES AND VIBRIOCINS

277

lated. Takeya etal.(1981) revealed that the FK phage which could also differentiate V. cholerae classical and V. cholerae El Tor strains was anti(1973) studied genically different from Mukerjee’s phage IV. Weston etal. the kinetics of serum neutralization of the phage VcAl and found that this phage resembled the coliphage T1 and T5 in so far as the slow neutralization by the specific antisera was concerned. The antigenic properties of vibriophages and particularly the quantitative aspects of serum neutralization have not been adequately investigated.

F. Mode of Actionof thePhage$149on the Classical and El TorBiotypes

Mukerjee’s group IV choleraphage $149 was reported to lyse all strains of V. cholerae classical, but none of the strains of V. cholerue El Tor (Mukerjee, 1963a). This was confirmed by Takeya and Shimodori (1963) and lysis by the group IV phage, in addition to the chicken erythrocyte agglutination (Finkelstein and Mukerjee, 1963) and the polymyxin B sen1965)tests, has since been used as a sitivity (Han and Khie, 1963;Roy etal., 0 : 1in most valuable test for differentiating the two biotypes of V. cholerae laboratories. In an attempt to understand the basis of resistance of V. cholerae El Tor to the group IV choleraphage $149, Maiti etal.(1977) reported that while the lipopolysaccharide (LPS) isolated from the V. cholerae classical could inactivate the group IV phage $149, that from the biotype El Tor failed to do so. Cholera and El Tor LPS were identical in respect of chemical composition except that the hexosamine content was significantly lower and galactosamine was absent in El Tor LPS. Subsequently, Maiti (1978a,b) reported that the cell walls isolated from cholera and El Tor strains were almost equally effective in adsorping the phage #149. Also the kinetics of adsorption of the phage #149 to the whole cells of cholera and El Tor strains were comparable. The nature of the receptors of the phage 4149 on El Tor strains has thus remained uncertain. The group IV choleraphage $149 in sufficiently high titer was reported to produce some clearing, partially resemblingphage lysis, when implanted on lawns of El Tor vibrios (Monsur etaL, 1965). While the phagepropagation in classical strains was highly effective, it was ineffective in El Tor strains although it killed the El Tor vibrios (Maiti and Chatterjee, 1971; Maiti, 1972). Although the efficiency of plating (EOP) of the phage #149 differed according to the V. cholerae classical hosts used as indicator, its EOP on the El Tor strain MAK 757 was nil (Maiti and Chaudhuri, 1980). The operation of host restriction was apparently complete in the El Tor strain MAK 757. Further details of the mode of action of the phage $149 on cholera and El Tor vibrios were reported by Maiti (1978b). Evidence

278

S. N. CHATTERJEE AND M. MAITI

was obtained with 32P-labeledbacteriophage 4149 for penetration of phage DNA into both bacterial strains. In the host strain OGAWA 154, the synthesis of the phage particles occurred normally. In the El Tor strain MAK 757 the phage DNA was not degraded but its expression was blocked. The killing effect of 4149 on the El Tor strain MAK 757 was presumably due to the damage of cytoplasmic membrane, which could not be repaired under the influence of phage information. This was indicated by the blockage of cellular respiration, RNA, and protein synthesis.

111. SENSITIVITY OF VIBRIOPHAGES TO PHYSICAL AND CHEMICAL AGENTS A. Heat Inactivation

The thermal death points of vibriophages were determined rather qualitatively by Mukerjee (1961b) and were considered as one of the parameters for their identification and classification. Systematic and quantitative studies on these phages were subsequently reported by different groups of workers (Maiti and Chatterjee, 1969a, 1971, 1972; Guice and Newman, 1969; Chanda and Chatterjee, 1975; Maiti and Chaudhuri, 1979). A first order inactivation kinetics (Fig. 7) described by the equation, S,/S, = exp(- kt ) where S,, Soare the survivals (plaque forming units/ml) at times t and 0, respectively, and k, the inactivation constant, was generally obeyed at different temperatures. The inactivation constant, k and the half life, were determined for different phages. The different-

I

-

0

20

40

TIME(rnin1

FIG. 7.

60

Kinetics of thermal inactivation of the choleraphage PL163/10 at 55°C (A), 60°C C( 0 )(Chanda and Chatterjee, 1975).

(A), 65°C (0),and 70

VIBRIOPHAGES AND VIBRIOCINS

279

thermodynamic parameters, viz. change in free energy of activation (AF), change in enthalpy of activation ( A H )and , change in entropy of activation (AS) were also determined by using the following relations where T is absolute temperature:

+ log T - log k ) (AH/2.3RT) + constant

A F = 4.58T (10.318 log k =

AS = (AH - AF)/T

(4)

(5) (6)

The estimated values of the inactivation and thermodynamic parameters of activation of different vibriophages are presented in Table 111. For comparison, this table also presents some inactivation data of the coliphage, T2. Many of the vibriophages have thermodynamic parameters similar to those of coliphage T2. The El Tor phage Ph-1 and the temperate phage PVc301 presented entropy (AS’) values much smaller than others, which possibly indicated that the proteins in these phages were of a different nature or of different degree of order. It may be noted in this respect that the El Tor phage Ph-1 exhibited a much greater degree of heterogeneity in size and shape than is usually done by any bacteriophage (Maiti and Chatterjee, 1969a, 1972). Weston et al. (1973) reported that the temperature phage VcAl was stable at 4°C but sensitive to temperatures above 20°C. Takeya et al. (1965b) also reported that the “kappatype” phage obtained from the El Tor vibrio strain JE5 was more heat sensitive than Mukerjee’s Vxholerae classical typing phages. The inactivation and thermodynamic parameters of these temperate phages (VcA1; “kappa”) were not reported.

B. Ultraviolet Inactivation and Photoreactivation UV inactivation characteristics of vibriophages have not been studied adequately. The inactivation kinetics of the cholera phage PL 163/10 was reported by Chanda (1977) and Chatterjee and Chanda (1976). The 254 nm UV light fast inactivated this phage and the inactivation apparently followed a single hit kinetics. The 37% survival dose and the velocity constant of the inactivation kinetics were 210 ergs/mm2 and 0.096 sec-’, respectively. No significant change in the inactivation kinetics resulted when the pH of the medium was changed from 6.0 to 9.0. Single hit inactivation kinetics was also exhibited by the temperate phage VcAl (Weston et al., 1973) and the kappa phage (Takeya et al., 1965b). Furazolidone protected the choleraphage PL 163/10 against UV inactivation (Chatterjee and Chanda, 1976, 1979). The UV protection increased with increasing concentration of furazolidone and with p H in-

280

S. N. CHATTERJEE AND M. MAITI TABLE I11

THERMODYNAMIC PROPERTIES OF DIFFERENT VIBRIOPHAGES Phage strains Virulent PL 163/10

42

4149

Ph-1

BPVcA BPVcD PVcATCC2 Temperate PVcll7 PVcl20 PVc301

Coliphage T2

Temperature ("C)

Halflife (min)

55 60 65 70 55 60 65 70 50 60 65 50 55 60 56 65 56 65 56 65

39 12 4.5 1.0 42.5 8.0 4.0 1.5 678.0 34.5 8.0 11.0 5.5 2.5 31.0 7.0 50.7 7.1 36.0 5.3

56 65 56 65 45 56 65

255 11.3 37.7 7.7 75.0 5.8 2.0

60 64 67 68 70 74

k (min-') 0.018 0.060 0.154 0.738 0.016 0.085 0.196 0.570 0.001 0.020 0.091 0.062 0.126 0.257

AH

AF

(cal/mole)

(cal/mole)

57050

52400

70024

30666 57000

55180 54990

55400 55700

42110

71700

AS

(cal/mole/ degree)

21910 21440 21140 20410 21960 21216 20983 20575 23440 22250 21530 20750 20580 20480 24420 24220 24800 24200 24600 23900

107.1 106.9 106.3 106.9 92.81 92.24 92.95 92.79 144.2 143.4 143.5 30.70 30.75 30.58 98b

25600 24500 24620 24260 24220 23400 25460

90b

25700 24800 24600 23800 23600 23600

92b 92b

94b

56b

138 139 139 140 140 139

References' 1 1 1 1 2 2 2 2 3 3 3 4

4 4 5 5 5 5 5 5

5 5 5 5 5 5 5 6 6 6 6 6 6

a (1)Chanda and Chatterjee (1975); (2) Maiti and Chaudhuri (1979); (3) Maiti and Chatterjee (1971); (4)Maiti and Chatterjee (1969a) and Maiti et ol.(1973); (5) Guice and Newman (1969); (6) Pollard and Reaume (1951). Average values presented.

VIBRIOPHAGES AND VIBRIOCINS

281

creasing between 7 and 9. The protection was also greater in 5 mM Tris-HC1 buffer than in phosphate-buffered saline at any pH between 7 and 9. Hg2+ and tilorone (broad spectrum antiviral compound having antitumor activity and binding to DNA with a specificity for A-T base pairs) also afforded protection to these phages against UV inactivation. When present along with furazolidone, Hg2+ or tilorone afforded extra protection which increased with increasing concentration of one or more of these agents. UV-inactivated choleraphage PL 163/10 underwent a maximum of about 36% photoreactivation within the host V. cholerae OGAWA 154 cells after 30 minutes of exposure to visible light and with pH of the medium ranging between 7.5 and 8.0 (Chanda and Chatterjee, 1976a). The degree of photoreactivation gradually decreased when the pH was changed to either side of the above range. Mg2+atconcentrations up to 0.3 M had no significant effect on the degree of photoreactivation. The dose reduction factor (DRF) due to photoreactivation of the UV-inactivated phage P L 163/10 was 0.68. These phages underwent not more than 5% liquid holding recovery within the host cells.

C. Photodynamic Inactivation Very few data are available on the photodynamic inactivation of vibriophages. Only the phage P L 163/10 has been investigated to some significant extent (Chatterjee and Chanda, 1976,1979). This phage is not inactivated either by visible light alone or by furazolidone or acridine orange in the dark. Furazolidone and also acridine orange led to the photodynamic inactivation of the phage PL 163/10 on exposure to white light. The inactivation increased with increasing concentration of furazolidone or acridine orange and also with pH increasing between 7 and 9. Mercury (Hg2+)or tilorone, a broad spectrum antiviral compound having antitumor activity and binding with DNA with a specificity for A-T base pairs (Chandra et al., 1972a,b,c),did not produce any photodynamic inactivation of these phages but afforded protection to them against furazolidone-induced photodynamic inactivation. The protection increased with increasing concentration of Hg2+ or tilorone a t a fixed concentration of furazolidone but decreased in the reverse cases. Chatterjee and Chanda (1979)estimated the dissociation constant, K ,of the active drug-phage complex and the velocity constant, 12, of the photodynamic action by using the relation derived by Yamamoto (1958) as

+

(7) kJk, = 1 KJD, where km is the maximum value of 4 and D, is the drug concentration.

282

S. N. CHATTERJEE AND M. MAITI

The dissociation constant of the furazolidone -phage complex was determined as 8.88 X M in phosphate-bufferedsaline. In 5 mMTris-HC1 buffer (ph 7.4) the value of the dissociation constant was reduced to 3.97 X M indicating a better association between the drug and the phage DNA and a higher photodynamic inactivation.

D. Inactiuation by Chemicals Very few of the vibriophages have been studied in respect of their sensitivity to different chemical agents. The chemicals, deoxyribonuclease (50 fig/ml), ribonuclease (50 pg/ml), mitomycin C (5 pg/ml), furazolidone (5 ,ug/ml), polymyxin B (5 pg/ml), berberine chloride (5 figlml), sodium deoxycholate (100 p M ) ,and chloroform (0.5 m1/10 ml) could not inactivate by any significant degree the phages PL 163/10, 42, 4149, and Ph-1 1

1

1

PL163/10a -

I

c

02b -

I

c ;-:

014gc Ph-l

-

*-

1

1

1

1

1

!A:

1

1

:H-o-l

*

I

PVc 30?

-

n

B P V c A'

-

w

-

1

2

1

-

0-i x

f 1

4

-

l-c+tw-x-*

-

-I

1

W -

PVc117'

vn

1

-

m

:

1

1

6

1

1

8

1

1

-

x-i

1

1

1 0 1 2

1

VIBRIOPHAGES AND VIBRIOCINS

283

after 1hour treatment at 37°C (Maiti and Chatterjee, 1971; Chanda and Chatterjee, 1975; Maiti et al., 1973; Maiti and Chaudhuri, 1979). While the phages PL163/10 and 4149 were insensitive to the action of Tris (100 p M ) ,Tris (100pM) and ethylemediamine tetracetate (50pg/ml), and Tris (100puM) lysozyme (50 pg/ml), the phages Ph-1 were significantly inactivated by these agents after 1hour treatment at 37°C (Maiti and Chatterjee, 1971; Maiti et al., 1973; Chanda and Chatterjee, 1975). On the other hand, treatment with sodium lauryl sulfate (0.05%, 1hour) at 37°C inactivated the phages PL163/10 and 4149 by more than 99%, but failed to inactivate the phages Ph-1 by any significant degree (Maiti and Chatterjee, 1971; Chanda and Chatterjee, 1975; Maiti et al., 1973). The V.parahaemolyticus phages exhibited varying sensitivities to organic solvents, e.g., ethyl ether, chloroform, and toluene. Most of these phages were more sensitive to ethyl ether and chloroform than toluene (Koga et al., 1981).

+

E. p H Inactivation The study of pH stability of vibriophages is important for microbiological interest and also for practical purposes relating to the possible therapeutic use of these phages. pH stability of some vibriophages is shown in Fig. 8. Vibriophages are usually more stable in the alkaline than acid side of the neutral pH. Some of them are fairly stable even at pH 12.0. The phage PVcll7 was quite stable even at pH 3.0.

IV. MORPHOLOGY AND OTHER PROPERTIES OF VIBRIOPHAGES A . Morphology Systematic morphological characterization of the vibriophages (Figs. 9 - 12) was reported for the first time by Chatterjee et al. (1965). Phages belonging to any one of Mukerjee’s four groups (Mukerjee, 1963a,b), when negatively stained by phosphotungstate following the method of Brenner and Horne (1959), presented characteristic morphology. The group I phages presented a polyhedral head of cross-diagonol dimensions 70.6 k 1.8 X 74.0 f 2.7 nm. The presence of any tail was uncertain although short rod-like projections at an apex of the head could be discerned. The group I1 phages possessed well-defined polyhedral head of dimensions 62.1 k 3.1 X 65.6 f 3.7 nm and a tail of length 81.0 -t- 3.2 nm and width 16.6 f 2.0 nm. The plate-like structures and prongs could be detected at the end of the tail. The group I11 phages also presented a polyhedral head of dimensions 61.1 k 5.0 X 64.4 f 5.3 nm and a short tail

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S. N. CHATTERJEE AND M. MAITI

typing phages, strain PL 163/10 (Mukejee’s group I) negatively FIG. 9. V. cholerae stained with sodium phosphotungstate. Inset, higher magnification reproduction. Bars in all figures represent 100 nm (Chattejee etal., 1965).

of length 17.8 k 2.0 nm and width 17.4 k 2.5 nm. Group IV phages hadon the other hand a polyhedral head of dimensions 73.8 k 3.3 X 83.6 f 4.0 nm and a long, thin, and flexible tail of length 152.8 f 8.2 nm and width 10.7 f 1.4 nm. A knob or plate-like structure of dimension about 13.0 nm was detected at the end of the tail. Subsequent studies by metal shadowing technique also failed to detect the presence of any tail-like structure in group I phages (Das and Chatterjee, 1966a). Vieu et al. (1965) also studied the morphology of some unclassified choleraphages by electron microscopy and revealed the presence of polyhedral heads and tails in these phages. Weston et al. (1973), revealed that the phage VcAl possessed a head of size 58.0 X 50.0 nm and a tail of length 87 nm and width 2.6nm. The El Tor phage Ph-I possessed a head of dimensions 72.6 X 72.0 nm and a short tail of about 20.0 nm in length (Maiti et al., 1973). Koga et al. (1982) classified 18bacteriophages lytic for V. parahaemolyticus into four distinct groups on the basis of their morphology. The group I phages

VIBRIOPHAGES AND VIBRIOCINS

285

Frc. 10. V.cholerae typing phages, strain $2 (Mukerjee’s group 11) negatively stained with sodium phosphotungstate (Chatterjee etal., 1965).

FIG.11. V. cholerae typing phages, strain $185 (Mukerjee’s group 111) adsorbed to the surface of a bacterial debris and lysed. Short tail structures are visible. Insets showing intact virions with short tails. Negatively stained with sodium phosphotungstate (Chatterjee etal., 1965).

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S. N. CHATTERJEE AND M. MAITI

FIG.12. V. cholerue typing phages, strain $149 (Mukerjee’sgroup IV) negatively stained with sodium phosphotungstate. Higher magnification indicates presence of appendages at the end of the tail (arrow) (Chatterjee etul., 1965).

possessed a hexagonal head and a tail with contractile sheath. All the phages of the other three groups had a relatively long, noncontractile tail, but differed in their head structures. Recently Ackermann et al. (1983) studied high resolution electron micrographs of 33 vibriophages (Fig. 13) and classified them into three families (Myoviridae, Styloviridae, and Podoviridae) and seven morphological types or species (X29, kappa, 11, IV, I, 111, and4996). Phages were all tailed

VIBRIOPHAGES AND VIBRIOCINS

287

FIG.13. El Tor phage 4 negatively stained with uranyl acetate. (Electron micrograph through the courtesy of H. W. Ackermann.)

and had icosahedral heads. These authors confirmed earlier observations (Chatterjee et al., 1965; Das and Chatterjee, 1966b) on the morphology of choleraphages belonging to Mukerjee’s groups I -IV, but could detect a tail in group I. The dimensions of the seven types of vibriophages (Ackermann et al., 1983) are summarized in Table IV.

B. Sedimentation Properties The buoyant density as determined by CsCl density gradient centrifuga, ~ the ) particle weight have tion, the sedimentation coefficient ( s ~ ~and been determined so far for the phage 4149 and only the buoyant density for the phages hv-1, a3a, and F1 (Table V). C. Properties of Phage Components 1. Nucleic Acids

Properties of the vibriophage nucleic acid are presented in Table V. The different phage strains studied so far possessed double-stranded DNA,

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S. N. CHATTERJEE AND M. MAITI TABLE IV

NEGATIVELY STAINED WITH PHOSPHOTUNGSTATE***~ DIMENSIONS (nm) OF THE VIBRIOPHAGES

Styloviridae

Myoviridae Phage componentd

X29

Kappa

I1

IV

Podoviridae

I

I11 ~

Head Tail Neck Core Extended sheath Contracted sheath Base plate Fibers or spikes

64x34

61 X 3 4

64x34

10x7 142x7 1 3 0 X 15 59x21 30x3 25x3

10x7 111x7 105X 15 4 3 X 19 25x3 22x3

10x7 79x7 6 8 X 14 20x30 30x3 17x3

83x40

71x37

6 1 X 32

-

-

-

159

13

15

-

-

-

15x3

-

-

27 x 5 120x3

4996 _ _ _ _ ~

~~

65 X 33

-

Main source of information: Ackermann etal.(1983). Myoviridae, Styloviridae, and Podoviridae are family names (see text). X29, kappa, I1 etc. are species namesproposedby H. W. Ackermannandassociates (see text; personal communication, 1984). Data presented as main dimension X side length (when available). a

except the filamentous phage F1 which contained single-stranded DNA as the genetic element. However, many important properties, e.g., presence of any unusual base, the linear or circular nature of the DNA, the hybridization study of different phage nucleic acids etc., have not been investigated for any of the vibriophages and, as a result, their classification has to depend, at present, chiefly on morphological characteristics. Transfection by vibriophage DNA has not been adequately investigated. Balganesh and Das (1979) reported transfection of V. cholerue classical strain OGAWA 154 by the phage $149 DNA, the cells becoming competent a t the mid-long phase of growth; 5 X lo4 infective centers per microgram of $149 DNA were obtained. Maiti etal. (1982) demonstrated that the phage 4 2 DNA infected the spheroplasts as well as the whole cells OGAWA 154. But when l.Opg/ml of $2 DNA couldproduce of V.cholerae 22 infective centers/ml only from the whole cells (- 108/ml),it produced at least lo7infective centers/ml from the spheroplasts (- 108/ml)of the same cells. 2. Proteins

Only two of the vibriophages, PL 163/10 and $2, have so far been investigated by the sodium dodecyl sulfate -polyacrylamide gel electrophoresis technique to resolve the presence and to estimate the molecular weight of different constituent polypeptide units. The presence of four polypeptide units of molecular weights 10,370 f 515 (I), 30,000 1303 (11),40,000 t 1049 (III),and 64,000 +_ 2433 (IV) was detected in the phage

*

289

VIBRIOPHAGES AND VIBRIOCINS TABLE V

PROPERTIES OF THE VIBRIOPHAGE COMPONENTS~ Phage strains Phage components

4149

42

hv-1

a3a

F1

Nucleic acid Type Number of strands (G + C)(%) Molecular weight ( X lo6)

DNA 2 46 36

DNA 2 42 93

DNA 55

DNA 2 44 34

DNA 1 -

Virion CsCl density (g/ml) Sedimentation coefficient

1.50 566

-

1.52 -

1.51 -

1.32 -

100

-

-

-

SZ0,W

Particle weight ( X lo6)

-

Main sources of information: Mitra andBasu (1968); Chaudhuri and Maiti (1980a); Chaudhuri and Maiti (1980b); Chaudhuri (1982); H. W. Ackermann and associates (see text; personal communication, 1984).

P L 163/10 (Chanda and Chatterjee, 1976b; Chanda, 1977). However, electrophoresis of the phage sample alkylated with iodoacetic acid resolved the presence of only one polypeptide chain of molecular weight 10,310 f 565. This unit was thus interpreted as the basic polypeptide unit of the phage PL 163/10 and the others were trimer, tetramer, and hexamer of this basic unit. The appearance of the polypeptide units 11,111,and IV could not be eliminated even by heating a t 100°C in presence of SDS and mercaptoethanol, indicating that these units were presumably formed by reaggregation subsequent to heating at 100°C. Such a strong tendency of viral proteins to reaggregate subsequent t o heating was also reported for other viruses (Haslam etal., 1969; Maizel, 1971;Ghabrial and Lister, 1974; Alvarez etal., 1972). It was presumed that alkylation with iodoacetic acid protected the sulfhydryl groups and prevented the reaggregation of the polypeptide. Electron microscopy resolved that the phage 42 possessed a much more complex structure than the phage PL 163/10. In conformity with this, a larger number of polypeptide units were detected in phage 4 2 (Chaudhuri and Maiti, 1980a, 1981). The estimated molecular weights of these units were 13,500 k 1000 (I), 21,000 f 2000 (11), 29,500 1000 (111), 37,500 f 1000 (IV), 53,000 f 3000 (V), 68,000 f 1000 (VI), 96,500 f 4000 (VII), and 143,000 k 8000 (VIII). The polypeptide of molecular weight 53,000 was the major protein unit and comprised 40.5% of the total viral protein. However, the presence or absence of multimeric units among the eight polypeptide units of the phage 42 was not investigated. Also, other

+

290

S. N. CHATTEWEE AND M. MAITI

aspects of viral proteins, e.g., identification of any functional protein including enzyme, number of protein subunits in the virion etc., have not been investigated for any of the vibriophages. OF VIBRIOPHAGES V. CLASSIFICATION

Ever since the discovery of cholera bacteriophages by Felix d’Herelle around 1920, different investigators (Asheshov, 1924; Pasricha etal., 1932a,b;Asheshov etal., 1933; Pollitzer, 1959) attempted to classify these phages in accordance with their biological properties. Mukerjee and his associates made a series of systematic studies in this respect and on the basis of the lytic patterns tested on 200 V. cholerae strains, the 30 choleraphages were classified into four distinct groups (I, 11,111,and IV) (Mukerjee etal., 1957; Mukerjee, 1960). This classification was subsequently confirmed by including data on plaque morphology, thermal death points, and, more important, the antigenic properties of these phages (Mukerjee, 1961a,b,c, 1962). Electron microscopy revealed distinct morphology for choleraphages belonging to any one of the four groups of Mukerjee (Chatterjee etal., 1965; Das and Chatterjee, 1966b). Mukerjee’s classification found useful application in the epidemiology of the disease cholera (Mukerjee, 1965,1967). Subsequently, with the introduction of V. cholerae El Tor strains as the causative agent of the disease, the El Tor phages were classified into 5 different groups (Basu and Mukerjee, 1968). Taxonomy of bacterial viruses has been presenting problems for over a long period of time and the need for development of a universal classification scheme has been keenly felt. It is being recognized that many of the biological properties of the phages, e.g., plaque morphology, adsorption characteristics, burst size etc., are of little or no taxonomic importance since these properties depend on the host and culture conditions as much as on the phage. Similarly, serological properties are of limited value but may be useful for species definition. Using the nature of nucleic acid [single stranded (ss) or double stranded (ds) DNA or RNA] and the electron microscopic morphology as the criteria, Bradley (1965,1967) classified the bacteriophages into six fundamental groups (A to F). Mukerjee’s phages were put under three groups, viz. C (phage I and III), A (phage II), and B (phage IV). Later, Ackermann and Eisenstark (1974) reviewed 1061 phages of different bacterial genera and expanded Bradley’s original scheme to include newly found phage groups. Also tailed phages weze subdivided according to head length. Thus Bradley’s six fundamental groups were expanded to 18 groups or types. The International Committee on Taxonomy of Viruses (ICTV) subsequently defined 10 clear-cut families which comprised all phages of known morphology (Matthews,

291

VIBRIOPHAGES AND VIBRIOCINS

1982). Following this recommendation, 33 vibriophages studied by Ackermann etal. (1983) were classified into three families, e.g., Myoviridae, Styloviridae, and Podoviridae, since all of them were tailed. Recently H. W. Ackermann, A. L. Furniss, S. S. Kasatiya, J. V. Lee, A. Mbiguino, F. S. Newman, J. F. Vieu, and A. Zachary (personal communication, 1983) extended the classification scheme and put forward species proposals for choleraphages chiefly on morphological basis. Any phage or group of phages that cannot be confounded with another entity was considered as a species. Accordingly the choleraphages belonging to four families (three for tailed phages and one filamentous phage) were subdivided into 15 distinct species (Table VI). Parahaemolyticus phages (Koga etal., 1982) TABLE VI CLASSIFICATION OF VIBRIOPHAGES ~~~~~~~~~~~

~

Family”

Speciesb

Some representative phage strains

MorphotypeC

Myoviridae

X29

Lab0 I(3), 7050, X29(4)

A1

Myoviridae Myoviridae Myoviridae

Kappa

El Tor 25, VcA2, kappa 42,13, 16, VcAl nt-1

A1 A1 A2

Myoviridae Styloviridae

06N-22P

IV

06N-22P $149

A2 B1

Styloviridae Styloviridae Styloviridae

hv-1 OXN-52P a3a

hv-1 OXN-52P a l , ~ ~ 2 , 0 3 a3a 6,

B1 B1 B2

Podoviridae Podoviridae Podoviridae Podoviridae Podoviridae

I OXN-l00P 4996 111 Berne 6/29

PL163/10, e3, e4 OXN-100P 4996 Ph-1, el, e2, e5,4185 Berne 6/29(7)

c1 c1 c1 c1

Inoviridae

V6

V6

F1

I1

nt-1

~

c2

~

~

~

Principal characteristics of the Morphotypes Head not elongated; tail contractile

Head elongated; tail contractile Tail long and noncontractile

Head elongated; tail noncontractile Tail short

Head elongated; tail short Filamentous

a Family names in accordance with the recommendation of the International Committee on the Taxonomy of Viruses (ICTV), Matthews (1982). Species names following the proposal of H. W. Ackermann and associates (seetext; personal communication, 1984). Morphotypes according to Ackermann and Eisenstark (1974).

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S. N. CHATTERJEE AND M. MAITI

subdivided into four groups could be apparently included into the Ackermann’s scheme as group I (family, Myoviridae; morphotype, Al), group I1 (family, styloviridae; morphotype, Bl), group I11 (family, styloviridae, morphotype B2), and group IV (family, styloviridae; morphotype, uncertain). Further studies of these phages particularly under high-resolution electron microscopy would lead to a better and more definite classification. However, many important data, e.g., physicochemical properties of the phage nucleic acid, nucleic acid homologies etc., are not available for many of the vibriophages and hence the taxonomy of these phages will be the subject of further studies in future.

VI. PRACTICAL USESOF VIBRIOPHAGES A. PhageTyping of Cholera Vibrios Several investigators (Nicolle etal., 1960, 1962; Newman and Eisenstark, 1964; Gallut and Nicolle, 1963; Mukerjee etal., 1957, 1959, 1960, 1963,1965) worked on the typing of different vibrio strains by noting their sensitivities to choleraphages. Such studies were very much dependent on the availability of large number of bacterial and phage strains for obvious reasons. Mukerjee etal. (1957) used four groups of choleraphages (I, 11, 111, IV) and classified the V. cholerae classical strains into five principal phage types. One of these types (type 2) could be further divided into three subtypes by phage adaptation (Mukerjee etal., 1960). Again the type 1 vibrio strains were subsequently divided into three subtypes in accordance with their susceptibility to lysis by two bacteriophages isolated from lysogenic V. cholerae strains (Mukerjee and Takeya, 1974). Phage typing of V. cholerae El Tor strains presented difficulties and the scheme initially presented by Mukejee (1964a) had to be discarded and modified (Basu and Mukerjee, 1968). The modified scheme included 3464 strains of V. cholerae El Tor isolated in epidemics between 1937 and 1966 and 5 phage groups. The V.cholerae El Tor strains could thus be classified into 6 phage types. A phage-typing scheme for NAG vibrios was subsequently proposed (Sil etal., 1972; Mukerjee, 1978) using 4 of the 6 new bacteriophages isolated from the sewage water of Calcutta. The NAG vibrios were classified into 10 phage types (Mukerjee, 1978). Drozhevkina and Arutyunov (1979) proposed a new phage-typing scheme using 7 bacteriophages including Mukerjee’s phages I - IV. The V. cholerae classical as the disease-causing agent has in recent years practically disappeared from the field and has been replaced by the biotype El Tor strains and accordingly the phage typing of these vibrios has drawn much attention.

VIBRIOPHAGES AND VIBRIOCINS

293

Lee and Furniss (1981) have proposed an improved phage-typing scheme serotype 0 :1 strains by using 14 bacteriophages including for V. cholerae phages I-IV of Mukerjee, 2 El Tor phages, and 8 new isolates. This El Tor into 24 phage types and scheme divided 1135 strains of V. cholerae has already found useful applications. The environmental nontoxigenic serotype 0 :1 have so far all been resistant to phages isolates of V. cholerae 13, 14, 16, and 24 indicating that phage typing may provide a means of differentiating potentially pathogenic and nonpathogenic organisms. Ackermann etal.(1983) have recently cautioned that the bacteriophages should not be used for phage typing without electron microscopic identity control, since many of the phages, including Mukerjee’s phages I- IV, now available to investigators are frequently contaminated.

B. Differentiation of theClassical andEl TorBiotypes Mukerjee’s phage IV was reported to lyse all strains of V. cholerae classical but none of the V. cholerae El Tor irrespective of their phage types and serotypes (Mukerjee, 1960,1963a). This important finding was immediately confirmed by Takeya and Shimodori (1963). The phage IV test, in addition to the chicken erythrocyte agglutination and polymyxin B sensitivity tests, has since been used for differentiation of the two vibrio biotypes in most laboratories and was also recommended by the World Health 1970). Recently, Takeya et Organization for such purposes (Barua etal., al. (1981) isolated a phage, FK phage, which was similar to the Mukerjee’s phage IV in its lytic spectrum but different from it in morphological and serological properties. All 25 strains of V. cholerae classical were lysed by 10 X RTD dose of FK phage, whereas all 56 strains of biotype El Tor and not belonging to the serotype 0 :1were resistant to 37 strains of V. cholerae it. The authors proposed the use of FK phage along with Mukerjee’s phage IV for differentiation of vibrio biotypes and also for phage-typing purposes.

C. Therapeutic and Prophylactic Use There had been much controversy about the effectiveness of using choleraphages for therapeutic and prophylactic purposes, an account of which was reviewed by Monsur and Marchuk (1974). Now that it is being realized that the disease asiatic cholera is caused by the exotoxins liberated by the actively growing vibrios, the possibility of using the phages for treatment of clinical cases obviously loses much significance. Also the phage prophylaxis has not been found superior to what can be achieved with tetracycline.

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S. N. CHATTERJEE AND M. MAITI

D. Epidemiological Use The phage typing has provided a valuable tool for epidemiological study of the disease cholera (Mukerjee and Takeya, 1974). Some of the significant findings include (1)rapid disappearance of the classical biotype and its replacement by the El Tor biotype in India, (2) detection of the four independent foci harboring different phage types of V. cholerae in the Indo-Pakistan subcontinent (Mukerjee, 1965)’ and (3) detection of the mode or modes of entry and spread of the disease caused by El Tor biotypes in the Indo-Pakistan subcontinent (Mukerjee and Takeya, 1974)etc. Lee and Furniss (1981) also reported similar useful applications of their phage-typing scheme. The phage typing and sero typing of the vibrio strains suggested that the disease occurring in Portugal in 1974 was brought from Angola or Mozambique rather than Portuguese Guinea. It is now believed that the classical biotype has almost disappeared from most parts of the world and has been replaced by the El Tor biotype. The phage-typing scheme of Lee and Furniss (1981) appears in this context very promising and may contribute toward further understanding of the epidemiology of the disease cholera. VII. LYSOGENIC VIBRIOPHAGES

A. Introduction Lysogeny is a hereditary property of producing bacteriophage without infection with external particles, and appears to be widely spread in nature. Although many strains of various species and genera (Salmonella, Pseudomonas, Bacillus, Staphylococcus etc.) exhibited lysogeny long ago, lysogeny in vibrios was detected for the first time in 1963 by Takeya and Shimodori in Japan. Prior to that time, V. cholerae El Tor had been usually considered as nonpathogenic or at least less pathogenic than V. cholerae classical. Takeya and Shimodori (1963) isolated pathogenic strains of El Tor vibrios almost all of which were lysogenic with a specific temperate phage. These authors proposed the terms “Celebes-type” for the pathogenic strains of El Tor vibrios and “Classic Ubon-type” for the nonpathogenic or less pathogenic ones (in accordance with names ofplaces where the respective strains were isolated). Takeya etal. (1965b)observed that the pathogenic or the “Celebes-type” El Tor vibrios and not the “Ubon-type” were lysogenic with the temperate phage. The phages produced by the pathogenic strains were almost identical with each other and were termed by these authors “kappa-type” phages (to be termed hereafter

VIBRIOPHAGES AND VIBRIOCINS

295

kappa phages). Neogy and Sanyal(l966) confirmed that the strains of El Tor vibrios isolated in Calcutta since 1964 were lysogenic with kappa phages. Also Chun etal. (1970) reported the presence of kappa phages (1970)further among the isolates in 1970epidemics of Korea. Parker etal. showed a positive correlation between the pathogenicity and the production of bacteriophage-related materials of V. cholerae.However, the concept of the positive correlation between virulence and lysogeny with kappa phages did not last long as further investigations (Takeya, 1976; Takeya etal., 1967b,c) revealed that even “cured” strains of El Tor vibrios were proved to cause severe disease. Cultures of many “cured” strains of “Celebes-type” El Tor vibrios were examined by electron microscopy after mitomycin C induction and were proved never to produce phage-related materials (Takeya etal., 1970). Gerdes and Romig (1975) also found that strains “cured” of kappa phage was not the pathogenicity of V. cholerae significantly altered relative to that of their “kappa” lysogenic parental strains. Besides lysogenic El Tor vibrios, the V. cholerae classical strain NIH 41 was also reported to produce temperate phages VcAl and VcA2 (Weston etal., 1973). Both these temperate phages could also lysogenize several El Tor strains.

B. Biological and OtherProperties 1. HostRange

The kappa phages have usually a very narrow host range. V. cholerue strain H218 has often been used as the indicator organism. A few other strains of cholera vibrio are also susceptible to kappa phages. Normally the kappa phages produce turbid plaques on H218 strain. However, kappa phage mutants producing clear plaques exhibit a broader host range. Another temperate phage, VcA1, also possessed a narrow host range (Weston etal., 1973). 2.Serological Properties

The kappa phages are serologically closely related and so also are the clear plaque mutants (Takeya etal., 1967~).The temperate phages VcAl and VcA2 are also serologically related to each other and to Takeya’s kappa phages (Johnson and Romig, 1981). Thirty-six V. cholerae classical strains including Bhaskaran’s mating strain 162 and the highly toxinogenic strain INABA 569B were found immune to the phage VcAl and all but 8 of them were immune to phage VcA2. Most of these strains produced, on induction, defective phage parts that were again serologically related to phages VcAl and VcA2.

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S. N. CHATTERJEE AND M. MAITI

The kinetics of serum neutralization of VcAl were typical and the velocity constant for the given antiserum preparation was 20min-'. VcAl phages thus resemble the coliphages T1 and T5 which are also neutralized slowly by their specific antiserum (Weston etal., 1973). 3.Morphology

Electron microscopic examination revealed that the kappa phages (Fig. 14) and their host range mutants were morphologically identical. The head showed an hexagonal outline 45-55 nm in diameter. The tail is 80-100 nm in length and 15 nm in width and exhibits cross striations (Mukerjee and Takeya, 1974). The phages VcAl and VcA2 were found morphologically slightly different (Weston et al., 1973; Johnson and Romig, 1981). 4.Liberation of KappaPhage

The kappa phages were found spontaneously liberated in broth cultures of the Celebes-type El Tor vibrios (Takeya etal., 1965b). The number of phage particles liberated in the culture increased in parallel with the growth of the vibrios and reached a maximum about 1 or 2 days after incubation at 37' C. Liberation of the kappa phages could also be induced by treatment of the El Tor vibrios with UV light, mitomycin C (Takeya et

FIG.14. Lysed kappa phages (left) with contracted sheath and protruding core of the tails. Intact kappa phage (right) with striated tail structure. (Electron micrographthrough the courtesy of S. Shimodori.) Negatively stained with sodium phosphotungstate.

VIBRIOPHAGES AND VIBRIOCINS

297

al., 1967b,c),or furazolidone (D. Mandal and S. N. Chatterjee, unpublished observation). Using a 15-W germicidal lamp at a distance of 120 cm, maximum induction was attained after exposure for about 20 seconds with the liberation of lo6- lo7 phage particles/ml.

5. Resistance The kappa phages are more heat sensitive but more resistant to UV radiation than are Mukerjee’s phages used for typing V. cholerae classical strains (Mukerjee and Takeya, 1974). The temperate phage VcAl was more UV resistant than the coliphage T1 (Weston et al., 1973). No systematic study on the pH stability of these kappa phages has yet been reported. The stability of the phage VcAl was however found unaffected by exposures to pH values between 5 and 10 for 1day (Weston et al., 1973).

C. Practical Use 1. Typing of El Tor Vibrios

Takeya and Shimodori (1963) proposed that the El Tor vibrios could be typed by noting the presence of the specific prophage. Accordingly a typing scheme was drawn and several reports were published on the typing of vibrio strains and its applications (Takeya et al., 1967a,b,c; Takeya, 1969). At least three different types, the “Classic-Ubon type,” the original “Celebes-type,” and the cured “Celebes-type” could be detected based on the liberation of kappa phages by or sensitivity to these phages of the vibrios under consideration. Similar typing schemes using temperate phages for V. cholerae classical and other vibrios have not been reported. 2.Diagnosis and Tracing of Cholera El Tor Carriers Since most of the “Celebes-type” El Tor vibrios were found to liberate serologically identical temperate phages, attempts were made to use this property for early diagnosis of these El Tor vibrios. The earlier method devised by Takeya et al. (1965a,b) consisted in enrichment culture of vibrio cells and detection of kappa phages liberated from them. Various numbers of cells of “Celebes-type” El Tor vibrio were inoculated in 10 ml of alkaline peptone water containing 0.5 g of human feces, and were incubated at 37°C. At l-hr intervals, the culture was centrifuged and the supernatant fluid was assayed for kappa phages using V. cholerae strain H218 as the indicator. Takeya et al. (1967a) subsequently used the phage enrichment method where the specimens from carriers were directly propagated on cells of the host strain andassayed. The propagating strain used in this revised method was the streptomycin-resistant V. cholerae strain

298

S. N. CHATTERJEE AND M. MAITI

H218 Sm’ (Zinnaka etal., 1966). This method was claimed to be superior and more effective than the earlier one and the phage was often detected even when the specimen was negative for vibrios. The method is expected to have further epidemiological use. 3.Phage-Induced Mutation orChangeof V. cholerae Strains

The temperate vibriosphages, VcAl and VcA2, were used to produce the toxin gene deletion mutants of V. cholerae (Mekalanos etal., 1982). The successful removal of the toxin gene was demonstrated by the analysis of gene sequences in the DNA isolated from these mutants. The possible use of these toxin gene deletion mutants as strains for the live oral cholera vaccine was proposed. Ogg etal. (1981) demonstrated transduction of several genetic markers (3 amino acid markers and 3 antibiotic resistance El Tor donor strain to V. cholerae classical characters) from a V. cholerae or El Tor recipient strains, the frequency ranging between and Further studies in this direction would help in the analysis of fine structure genome. and mapping of V. cholerae Besides the serological properties and interrelationship of the temperate phages, the effect of these phages on the biology of the vibrios has not been adequately investigated. Ogg etal. (1978) reported the conversion of a V. cholerae classical strain from serotype OGAWA t o serotype Hikojima, which also gained the ability to synthesize the antigenic factor C. The conversion was shown to be stable and the converted strain was lysogenic and released the phages having a host range similar to the phage of the donor strain. Siddiqui and Bhattacharyya (1982) however could not detect any change in serotype or classical phage sensitivity after lysogenizaclassical with temperate phages. However a phage-type tion of V. cholerae change in the El Tor strains, i.e., conversion from sensitivity to resistance to the El Tor typing phage E3, was noted. There is scope for further studies on the serotype and phage-type changes induced by lysogenization with temperate phages because of its epidemiological importance. VIII. VIBRIOCINS

A. Introduction Gratia (1925) discovered a highly specific antibiotic (principe V ) proand active against another strain of the same duced by one strain of E.coli species. He described many of the basic features of what was later realized to be a group of similar antibiotics produced by various members of the Enterobacteriaceae and for which the generic name “colicine” was pro-

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posed by Gratia and Frbdkricq (1946). The more general term “bacteriocine” (the final “e” being dropped later) was proposed later by Jacob etal. (1953) for highly specific antibacterial proteins produced by certain strains of bacteria and active mainly against some other strains of the same species. or vibriocins (Farkas-Himsley and SeyThe bacteriocins of V. cholerae fried, 1962) were discovered much later. Bhaskaran (1960) observed a peculiar lethal factor which appeared closely related to sex factor. Working with genetic recombination of V. cholerae strains with and without fertility factors (P+and P-), Bhaskaran (1960,1964) found that P+ strains produced a bacteriocin-like substance which was distinct from bacteriophage. Nicolle etal. (1962) and Barua (1963) however failed to demonstrate such a bacteriocin in V. cholerae strains examined by them. Farkas-Himsley and Seyfried (1962,1963a,b) were the first to report the presence of vibriocin in V. cholerae. They found that anaerobiosis induced a lysogenic streptomycin-sensitive strain of V. cholerae (ATCC 9168) to lethal biosynthesis of a substance lethal to its streptomycin-resistant mutant when tested aerobically. Subsequently Wahba (1965) succeeded in demonstrating the presence of vibriocins in 13of 16 strains of V. cholerae. Takeya and Shimodori (1969) also detected such an antibiotic substance by a “lacuna” forming method. The initial controversies on the existence of vibriocins were largely due to the nonavailability of standardized methods for production and detection of such antibiotic substances.

B. Production and Detection Farkas-Himsley and Seyfried (1962) used a streptomycin-sensitive strain of V. cholerae (ATCC 9168) and could induce vibriocin production by (1)anaerobiosis (using thioglycollate, 0.15% in nutrient broth culture medium), (2) UV irradiation, or (3) cold shock. Streptomycin-resistant mutants of this strain were sensitive to the vibriocins when tested aerobically and thus served as a vibriocin indicator strain. Later, mitomycin C (0.3 pg/ml for 10 minutes) was also found to induce vibriocin production (Jayawardene and Farkas-Himsley, 1969a; Parker etal., 1970; Farkas1971). The conditions for vibriocin production were thus Himsley Ptal., closely related to those for bacteriophage induction. Wahba (1965) also used the cold shock method for production of vibriocin. It was presumed that the cold shock helped not only in the release but also in the diffusion of vibriocins before addition of indicator vibrios. Reeves (1965) observed that although the bacteriocinogenic strains possess the stable genetic ability to produce a bacteriocin, they do not do so all the time or under all conditions. The factors controlling the synthesis are imperfectly under-

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stood. This holds true for vibriocin production. Farkas-Himsley and Seyfried (1963b) observed large fluctuations in the production of colicins and vibriocins with different batches of nutrient broth. Using nutrient broth (Oxoid) they showed that the vibriocins were produced only if cultures in the logarithmic phase of growth, and containing between 7 X lo7 and 2 X lo8 bacteria per ml were induced. No production occurred when early logarithmic or stationary phase cultures were used. Also the redox potential of the medium had to be below - 78 mV for any production at all to occur, optimal conditions being between - 265 and -270 mV. A complex growth medium was much more effective than a simple chemically defined one (Jayawardene and Farkas-Himsley, 1969a). It is generally believed that the timing of bacteriocin harvest must be determined empirically for each different organism, method, and set of conditions (MayrHarting etal., 1972). Farkas-Himsley and Seyfried (1963a,b) observed that the vibriocin levels remained the same after 4, 12, and 24 hours incubation of the culture a t 37°C. However, the time needed to achieve the maximum yield is usually much reduced if the culture is induced. Chakraborty etal. (1970),however, outlined the essential requirements of (1)citrate phosphate buffer at a concentration of 0.5 - 0.7% for each chemical (2) pH between 7.5 and 7.6, and (3) cold shock for 18hours for obtaining reproducible yields of vibriocin. For the detection of vibriocin activity, a streptomycin-resistant mutant of strain 9168 was used and high sensitivity was achieved only under aerobic condition (Farkas-Himsley and Seyfried, l962,1963a,b; Jayawardene and Farkas-Himsley, 1969b). The producer organisms in broth culture were separated by centrifugation and the supernatant was passed through Millipore filters (0.45 pm pore size). Three methods were used to detect the vibriocin activity in the filtrate: (1)a drop of the filtrate was placed on the surface of nutrient agar plate seeded with the indicator strain, (2) an aliquot of the filtrate was added to the broth culture of the indicator strain and turbidity measurements were done; the vibriocin titer was expressed as the reciprocal value of the highest dilution of the filtrate leading to growth inhibition, and (3) the inhibition zones around single colonies of the producer strains were demonstrated by using a modified plate overlay method of Frhdhricq (1954). The number of lethal units was determined by measuring the fraction of indicator cells remaining viable after 30 minutes incubation with a vibriocin sample at 37°C. The colorimetric method for bacteriocin assay as devised by Shannon and Hedges (1970) was rather simple, rapid, and reliable. Kageyama etal. (1964) observed that the release from sensitive indicator cells of materials specifically absorbing light in the UV region (260 nm) was proportional to pyocin concentration under defined conditions with indicator cells in excess. This principle was used by Krol and

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Farkas-Himsley (1970) in devising a rapid and quantitative assay for vibriocin activity.

C. HostRange The bacteriocins, in general, were earlier considered as antibiotics with a narrow bacterial host range and of activity depending on their accessibility to suitable surface receptors on the sensitive bacteria (Frkdkricq, 1946). This idea has however changed significantly by now. Vibriocins were not found to be species specific and Farkas-Himsley and Seyfried (196313) had shown that a number of strains of E.coli, P. aeruginosa, and P. fluorescence were sensitive to vibriocin. Datta and Prescott (1969a) also reported that strains vibriocin inhibited a wide range of enterobacteriae. Besides E.coli, S.flexneri, and V. cholerae were used as indicators of vibriocin of S. sonnei, by Chakraborty etal. (1970). An inverse relationship between sensitivity to vibriocin and sensitivity to streptomycin was observed (Farkas-Himsley and Seyfried, 1963b). Among 23 streptomycin-resistant strains, 19 (83%) were sensitive to vibriocin, whereas of 17 streptomycin-sensitive cells, 13 (78%)were resistant. Vibriocin was also found to inhibit effectively the growth of the established human cell line, HeLa and murine fibroblasts (Farkas-Himsley, 1974). Three bacteriocins, derived from unrelated bacterial families (colicin, pyocin, and vibriocin), were equally effective in inhibiting the L60T mouse fibroblasts (Farkas-Himsley, 1980). It was further observed that two human adenocarcinoma established cell lines from the colon and rectum were more sensitive to vibriocin than normal human embryonic intestinal cells (Farkas-Himsley etal., 1975). These and other similar observations prompted Farkas-Himsley (1980) to observe that bacteriocins can no longer be regarded strictly as antibiotics which possess narrow bacterial host specificity. They may be more properly viewed as broad-spectrum inhibitors with the capability of affecting several types of eukaryotic as well as prokaryotic cells.

D. Morphology Several authors studied the morphology of vibriocins (Fig. 15) by electron microscopy using the negative staining technique. Their observations were similar in so far as vibriocins were described as bacteriophage tail-like structures. Jayawardene and Farkas-Himsley (1968) described vibriocins as a double hollow cylindrical structure with a knob of diameter 110 nm at one end. These structures contained an outer sheath of width about 24 nm and an inner core of diameter 9- 10 nm. Both contracted and extended froms of the sheath with empty or filled inner cores were observed. These observations were in general confirmed by Lang etal.

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FIG.15. Tubular vibriocin particles (left) with empty or partially filled cores (obtained through the courtesy of D. Lang). Vibriocin particle (middle) exhibiting polysheaths (PS) with exposed core (C) and an opening (arrow) at one end (obtained through the courtesy of H. Farkas-Himsley). Tubular particles (right),termed rhapidosomes obtained by autolysis of El Tor vibrio MAK 757 (Adhikari and Chatterjee, 1972). Similarity of these particles with vibriocins is evident. Electron micrographs were obtained after negative staining with sodium phosphotungstate.

(1968). Parker etal.(1970) also observed the phage tail-like structures from 12 vibriocin-producing strains. The extended sheath of these structures measured 104 X 21 nm. The sheath in the contracted form measured 45 X 24 nm and the dimensions of the protruding core were 10.4 X 9 nm. These structures exhibited a distinct neck a t one end and fibers or appendages at the other. Similar observations were also recorded by Takeya etal.(1970). Lang etal. (1968) also detected the presence of segmented double hollow tubes arranged end to end in vibriocin preparations. The distance between the segments was between 40 and 130 nm and the overall particle lengths were 300 nm or more. The presence of some interesting tubular structure in the autolysate of V. chalerue El Tor strain MAK 757 was reported by Adhikari and Chatterjee (1972). The lengths and widths of these structures varied between 156-289 and 2233 nm, respectively. Although the authors termed these particles rhapidosomes, their morphology appears very similar to that of a vibriocin.

E. Physical and Chemical Properties

Not much work has so far been reported on the physical and chemical properties of vibriocins. Also very few authors have attempted to obtain a

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purified preparation of vibriocin for such studies. Farkas-Himsley and Seyfried (1965) fractionated the supernatant of a vibriocin-producing culture by sucrose gradient centrifugation. Subsequently Jayawardene and Farkas-Himsley (1968,1969a) purified the vibriocins, released in buffered saline after incubation with mitomycin C, by gel filtration on Sephadex G-200 and by ultracentrifugation. The active fractions obtained earlier after sucrose density gradient centrifugation contained protein, nucleic acids, and phospholipids. The protein nature of these particles was confirmed since these were inactivated by trypsin (0.05%, w/v) at 37°C and pH 8.0 and also by other proteolytic enzymes. These particles could withstand a fairly wide change in pH and were not inactivated after exposures for up to 30 minutes to pH levels ranging between 1.3 and 12.5 at 30°C (Farkas-Himsley and Seyfried, 1963a). The vibriocins were also comparatively more heat resistant than other bacteriocins of comparable size. The particles were not inactivated by exposure to 56°C for 5 hours or by boiling for 1 minute, but were inactivated by boiling for 10 minutes. Although the molecular weight of these particles has not so far been determined by conventional techniques, their electron microscopic photographs indicated that they should correspond to Bradley’s (1967)high-molecular-weight type of bacteriocins. But unlike the high-molecular-weight bacteriocins described by Bradley (1967), the vibriocins are trypsin sensitive and heat resistant.

F. Mode of Action Farkas-Himsley and her collaborators extensively studied the mode of action of vibriocins (Jayawardene and Farkas-Himsley, 1969a,b, 1970b; Krol and Farkas-Himsley, 1971a,b). Like the action of many other bacteriocins, the reaction of vibriocin with a sensitive cell is expected to be biphasic in nature. The first phase involves the adsorption of the particles to specific receptors at the cell surface (adsorption phase). This is followed somewhat later by the second phase when pathological changes leading to death of the cell (lethal phase) occurs. Vibriocin adsorption on sensitive cells was found temperature independent but the lethal action occurred only in an appropriate narrow temperature range and required oxidative phosphorylation. The susceptibility of sensitive bacteria to the bactericidal action of vibriocin was greatly diminished if the cells were pretreated with sodium azide and 2,4-dinitrophenol, the inhibitors of oxidative phosphorylation. Inactivation of the sensitive cells followed a single hit kinetics indicating that adsorption of one particle is enough to kill the cells. As in the case of some other bacteriocins, colicins in particular, the lethal action of vibriocin is reversed by trypsin added within 7 - 10

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minutes after adsorption. Also the cells are most sensitive to vibriocin action in their exponential growth phase and are tolerant in their stationary growth phase or under anaerobic condition. Chloramphenicol(5 pg/ ml) inhibits the lethal action of vibriocins indicating that protein synthesis is essential for vibriocin action. A t the molecular level, vibriocin inhibits DNA synthesis in sensitive cells and promotes DNA degradation leading to leakage of nucleotides. Within 5 minutes of the addition of vibriocin, DNA synthesis stopped, RNA synthesis was severely inhibited, while protein synthesis continued at one-half to two-thirds the normal rate (Jayawardene and Farkas-Himsley, 1970a). The mode of action of vibriocin also resembled that of colicin E, causing changes in membrane permeability which led to enhanced potassium efflux (Jayawardene and Farkas-Himsley, 1970a) and inhibited uptake of metabolic precursors for DNA and RNA synthesis. The uptake of amino acids was much less affected (Krol and Farkas-Himsley, 1972). More recent studies revealed that vibriocin effectively inhibited the growth of the established human cell line, HeLa and murine fibrioblasts (Farkas-Himsley, 1974). Similar findings were reported from studies with colicin E, and E, (Smarda etal., 1975; Smarda and Obdrzalek, 1977; Farkas-Himsley, 1980). Vibriocins, pyocins, and colicins were equally effective in inhibiting the L60T mouse fibroblasts, the inhibition being most effective in the G2 phase of the cell cycle (Farkas-Himsley and Cheung, 1976). Studies with pyocins 1-4, colicin HSC 10, and different types of cells suggested that the bacteriocin preparation might be nontoxic to normal cells but had an affinity for the tumor cells (Farkas-Himsley, 1980). Two human adenocarcinoma established cell lines from the colon and rectum were found more sensitive to vibriocin than normal human embryonic intestinal cells (Farkas-Himsley etal., 1975). These and other similar findings prompted Farkas-Himsley (1980) to observe that the bacteriocins might find use as a diagnostic tool for sensitive cancer cells and also might have therapeutic value as nontoxic inhibitors of neoplasia. In fact, an exciting field of study has been initiated where many important and interesting developments can be expected in the near future.

G. Practical Use 1. Vibriocin Typing

Typing of bacteria within a species using bacteriocin production as a marker has been successfully applied to many species including P. aeruginosa(Holloway, 1960), S. sonmi (Abbott and Shannon, 1958), etc. Extensive tests were carried out by McGeachie (1965) and Naito etal. (1966) on the reproducibility of bacteriocin-typing methods, which re-

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vealed that typing by colicin production was more stable than typing by sensitivity. Although bacteriocinogenic strains are believed to possess the stable genetic ability to produce bacteriocins (Reeves, 1965),they do not do so all the time or under all conditions. This is particularly true for vibriocin production and largely because of this difficulty, typing of vibrios by vibriocin production has not been reported by many authors. Wahba (1965) attempted unsuccessfully to determine whether a relationship exclassical and V. cholerae El Tor in terms of isted between V. cholerae vibriocin production. The possibility of using vibriocins as a taxonomic classical and V. cholerae El Tor was also tool in differentiating V. cholerae examined by Datta and Prescott (1969b) without any success. FarkasHimsley (1973) used some of the strains already tested by Wahba (1965), Datta and Prescott (1969b), and Chakraborty etal.(1970), but obtained different patterns of inhibition. Chakraborty etal.(1970, 1971a) in this background specified the use of certain factors controlling the vibriocin production and claimed to have succeeded in demonstrating abundant and reproducible yield of vibriocin. The authors followed in general the typing procedure described by Abbott and Shannon (1958) and used sensitive E.coli Row, S. sonnei, and S. flexneri as indicator strains of V. cholerae, organisms. By using 8 indicator strains, 11 bacteriocin types were recognized among 425 strains (87% of the strains were typable). Subsequently 10 vibriocin types were recognized among 215 strains of NAG vibrio (Chakraborty etal., 1971b). Recently Mitra etal. (1980) slightly modified the typing medium of Chakraborty etal. (1970) and subjected a total of 743 strains of 0 :1 “agglutinable” and 293 strains of 0 :1 “nonagglutinable” vibrios to bacteriocin typing. Among the agglutinable strains, 11of the earlier (Chakraborty etal., 1970) reported types and 6 newer types were recognized. Likewise 18 types were identified among the “nonagglutinable” vibrios. It appears that the vibriocin typing procedure of Chakraborty etal. has not been used by other workers hitherto. But vibriocin typing will certainly provide a useful tool of epidemiological significance and hence there is considerable scope for more intensive studies on this line. Particularly, there is scope for development of a method which will be accurate, reproducible, and will not depend on subjective factors. These considerations led Farkas-Himsley and Page1 (1977) to propose another bacteriocin typing method based on the quantitative spectrophotometric detection of UV absorbing materials in supernatants of bacteriocin-sensitive cultures. It was claimed that this method yielded results identical with that obtained by the “Scrape and Streak” technique of Gillies (1963) in 275 tests of pyocin and colicin typing. The general applicability of this method to vibriocin typing or typing by different bacteriocins should be investigated further.

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2. Diagnostic and Therapeutic Use

Interesting observations have recently been recorded on the interaction of vibriocins or bacteriocins in general with the mammalian cells and cancer cells in particular. The interaction of vibriocin with eukaryotic cells was found to have a specificity that would permit recognition of malignant rather than benign cells. The vibriocins or the bacteriocins in general thus have the potentialities as a diagnostic tool for sensitive cancer cells and also might be used therapeutically as nontoxic inhibitors of neoplasia (Farkas-Himsley, 1980).

IX. CONCLUDING COMMENTS This article has aimed at presenting available information on the physical, chemical, and biological properties of the vibriophages and vibriocins. The lacunae in our knowledge have also been indicated as far as practicable. An idea of the directions in which further research on vibriophages and vibriocins is likely to proceed in the future may be delineated from what has been recorded here. More systematic studies on the nucleic acids and proteins of the vibriophages should be taken up particularly for greater taxonomic interest. The genetics of the host bacteria, the vibrios, will present a very interesting, important, and useful field of study. In this respect, the role of transducing phages deserves special mention. Of particular interest will be their role in the definition and characterization of the toxin (choleragenic) gene and the production of nontoxinogenic mutants suitable for use in the live oral cholera vaccine to be developed therefrom. Phage conversion or the alteration of bacterial properties mediated by the phage genome has so far not been extensively investigated in relation to the vibrio-phage interactions. In this respect the synthesis of diphtheria toxin by Corynebacterium diphtheriae following infection by j?phage and related phages (Freeman, 1951; Barksdale, 1970; Grange and Redmond, 1978) should provide a good guideline to vibriophage workers. Further studies on the phage typing and vibriocin typing of vibrios will continue to be of absorbing interest to epidemiologists. Studies on the vibriocins with greater emphasis on the application of biochemical and biophysical approaches are likely to pay a rich dividend in the near future. Much excitement and/or controversies may arise during further investigations on the possible use of the vibriocins in the diagnosis and therapy of neoplasia. This aspect certainly deserves much greater attention for all concerned.

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ACKNOWLEDGMENTS One of us (S.N.C) is indebted to the Indian Council of Medical Research for the award of research grants for many years and for the opportunity to act as a Member of the Cholera Expert Committee for several years, which enabled him to gain valuable experience and to do much of the work reproduced in this article. We are thankful to Drs. H. W. Ackermann, D. Lang, S. Shimodori, H. Farkas-Himsley, H. Drexler, and J. V. Lee for kindly making available to us copies of their published works and electron micrographs. We are particularly indebted to Dr. H. W. Ackermann for kindly allowing us to go through the preprints of his unpublished works and for providing other valuable information. Permissions to reproduce the following figures were granted by The American Society for Microbiology, Washington for Figs. 1, 3, 5, and 6; Institut Pasteur, Paris for Fig. 2, The Faculty Press, Cambridge, England for Fig. 15 (middle),and The Indian Journal of Medical Research for portions of Figs. 9, 10, 11, and 12.

REFERENCES Abbott, J. D., and Shannon, R. (1958). J. Clin. Pathol. 11,71-77. Ackermann, H. W. (1969). Pathol. Biol. 17,1003-1024. Ackermann, H. W. (1976). Pathol. Biol. 24, 359-370. Ackermann, H. W. (1984). In “Current Problems in Virus Taxonomy” (R. E. F. Matthews, ed.). CRC Press, Boca Raton, Florida (in press). Ackermann, H. W., and Eisenstark, A. (1974). Intervirology 3,201-219. Ackermann, H. W., Audurier, A., Berthiaume, L., Jones, L. A., Mayo, J. A.,andVidaver, A. K. (1978). Adu. Virus Res. 23, 1-24. Ackermann, H. W., Furniss, A. L., Kasatiya, S. S., Lee, J. V., Mbiguino, A., Newman, F. S., Takeya, K., and Vieu, J. F. (1983). Ann.Virol. (Znst. Pasteur) 134E,387-404. Adams, M. H. (1959). “Bacteriophages.” Wiley (Interscience), New York. Adhikari, P. C. (1971). Ph.D. Thesis, University of Calcutta, Calcutta, India. Adhikari, P. C., and Chatterjee, S. N. (1972). Can. J. Microbiol. 18, 541-542. Alvarez, G., Salas, E., Perez, N., and Celis, J. E. (1972). J. Gen.Virol. 14, 243-250. Anderson, L. A. P. (1935). In “Report on the Scientific Advisory Board of the Indian Research Fund Association for the Year 1934-35,” pp. 65-96 (cited by Pollitzer, 1959). Asheshov, I. N. (1924). J. Infect. Dis. 34,534-542. Asheshov, I. N., Asheshov, I., Khan, S., and Lahiri, M. N. (1930). Indian J . Med. Res.17, 971-984. Asheshov, I. N., Asheshov, I., Khan, S., Lahiri, M. N.,andChatterjee, S. K. (1933). IndianJ. Med. Res. 20,1127-1157. Balganesh, M., and Das, J. (1979). Biochem. Biophys. Res.Commun. 90, 726-733. Barksdale, L. (1970). Bacteriol. Rev. 34, 378-422. Enuiron. Microbiol. 36,492-499. Baross, J. A., Liston, J., and Morita, R. Y. (1978). Appl. Barua, D. (1963). Nature(London) 200, 710-711. Barua, D., and Burrows, W. (1974). “Cholera.” Saunders, Philadelphia, Pennsylvania. Barua, D., Burrows, W., and Gallut, J. (1970). Public Health Pap. 40, 128-129. Basu, S., and Mukerjee, S. (1968). Experientia 24, 299-300. Basu, S., andMukerjee, S. (1970). Bull. W. H.0. 43, 509-512. Bhaskaran, K. (1960). J. Gen. Microbiol. 23, 47-54. Bhaskaran, K. (1964). Bull. W. H. 0. 30, 845-853.

308

S. N. CHATTERJEE AND M. MAITI

Bhattacharya, F. K., Narayanaswami, A., and Mukerjee, S. (1971). In “Immunity and Immunoprophylaxis in Cholera,” Tech. Rep. Ser. No. 9, pp. 55-57. Indian Council of Medical Research, New Delhi. Bradley, D. E. (1965). J . R. Microsc. SOC.8 4 , 257-316. Rev.31, 230-314. Bradley, D. E. (1967). Bacteriol. Brandis, H. (1978). In“Methods in Microbiology” (T. Bergan and J. R. Norris, eds.), Vol12, pp. 117-126. Academic Press, New York. Brenner, S., and Horne, R. W. (1959). Biochim. Biophys. Acta34, 103-110. Immun. 1, Chakraborty, A. N., Adhya, S., Basu, J., and Dastidar, S. G. (1970). Infect. 293-299. Chakraborty, A. N., Dastidar, S. G., and Adhya,S. (1971a). In“Immunity and Immunoprophylaxis in Cholera,” Tech. Rep. Ser. No. 9, pp. 190-193. Indian Council of Medical Research, New Delhi. Chakraborty, A. N., Sil, J., and Mukerjee, S. (1971b). In “Immunity and Immunoprophylaxis in Cholera,” Tech. Rep. Ser. No. 9, pp. 161- 167. Indian Council of Medical Research, New Delhi. Chanda, P. K. (1977). Ph.D. Thesis, University of Calcutta, Calcutta, India. Chanda, P. K., and Chatterjee, S. N. (1975). ActaVirol. (Engl. Ed.)19,197-203. Chanda, P. K., and Chatterjee, S. N. (1976a). Can.J. Microbiol. 22, 1186-1187. Chanda, P. K., and Chatterjee, S. N. (1976b). ActaVirol. (Engl. Ed.)20,291-296. Chandra, P., Zunino, F., Gotz, A., Gericke, D., Thorbeck, R., and Di Marco, A. (1972a). FEBS Lett. 21,264-268. 22, 161-164. Chandra, P., Zunino, F., and Gotz, A. (1972b). FEBS Lett. Chandra, P., Zunino, F., Gaur, V. P., Zaccara, A., Woltersdorf, M., Luoni, G.,and Gotz, A. 28, 5-9. (1972~).FEBS Lett. Chatterjee, S. N., and Chanda, P. K. (1976). Int. J. Radiut. Biol. 30, 79-82. Chatterjee, S. N., and Chanda, P. K. (1979). Indian J. Biochem. Biophys. 16, 320-325. Chatterjee, S. N., and Maiti, M. (1973a). Indian J.Exp.Biol. 11,134-136. Chatterjee, S. N., and Maiti, M. (197313). J. Virol. 11,872-878. Chatterjee, S. N., and Raychaudhuri, C. (1971). IndianJ. Exp.Biol. 9, 270-271. Chatterjee, S. N., Das, J., and Barua, D. (1965). Indian J. Med.Res.53, 934-937. Chatterjee, S. N., Adhikari, P. C., and Raychaudhuri, C. (1971). In “Immunity a n d h m u n o prophylaxis in Cholera,” Tech. Rep. Ser. No. 9, pp. 198-208. Indian Council of Medical Research, New Delhi. Chaudhuri, K. (1982). Ph.D. Thesis, University of Jadavpur, Calcutta, India. Chaudhuri, K., and Maiti, M. (1980a). Indian J. Biochem. Biophys. 17,207-212. 49, 433-436. Chaudhuri, K., and Maiti, M. (1980b). J. Gen.Virol. Chaudhuri, K., and Maiti, M. (1981). Can. J . Microbiol. 27, 72-75. Chun, D., Chung, J. K., Lew, S. T., and Chang, M. W. (1970). J. KoreanMed.Assoc. 13, 53 - 58. Das, J., and Chatterjee, S. N. (1966a). IndianJ. Med.Res.54, 531-534. Das, J., and Chatterjee, S. N. (1966b). Proc.Int.Congr. Electron Microsc. 6th, 1966pp. 139- 140. 98, 849-850. Datta, A., and Prescott, L. M. (1969a). J. Bacteriol. Datta, A., and Prescott, L. M. (1969b). Indian J. Med.Res.6 7 ,1402-1408. d’Herelle, F. (1922). “The Bacteriophage -its Role in Immunity” (cited in Pollitzer, 1959). d’Herelle, F. (1923). C. R.Seances SOC.Biol. SesFil. 88, 723-725. d’Herelle, F., and Malone, R. H. (1927). IndianMed.Guz. 62,614-618. 50, 151-170. Delbriick, M. (1945). J. Bacteriol. Doermann, A. H. (1951). Fed.Proc., Fed.Am. SOC.Exp.Biol. 10,591-594.

VIBRIOPHAGES AND VIBRIOCINS

309

Doermann, A. H. (1952). J. Gen. Physiol. 35,645-649. Drozhevkina, M. S.,and Arutynnov, Yu. I. (1979). J. Hyg., Epidemiol., Microbiol., Immunol. 23,340-347. Ellis, E. L., and Delbriick, M. (1939). J. Gen. Physiol. 22, 365-384. Farkas-Himsley, H. (1973). Cited by Brandis (1978), pp. 50- 115. Farkas-Himsley, H. (1974). IRCS Libr. Compend. 2,1117. Farkas-Himsley, H. (1980). J. Antimicrob. Chemother. 6, 424-427. Farkas-Himsley, H., and Cheung, R. (1976). Cancer Res. 36,3641-3649. Farkas-Himsley, H., and Pagel, P. (1977). Infect. Immun. 16, 12-19. Farkas-Himsley, H., and Seyfried, P. L. (1962). Nature (London) 193,1193-1194. Farkas-Himsley, H., and Seyfried, P. L. (1963a). Can. J. Microbiol. 9, 329-338. Farkas-Himsley, H., and Seyfried, P. L. (1963b). Can. J. Microbiol. 9,339-343. Farkas-Himsley, H., and Seyfried, P. L. (1965). Zentralbl. Bakteriol., Purasitenkd., Mfektionskr. Hyg., Abt. 1 :Orig. 196, 298-302. Farkas-Himsley, H., Kormendy, A,, and Jayawardene, A. (1971). Cytobios 3,97-116. Farkas-Himsley, H., Cheung, R., and Tompkins, W. A. F. (1975). IRCS Med. Sci.: Libr. Compend. 3,148. Feary, T. W., Fisher, E, Jr., and Fisher, T. N. (1964). J. Bacteriol. 87, 196-208. Exp. Biol.Med. 112,355-359. Finkelstein, R. A., and Mukerjee, S. (1963). Proc. SOC. 10, 196. Flu, P. C. (1924). Tijdschr. Vergelijkd. Geneesk. Flu, P. C. (1925). Arch. Schzfls-Trop.-hyg. 29, Suppl. I, 99; summarized in Trop. Dis. Bull. 23, 190 (1926). FrBdBricq, P. (1946). C. R. Seances SOC.Biol.Ses Fil. 140, 1189-1191. FrBdBricq, P. (1954). C. R. Seances SOC.Biol.Ses Fil. 148, 399-402. Freeman, V. J. (1951). J. Bacteriol. 6 1 , 675-688. Gallut, J. (1971). Bull. SOC.Pathol. Exot. SesFil.64, 551-560. Gallut, J., and Nicolle, P. (1963). Bull. W. H. 0. 28,389-393. Gardner, A. B., and Venkatraman, R. V. (1935). J . Hyg. 35, 262-282. Gerdes, J. C., and Romig, W. R. (1975). Infect. Zmmun. 11,445-452. Ghabrial, S . A., and Lister, R. M. (1974). Virology 57, 1-10, Gillies, R. R. (1963). J. Hyg. 62, 1- 10. Grange, J. M., and Redmond. W. B. (1978). Tubercle 59, 203-225. Gratia, A. (1925). C. R. Seances SOC.Biol. Ses Fil. 9 3 , 1040- 1041. Gratia, A., and FrBdBricq, P. (1946). C. R. Seances SOC.Biol.Ses Fil. 140,1032- 1033. Guice, M. B., and Newman, F. S. (1969). J . Infect. Dis.119, 2-12. Han, G. K., and Khie, T. S. (1963). Am. J . Hyg. 7 7 , 184-186. Haslam, E. A., Cheyne, I. M., and White, D. D. (1969). Virology 39,118-129. Holloway, B. W. (1960). J. Pathol. Bacteriol. 80, 448-450. Hugh, R. (1965a). Znt. Bull. Bacteriol. Nomen. Taxon. 15, 61-67. Hugh, R. (1965b). Proc. Cholera Res. Symp., Ist, 1965 pp. 1 - 3. Hugh, R., and Feeley, J. C. (1972). Int. J. Syst. Bacteriol. 22, 123. Jacob, F., Lwoff, A., Siminovitch, L., and Wollman, E. (1953). Ann. Inst. Pasteur, Paris 84, 222-224. Jayawardene, A., and Farkas-Himsley, H. (1968). Nature (London) 219,79-80. Jayawardene, A., and Farkas-Himsley, H. (1969a). Microbios l B , 87-98. Jayawardene, A., and Farkas-Himsley, H. (1969b). Microbios 4 , 325-333. Jayawardene, A,, and Farkas-Himsley, H. (1970a). J. Bacteriol. 102,382-388. Jayawardene, A,, and Farkas-Himsley, H. (1970b). Infect. Immun. 2 , 519-520. Jesaitis, M. A., and Goebel, W. F. (1955). J. Exp. Med. 102, 733-752. Johnson, S. R., and Romig, W. R. (1981). J. Bacteriol. 146, 632-638.

310

S. N. CHATTERJEE AND M. MAITI

Jotten, K. W. (1922). Klin. Wochenschr. 1, 2181. Kageyama, M., Ikeda, K., and Egami, F. (1964). J. Biochem. (Tokyo) 66,59-64. Keeler, R. F., Ritchie, A. E., Bryner, J. H., and Elmore, J. (1966). J. Gen. Microbwl. 43, 439-454. Koga, T., Toyoshima, S., and Kawata, T. (1982). Appl. Enuiron. Microbiol. 44,466-470. Krol, P. M., and Farkas-Himsley, H. (1970). Microbios 2, 287-289. Krol, P. M., and Farkas-Himsley, H. (1971a). Infect. Immun. 3,184-186. Krol, P. M., and Farkas-Himsley, H. (1971b). Microbios 3, 183-186. Krol, P. M., and Farkas-Himsley, H. (1972). Microbios 6, 199-211. Kropinski, A. M. B., and Warren, R. A. J. (1970). J. Gen. Virol. 6,85-93. Lang, D., McDonald, T. O.,and Garden, E. (1968). J. Bacteriol. 95, 708-709. Lederberg, E. M., and Lederberg, J. (1952). Genetics 38,51-64. Lee, J. V., and Furniss, A. L. (1981). I n “Acute Enteric Infections in Children” (T. Holme, J. Holmgren, M. H. Merson, and R. Mollby, eds.), pp. 119-122. Elsevier/North-Holland Biomedical Press, Amsterdam. Lindberg, A. A. (1967). J.Gen. Microbiol. 48, 225-233. McGeachie, J. (1965). Zentrulbl. Bukteriol., Purasitenkd., Infektionskr. Hyg., Abt. 1 :Orig. 196,377-384. Maiti, M. (1972). Ph.D. Thesis, University of Calcutta, Calcutta, India. Maiti, M. (1978a). I n “Receptors in Biological Systems,” pp. 127-144. Dept. of Atomic Energy, Govt. of India. Maiti, M. (1978b). Can. J.Microbiol. 24, 1583-1589. Maiti, M., and Chatterjee, S. N. (1969a). Bull. Calcutta Sch. Trop. Med. 17, 77-79. Calcutta Sch. Trop. Med. 17, 119-120. Maiti, M., and Chatterjee, S. N. (1969b). Bull. Maiti, M., and Chatterjee, S. N. (1970). Bull. Calcutta Sch. Trop. Med. 18, 19-22. Maiti, M., and Chattejee, S. N. (1971). J. Gen. Virol. 13, 327-330. Maiti, M., and Chatterjee, S. N. (1972). Indian J. Exp.Biol. 10, 114-118. Maiti, M., and Chaudhuri, K. (1979). ZndianJ. Exp.Biol. 17, 628-631. Maiti, M., and Chaudhuri, K. (1980). IndianJ. Biochem. Biophys. 17,72-73. Maiti, M., Chanda, P. K., and Chatterjee, S. N. (1973). Indian J. Exp.Biol. 1 1 , 169-174. Maiti, M., Sur, P., and Chatterjee, S. N. (1977). Ann. Microbiol. (Paris)128A,35-39. Maiti, M., Chaudhuri, K., and Bairagi, A. K. (1982). I n “Annual Report 1981-82,” pp. 54-56. Indian Institute of Chemical Biology, Calcutta. Maizel, J. V. (1971). Methods Virol. 5 , 179-246. Matthews, R. E. F. (1982). Interuirology 17, 1-99. Mayr-Harting, A., Hedges, A. J., and Berkeley, R. C. W. (1972). I n “Methods in Microbiology” (J. R. Norris and D. W. Ribbons, eds.), Vol. 7A, pp. 315-422. Academic Press, New York. Mekalanos, J. J., Moseley, S. L., Murphy, J. R., and Falkow, S. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 151-155. Mitra, S., and Basu, S. (1968). Biochirn. Biophys. Acta 156,143-149. Mitra, S., Balganesh, T. S., Dastidar, S. G.,and Chakrabarty, A. N. (1980). Infect. Immun. 30,74-77. Monsur, K. A,, and Marchuk, L. M. (1974). I n “Cholera” (D. Barua and W. Burrows, eds.), pp. 273-280. Saunders, Philadelphia, Pennsylvania. Monsur, K. A., Rizvi, S. S. H., Huq, M. I., and Benenson, A. S. (1965). Bull. W. H. 0. 32, 211-216. Morrison, J. (1933). I n “Report of the Tenth Conference of Medical Research Workers, 1932,” p. 145. Simla (quoted by Pasricha etal., 1936).

VIBRIOPHAGES AND VIBRIOCINS

311

Mukerjee, S.(1960). Ann. Biochem. Exp.Med. 19,9-12. Mukerjee, S. (1961a). Ann. Biochem. Exp. Med. 21,257-264. Mukerjee, S. (1961b). Ann. Biochem. Exp. Med.21, 265-266. Mukerjee, S. (1961~).Ann. Biochem. Exp.Med. 21, 267-270. Mukerjee, S.(1962). Ann. Biochem. Ezp. Med. 22,5-8. Mukerjee, S. (1963a). Bull. W. H.0. 28, 333-336. Mukerjee, S. (196313). Bull. W. H. 0. 28, 337-345. Mukerjee, S. (1964a). Indian J. Med. Res.5 2 , 331-354. Mukerjee, S. (196413). Indian J. Med. Res. 52,871-881. Mukerjee, S. (1965). Proc. Cholera Res. Symp., Ist, 1965pp. 9- 16. Mukerjee, S. (1967). Indian J . Med. Res. 5 5 , 308-313. Mukerjee, S. (1978). I n “Methods in Microbiology” (T.Bergan and J. R. Norris, eds.), Vol. 12, pp. 50-115. Academic Press, New York. Mukerjee, S.,and Takeya, K. (1974). In “Cholera” (D. Barua and W. Burrows, eds.), pp. 61-84. Saunders, Philadelphia, Pennsylvania. Mukerjee, S., Guha, D. K., and Guha Roy, U. K. (1957). Ann. Biochem. Erp.Med. 17, 161- 176. Mukerjee, S., Guha Roy, U. K., Guha, D. K., and Rudra, B. C. (1959). Ann. Biochern. Exp. Med. 1 9 , 115-124. Mukerjee, S., Guha Roy, U. K., and Rudra, B. C. (1960). Ann. Biochem. Exp.Med. 20, 182-203. Mukerjee, S., Guha Roy, U. K., and Rudra, B. C. (1963). Ann. Biochem. Ezp. Med. 23, 201 - 208. Mukerjee, S., Basu, S., and Bhattacharya, P. (1965). Br. Med.J. 11,837-839. Naito, T., Kono, M., Fujise, N., Yakushiji, Y., and Aoki, Y. (1966). Jpn. J . Microbiol. 10, 13-22. Neogy, K. N., and Sanyal, S. N. (1966). Bull. Calcutta Sch. Trop. Med. 14,94-95. Newman, F. S., and Eisenstark, A. (1964). J.Infect. Dis. 114,217-225. Nicolle, P., Gallut, J., and LeMinor, A. (1960). Ann. Znst. Pasteur, Paris 9 9 , 664-671. Nicolle, P., Gallut, J., Ducrest, P., and Quiniou, J. (1962). Reu.Hyg. Med. SOC.10,91-126. Nobechi, K. (1926). C. R. Seances SOC. Biol. Ses Fil.95, 1250-1252. Ogg, J. E., Shrestha, M. B., and Poudayl, L. (1978). Infect. Immun. 19, 231-238. Ogg, J. E., Timme, T. L., and Alemohammad, M. M. (1981). Infect. Zmmun. 31,737-741. Parker, C., Richardson, S. H., and Romig, W. R. (1970). Infect. Immun. 1,417-420. Pasricha, C. L. (1933). Annu. Rep. Calcutta Sch. Trop. Med.,1932pp. 109-114. Pasricha, C. L., De Monte, A. J., and Gupta, S. K. (1932a). Indian Med. Gaz. 67,262-264. Pasricha, C. L., De Monte, A. J., and Gupta, S. K. (1932b). Indian Med. Gaz. 67,487-490. Pasricha, C. L., De Monte, A. J., and Gupta, S. K. (1936). Indian Med. Gaz. 71,194-196. Pasricha, C. L., Lahiri, M. N., and De Monte, A. J. (1941). Indian Med.Gaz. 76,218-225. Pollard, E., and Reaume, M. (1951). Arch. Biochem. 32, 278-287. Pollitzer, R. (1959). “Cholera.” World Health Organization, Geneva. Raychaudhuri, C . ,Chatterjee, S. N., and Maiti, M. (1970). Biochim. Biophys. Acta 222, 637- 646. Reeves, P. R. (1965). Bacteriol. Reu.2 9 , 24-45. Ribi, E., Anacker, R. L., Brown, R., Haskins, W. T., Malmgren, B., Milner, K. C., and Rudbach, J. A. (1966). J. Bacteriol. 9 2 , 1493-1509. Rizvi, S. H., and Monsur, K. A. (1965). Proc. Cholera Res. Symp., Ist, 1965pp. 19-24. Roy, C., Mridha, K., and Mukerjee, S. (1965). Proc. SOC.Exp. Biol.Med. 1 1 9 , 893-896. Sakazaki, R. (1968). Jpn. J . Med. Sci. Biol. 2 1 , 359-362.

312

S. N. CHATTERJEE AND M. MAITI

Sakazaki, R. (1970). Public Health Pap. 40, 33-67. Sakazaki, R., and Shimada,T. (1975). Proc. Znt. Symp. Vibrionuceae OtherAgents Enterotoricosis p. 16. Sakazaki, R., Iwanani, S., and Fukami, H. (1963). Jpn. J. Med. Sci. Bid. 16,161-188. Sakazaki, R., Tamura, K., Gomez, C. Z., and Sen, R. (1970). Jpn. J. Med. Sci. Biol. 23, 13-20. Schlesinger, M. (1932). 2.Hyg. Infektionskr. 114,746-753. SBchaud, J., and Kellenberger, E. (1956). Ann. 1st. Pasteur, Paris 90, 102-106. Shannon, R., and Hedges, A. J. (1970). J.Appl. Bucteriol. 33,555-565. Siddiqui, K. A. I., and Bhattacharya, F. K. (1982). Infect. Zmmun. 37, 847-851. Sil, J., Dutta, N. K., Sanyal, S. C., and Mukerjee, S. (1972). Indian J. Microbiol. 12, 110- 113. Smarda, J., and Obdrzalek, V. (1977). ZRCS Med. Sci.: Libr. Compend. 5 , 524. Smarda, J., Ebringer, L., and Mach, J. (1975). J. Gen. Microbiol. 86,363-366. Stent, G. S. (1963). “Molecular Biology of Bacterial Viruses.” Freeman, San Francisco, California. Sur, P., and Chatterjee, S.N. (1970). Bull. Calcutta Sch. P o p . Med. 18, 76-78. Sur, P., Maiti, M., and Chatterjee, S. N. (1974). Indian J. Exp. Bid. 12, 479-483. Takeya, K. (1967). Jprz. J. Trop. Med. 8 , 5-9. Takeya, K. (1969). Mod. Media 18.69-74. Takeya, K., and Shimodori, S.(1963). J. Bacteriol. 85, 957-958. Takeya, K., and Shimodori, S. (1969). J . Bacteriol. 99, 339-340. Takeya, K., Shimodori, S., and Zinnaka, Y. (1965a). J. Bacteriol. 90,824-825. Takeya, K., Zinnaka, Y., Shimodori, S., Nakayama, Y., Amako, K., and Iida, K. (1965b). Proe. Cholera Res. Symp., lst, 1965 pp. 24-29. Takeya, K., Shimodori, S., andzinnaka, Y. (1967a). Bull. W. H. 0. 36,990-992. Takeya, K., Shimodori, S., and Gomez, C. Z. (1967b). Bull W.H. 0. 37,806-810. Takeya, K., Shimodori, S., and Zinnaka, Y. (1967~).Proc. ChokraRes. Symp., 3rd, I967pp. 61-66. Takeya, K., Shimodori, S., and Amako, K. (1970). Proc. Cholera Res.Symp., 6th, 1970pp. 23 - 26. Takeya, K., Otohuji, T., and Tokiwa, H. (1981). J.Clin. Microbiol. 14,222-224. Vieu, J. F., Nicolle, P., and Gallut, J. (1965). Proc. Cholera Res.Symp., Ist, 1965 pp. 34-36. Wahba, A. H. (1965). Bull. W. H. 0. 33, 661-664. Weidel, W. (1958). Annu. Reu. Microbiol. 12,27-48. Weston, L., Drexler, H., and Richardson, S.H. (1973). J. Gen.Virol. 23, 155-158. Westphal, O., Luderitz, O., and Bister, F. (1952). 2. Naturforsch., B: Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 7B,148-155. White, P. B. (1936). J. Pathol. Bacteriol. 43, 591-593. White, P. B. (1937). J. Pathol. Bacteriol. 44, 276-278. Yamamoto, N. (1958). J . Bacteriol. 75, 443-448. Zinnaka, Y., Oshikata, H., and Takeya, K. (1966). Med. Bid. 72, 220-223.

ADVANCES IN VIRUS RESEARCH, VOL. 29

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION AND RESISTANCE OF PLANTS TO VIRUSES J.

G.Atabekov and Yu. 1. Dorokhov

Department of Virology and Laboratory of Molecular Biology and Bioorganic Chemistry Moscow State University Moscow, USSR

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Transport of Virus Genome from Infected to Healthy Cells: An Active Virus-Coded Function . . . . . . . . . . . . . . . . . . . . . . . . A. The Phenomenon of Transport and the Role of Cell Plasmodesmata . . B. Ways of Determining the Efficiency of Transport . . . . . . . . . . C. The Structure of Tobacco Mosaic Virus Genome and the Virus-Coded Transport Function . . . . . . . . . . . . . . . . . . . . . . . 111. Resistance of Plants to Viruses as a Problem of Transport of the Virus Genome from Infected to Healthy Cells. . . . . . . . . . . . . . . . . A. Extreme and Facultative Resistance of Plants to Viruses . . . . . . . B. Role of Transport Function in Virus Host Range Control . . . . . . . C. Two Conceivable Means of Cell-to-Cell Transport: Modification of Plasmodesmata and Suppression of the Plant Defense Reactions. . . . D. The Subliminal Symptomless Infections . . . . . . . . . . . . . . IV. The Transport Form of Viral Infection . . . . . . . . . . . . . . . . . A. Mature Virions Do Not Appear to Participate in Infection Transport. . B. Detection and Properties of Virus-Specific Informosome-Like Ribonucleoprotein (vRNP) . . . . . . . . . . . . . . . . . . . . C. Structure of vRNP . . . . . . . . . . . . . . . . . . . . . . . . D. Functions of vRNP . . . . . . . . . . . . . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION At the moment of inoculation of a plant with a virus only a very small number of cells become infected. The virus replicates in these primarily infected cells and then moves to the neighboring healthy cells. It has been widely accepted that the transport of infection (the cell-to-cell movement and systemic spreading of the virus) is a passive process, i.e., that the infective material accumulates in the primarily infected cells and, as its concentration increases, migrates then to the surrounding healthy cells. Almost 30 years ago it had been assumed that “the rate of intercellular 313 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039829-X

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virus spread may depend solely upon the rate of production of infectious units within a cell, the velocity of cyclosis and the number of available cell exits in the form of plasmodesmata” (Rappaport and Wildman, 1957). The notion on the transport of viral infection as a passive, i.e., not a virus-coded, process has persisted until recently (see, e.g., reviews by De Zoeten, 1981;Matthews, 1981). However, considerable evidence collected during the last years allows us to consider in the present review the transport process as a distinct virus-specific function- transport function (TF)-performed by virus-coded (or virus-induced) protein(s). Until not long ago the virus genome was held to be transported either in mature virion particles or in the form of free RNA. Recently special virus-specific ribonucleoprotein particles (vRNP) were found to be formed in the virus-infected plant. They differ from the virion in structure and contain substantial amounts of subgenomic and a relatively small quantity of genomic viral RNA(s); besides, vRNP contains virus-specific proteins (Dorokhov etal., 1983a). Evidence is available favoring that vRNP plays the part of the transport form of viral infection (Dorokhov etal., 1983b), and contains the subgenomic RNA coding for the transport protein as well as probably the transport protein itself. If the transport event does not take place (i.e., the virus cannot be transported to the surrounding healthy cells) the plant remains healthy as the portion of the primarily infected cells containing the virus progeny is infinitesimal. In those cases when the TF cannot be performed the plant behaves as resistance to the virus: virus propagation is restricted to the primarily infected cells owing to the blockage of the TF in the particular virus - host combination. In certain cases the virus infection does not spread systemically over the infected plant but is localized within (or near from) the site of entry-the virus replication is inhibited by the defense reactions of the host. Although considerable success has been reached in the study of different forms of resistance of plants to virus infection, it would be beyond the scope of this review to discuss the phenomena of hypersensitivity, acquired resistance, and cross-protection covered by other reviewers: the reader is referred to recent reviews of Kassanis (1981), Sela (1981), Fraser (1982), (1982), and Van Loon (1982, Gianinazzi (1982, 1983), Loebenstein etal. 1983). Only the aspects of the problem of localized resistance will be considered, which are related to the phenomenon of movement (transport) of the virus genetic material over the infected plant.

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11. TRANSPORT OF VIRUSGENOME FROM INFECTED TO HEALTHY CELLS: AN ACTIVE VIRUS-CODED FUNCTION A. The Phenomenon of Transport and the RoleofCell Plasmodesmata During the primary infection of the plant the virus particles penetrate through microinjuries into the cells of the epidermis and probably into occasional cells of the mesophyll (Sulzinski and Zaitlin, 1982). Further systemic spreading of infection takes place by two ways: (1)slow cell-tocell movement (short distance transport) in the parenchyma and (2) rapid migration over long distances via the conducting tissues (long-distance transport). The long-distance transport of plant viruses occurs usually in the phloem, and in a few cases in the xylem. The phenomenology of shortand long-distance transport has been extensively discussed in a number of reviews (Bennett, 1956;Esau, 1956; Schneider, 1965; Gibbs and Harrison, 1976; Zhuravlev, 1979; Matthews, 1981). The plant cell protoplasts are interconnected with strands of cytoplasm, thus forming a united system, the symplast. The cytoplasmic bridges connecting the adjacent cells are known as plasmodesmata. It is universally believed that it is the plasmodesmata that play the role of the transport channels through which the infective principle is transferred from cell to cell, although the virus particles have been visualized in sieve pores as well (for review, see Bennett, 1956; Matthews, 1981). The diameter of plasmodesmata is estimated differently by different authors and depends on a number of factors (see reviews by Robards, 1975;Zhuravlev, 1979). In some cases the plasmodesmata may be quite sizable, reaching 200 and even 500 nm. Their number also varies in different tissues and depends on the plant species. There have been numerous reports on the presence of the virions of plant viruses inside the plasmodesmata of infected tissues; these observations by no means signify that the virion is the obligate transport form of viral infection (see further), but give an idea about the relative dimensions of plasmodesmata. In recent years some information has been obtained on the structure of plasmodesmata. They turned out to be not simply homogeneous cytoplasmic strands joining adjacent cells; their composition includes a desmosoma1 element (desmotubules) resembling microtubules (Robards, 1975). By virtue of the existence of the system of plasmodesmata, a joint continuous meshwork is formed of the endoplasmic reticulum of neighboring cells in the symplast. This, however, does not mean that the first act of infection of a single cell would suffice for the subsequent systemic spreading of infection throughout the symplast.

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The systemic cell-to-cell spreading is a discrete process in the sense that virus replication in the primarily infected cell and transport of its genome into the adjacent healthy cell are two separate events. Though the infective principle is transferred via the plasmodesmata, it nevertheless seems likely that the plasmodesmata of the originally infected cells are "closed" for the virus, i.e., do not permit the infection to pass into the adjoining healthy cells. The virus must act on the cells so as to modify them and thereby open the gates for the migration of the virus genetic material to the healthy cells. The rate of virus transport in the infected tissue appears to be determined by the number of plasmodesmata connecting the neighboring cells (Wieringa-Brants, 1981); the increase in the number or size or structural alterations of plasmodesmata may favor enhanced transport of infection (Shalla etal., 1982; Sulzinski and Zaitlin, 1982). In other words, it is possible that the transport function consists in a certain modification of the infected cell (e.g., proteolytic degradation of tubular elements, increase of the number or size of plasmodesmata). The latter suggestion is supported by some observations on the modification of plasmodesmata in virus-infected plants (Esau etal., 1967;Davison, 1969; Kitajima and Lauritis, 1969; Esau and Hoefert, 1972; Kim and Fulton, 1973; Chamberlain et aL, 1977) and is appealingly simple. Unfortunately, nothing is yet known about the molecular mechanism of the modification of plasmodesmata during the transport of infection. The above-mentioned speculation concerning the proteolytic degradation of the tubular elements of plasmodesmata by a virus-specific transport protein cannot be ruled out by the experimental data now a t hand. Several viruses were shown to code for proteolytic activities cleaving the precursor proteins, the translation products of the viral RNA. Cowpea mosaic virus (CPMV) has been reported to code for such protease(s) (Franssen etal., 1982; Goldbach and Krijt, 1982; Goldbach etal., 1983). The proteolytic activity is encoded in B-RNA of CPMV, which can replicate independently but needs expression of M-RNA for efficient cell-tocell transport (Rezelman etal., 1982). The results of Rezelman etal. (1982) allow one to suggest that the transport coded for by M-RNA of CPMV is performed by particular polypeptide(s) formed upon the cleavage of the primary translation product. Such polypeptide(s) can be thought to have proteolytic activity. As a result of a number of consecutive acts of short-distance transport the infection moves through the plasmodesmata from cell to cell within the parenchyma tissue. In each of the newly involved parenchymal cells the virus replicates to form a progeny of daughter particles. The systemic infection developing on the basis of short-distance transport at a certain

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stage affects the cells of phloem parenchyma, companion cells, occupies the sieve tubes, and further proceeds by the pathway of assimilate transport through the phloem. Penetration of the infective principle (the transport form of the virus) into the phloem cells signifies the transition from short- to long-distance transport of infection. In the long-distance transport the infective agent is carried passively in the flow of metabolites. The critical step in this process is not the long-distance transport through the vessels as such, but the release of the infective material into the conducting system, its transport from the mesophyll cells to the sieve tubes of the phloem. The transfer to the sieve tubes can be supposed to take place via the plasmodesmata connecting the cells of the parenchyma and the conducting bundle, i.e., to be the concluding act of short-distance transport. Further, the infective material is “ejected” into the conducting tissue and passively transported in the flow of metabolites. In other words, the short- and the long-distance transport are two intimately connected processes, and the virus genetic material most probably participates in both as a physically invariant “transport form” (see below). By the distance and rate of migration Schneider (1965) discerns a third mode of the transport of viral infection, intermediate-distance transport. In elongated cells of phloem parenchyma the virus is transported twice as fast as in the cells of the leaf mesophyll (Weintraub etal., 1976). There are also carrier cells positioned between the parenchyma cells and conducting elements (Hiruki etal., 1976). The carrier cells are modified sieve elements and have in their walls specific protrusions which enhance the efficiency of their exchange with the surrounding tissue (Esau, 1972,1978; Cronshaw, 1974). The carrier cells are connected by numerous plasmodesmata with sieve elements, parenchyma cells, and with each other. They appear to link the rapid and the slow transport. Obviously the phloem cells have a well-developed system of connections with the parenchyma cells, which ensures the virus migration from the vascular system to the mesophyll and back. The viruses appear to be distinctly specific in the manner in which they are transported within the plant: 1.Typical viruses of the so-called “mosaic group” are introduced into the host by mechanical inoculation and produce symptoms like a combination of yellow and green patches on leaves. Typical mosaic viruses are not restricted to any specific tissue, and the virus (e.g., tobacco mosaic virus, TMV) multiplies in all types of living tissues of the host and is distributed through all tissues including the phloem and even the xylem (Esau, 1956). These viruses multiply primarily in the parenchyma and then enter the vascular bundles of the inoculated leaf.

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Thus the following transport events take place during the spreading of a mosaic virus over the host plant. First, the cell-to-cell transport occurs within the parenchyma of the originally infected leaf. Then the virus penetrates the vascular system, moving from the parenchyma into the phloem of the same leaf. The infection is then passively carried through the sieve elements of the stem downward to the roots and upward to the upper leaves. The final event is the penetration of the virus from the phloem into the parenchyma of a secondarily infectedleaf (and subsequent cell-to-cell transport within it). Thus, the typical mosaic viruses like TMV can effectively move in two directions: from parenchyma into phloem and back. On the other hand, they fail to cause systemic infection when experimentally introduced into the vascular system of the host plant (Caldwell, 1931; Matthews, 1981; Taliansky et al., 1982a,b) through the cut stem or petioles. It can be suggested that normally no movement of the virus occurs in the xylem vessels although TMV probably can penetrate the xylem (Esau and Cronshow, 1967). In any case, having been experimentally introduced into the xylem the mosaic virus is unable to leave the vessels and move into the parenchyma. A certain barrier can be assumed to operate on the way of transport of such viruses as TMV and turnip yellow mosaic virus (TYMV) from xylem to parenchyma. 2. In contrast to the first group, a certain group of viruses is distinguished by their ability to move from the tracheary elements into the mesophyll cells. Schneider and Worley (1959a,b) working with the southern bean mosaic virus (SBMV) obtained results that are best explained by the movement of the virus particles in the water stream in the xylem with their subsequent transport and replication in the surrounding living cells of the undamaged leaf. In this case the transport of the virus from the xylem to parenchyma cells seems not to be barred. 3. The third group may be represented by the beet yellows virus (BYV) which appears to primarily affect the phloem tissues; hence it is able to pass into the mesophyll (Esau, 1960a). Probably BYV does not multiply in the enucleated cells of mature sieve elements and its replication is confined to the young ones (Esau, 1960b). 4. The fourth group can be recognized as phloem-limited viruses. These species are associated with the phloem, i,e., multiply and move there and remain restricted to the phloem invariably. For example, geminiviruses and luteoviruses are confined to the phloem tissue, namely sieve tubes, phloem parenchyma, and companion cells (Kubo, 1981). The phloemlimited viruses are normally unable to move from the phloem into parenchyma cells, though they can multiply in the isolated mesophyll protoplasts (Imazumi and Kubo, 1980a) as well as in the primarily inoculated epidermal cells of the host plant (Imazumi and Kubo, 1980b).

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It has for a long time been known (for review, see Bennett, 1956; Esau, 1956) that some plant viruses are restricted to specific tissues of the infectedplant, but this phenomenon has never been discussed in terms of the blockage of an active virus-specific transport function which should be performed to release the virus beyond the tissue to which the virus is confined.

B. Ways ofDetermining the Eficiency ofTramport In the studies of the systemic spreading of viruses this phenomenon is usually assessed only qualitatively, without applying quantitative criteria of transport efficiency. Not infrequently the rate of virus expansion through the infected plant is determined by assaying in time the infectivity of extracts from leaves not subjected to mechanical inoculation and situated above (or below) the virus-inoculated leaf (for review, see Matthews, 1981). With such an approach, the observedoverall rate of transport is the resultant of the rates of slow cell-to-cell transport within the inoculated leaf, rapid long-distance transport through the vessels during the spreading of the infection beyond the inoculated leaf, and the final movement of the infection from the phloem again to the parenchyma. This approach does not seem to be always correct since the rates of the short- and longdistance transport may depend on different factors (see below). It is known that local lesions caused by different TMV strains in Ngene-carrying hosts may substantially vary in size. It seemed quite reasonable to use the kinetics of lesion growth for the comparative assessment of TF in different virus strains and mutants (Rappaport and Wildman, 1957;Siegel, 1960). Indeed, the TMV mutants Lsl and Ni2519 form small lesions on the necrotic host plant, unlike the normally spreading strains. It is, however, doubtful that this feature can be used as a universal criterion for detecting transport mutants. For instance, it is known that the bean TMV strain (dolichos enation mosaic virus, DEMV) produces small lesions in Nicotianaglutinosa L plants, which does not appear to prevent this virus from normally spreading in systemically infected bean plants. Some TMV mutants defective in the coat protein are also known to form small (as compared to uulgare) lesions on necrotic hosts (Sehgal, 1973); yet the decrease in the lesion size is not directly related to this property, as other defective mutants of this type produce large lesions (Kapitsa et at., 1969; Bhalla and Sehgal, 1973). The doubtfulness of assessing the virus-specific T F by the size of lesions becomes especially obvious on admitting that the lesion size and the virus transport depend not only on the virus itself but also on the strength of the defense reactions of the N-gene-carrying host. The necrotization of virus-infected cells is not a prerequisite for the

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localization of virus infection (Cohen and Loebenstein, 1975; Roberts, 1982), i.e., other active antiviral reactions should be responsible for the virus localization (for reviews, see Van Loon, 1982, 1983; Loebenstein et al., 1982). It was concluded by Van Loon (1982) that “the localizing mechanism is operative in neighboring not yet infected cells rather than in the cells in which the virus is actively multiplying”; however, it is widely accepted that the resistance localizing the spreading of the virus is based on the inhibition of virus replication but not on the suppression of the TF as such. The lesion size is probably determined by several factors: (1)the efficiency of the virus-specific TF as the performance of the transport gene; (2) the efficiency of the virus in inducing the defense reactions of acquired resistance (AR) in the host plant; (3) the efficiency of the plant antiviral reactions; and (4) the rate of necrotization and callose accumulation in the infected cells. In this connection an obvious advantage is offered by the fact that AR and antiviral defense, as well as the very lesion formation in N-gene-carrying plants, are temperature-sensitive; no lesions are formed a t 32 -33°C and the infection spreads systemically (Samuel, 1931; Jockusch, 1966~). Fraser (1983) reported that, similarly to the N gene, the hypersensitive reaction of the N’ gene is temperature-sensitive. This circumstance makes it possible to estimate the efficiency of virus transport under switched-off reactions of AR and lesion formation (32-33°C) with subsequent transfer of the plant to 22- 24°C to restore the necrotic reaction (Jockusch, 1966a). The temperature-shift treatment (TST) permits, after the formation of the primary lesion (3 days at 22”C),necrotization to be suppressed (2 days a t 32-33°C) so as to allow the radial systemic spreading of the infection. Upon subsequent transfer of the plant back to 22°C the necrotization is again switched on, entailing an appreciable expansion of the lesion owing to the formation around the primary lesion of an aureole-an additional zone of cells taken over by the wave of virus transport a t 32-33°C and necrotized upon the return to 22°C. TST is a convenient means of testing the t s phenotype in transport. By the use of TST it is easy to establish, for instance, that the Nil18 TMV mutant ts in the coat protein is trin the transport function (active in spreading under switched-off necrotization at 32 -33°C). On the other hand, Ni2519 and Lsl behave in this test as ts in transport (lack of lesion growth after 32-33°C) (Figs. 1and 2). The efficiency of short-distance transport can be also assessed by immunofluorescence microscopy. In this case the spreading of the viral infection from cell to cell is visualized by detecting the viral antigen with fluorescent antibodies. Under blocked infection transport this method

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FIG.1. Lesions produced in an inoculated leaf of Xanthi-nc plants kept for 5 days in a greenhouse at 22 to 30°C. The right half was inoculated with Lsl(O.1 pg/ml) and the left half with L (0.1 pg/ml). Courtesy of Dr. M. Nishiguchi. FIG.2. Lesions produced in a leaf of a Xanthi-nc tobacco plant kept a t 22°C for 3 days, then at 32°C for 2 days, and finally a t 22°C for 1day. The upper half was inoculatedwith Lsl (0.1 pg/ml) and the lower half with L (0.1pg/ml).

reveals mainly individual infected (antigen-containing) cells; on the other hand, under conditions of active transport the fluorescent antibodies stain groups of infected cells (Nishiguchi etal., 1980). The most proper procedure to estimate the efficiency of short-distances (1978). The leaves transport in infected tissue is that of Nishiguchi etal., of a systemic host are inoculated with the virus and kept under conditions optimal for the onset of infection and initial transport. Then disks are cut out of the inoculated leaves and incubated for some more time; the controls are disks without such incubation. The efficiency of virus transport in infected tissues is judged from the increment in the percentage of virus-infected (antigen-containing) cells in a disk. The determination of the short-distance-transportrate of the virus on the basis of the increase in the

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number of infected cells in the disk per unit of time is a quite proper technique, yet it is desirable to combine it with the assay of infectivity under the same conditions related to the number of infected cells. C. The Structure of Tobacco MosaicVirusGenome and theVirus-Coded Transport Function 1. Molecular Organization of TMV Genome

The structure and expression of the TMV genome have been discussed in detail in a number of reviews (Atabekov and Morozov, 1979; Davies, 1979;Hirth and Richards, 1981;Davies and Hall, 1982;Van Vloten-Doting and Neelman, 1982). This issue is considered below only in the aspect of the transport of the virus genetic material from infected to neighboring healthy cells as a function encoded in the genome of the virus itself. In addition to normal host products preexisting in the healthy cell, two types of products participating in virus replication arise owing to the viral infection and expression of the virus genetic material: (1)virus-specific proteins, i.e., the proteins coded by the viral genome, and (2) virus-induced proteins, i.e., those coded for by the cell genome but activated upon viral infection. It can be assumed that the synthesis of virus-induced proteins is either stimulated or derepressed by infection but they are invariably coded for by the host cell. Induction of this type of products can be exemplified with interferon formation in virus-infected animal cells and with the production of the so-called pathogenesis-related proteins in virus-infected plants (for review, see Van Loon, 1983). Unfortunately, it is difficult to discriminate experimentally between virus-coded and virusinduced proteins. The genomic TMV RNA directs in cell-free systems from wheat embryos or rabbit reticulocytes the synthesis of two polypeptides (MW 130 X lo3 and 165 X lo3) with overlapping amino acid sequences (Knowland etal., 1975; Beachy etal., 1976; Pelham and Jackson, 1976; Pelham, 1978). No synthesis of the TMV coat protein (MW 1978; Scalla etal., 17,500) is observed upon translation of the genomic RNA (Hunter etal., 1976;Zaitlin etal., 1976);its synthesis inuiuorequires formation of a short subgenomic RNA (LMC, low-molecular-weight component), which directs the coat protein synthesis in a cell-free system as well (Jackson etal., 1972; Siege1 etal., 1973, 1976; Hunter etal., 1976). In infected leaf tissues, besides LMC and genomic TMV RNA, a heterogeneous population of RNA molecules is synthesized with molecular weights ranging from 0.6 to 1.6 X lo6,which is at least partly present in the total RNA isolated from TMV virions (Whitfeldand Higgins, 1976;Zaitlin

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etal., 1976;Beachy and Zaitlin, 1977). The subgenomic RNA with molecular weight around 0.68 X lo6is designated as intermediate-size RNA 2 (I, RNA), and the set of RNAs with molecular weights of 0.9- 1.6 X lo6as I, RNA (Zaitlin etal., 1976; Beachy and Zaitlin, 1977). LMC, I,, and I, RNAs were shown to have a common 3’-terminal nucleotide sequence with the genomic TMV RNA (Zaitlin etal., 1976;Beachy and Zaitlin, 1977). In a cell-free protein-synthesizing system RNAs I, and I, direct the synthesis of a 30,000-MW polypeptide having no common 1976; amino acid sequence with the TMV coat protein (Beachy etal., 1976; Beachy and Zaitlin, 1977). This Bruening etal., 1976; Zaitlin etal., polypeptide is also revealed in the TMV-infected leaf tissue (Leonard and 1983) and protoplasts (Watanabe etaL,1983; Zaitlin, 1982; Joshi etal., Ooshika etal., 1983). Thus, TMV can produce in uiuoat least four polypeptides with molecular weights of 165,000 and 130,000, 30,000, and 17,500 (coat protein), whose synthesis is directed by genomic RNA, subgenomic I, and I, RNAs, and LMC, respectively (Fig. 2). It is not excluded that in virus-infected plant cells “chymeric” proteins may be synthesized, of which the amino acid sequence is coded partly by the viral and partly by the host-cell genome. This fantastic assumption stems from the analysis of the so-called H protein (MW 26,500) found in the TMV preparation in the amount of a single molecule per virion (Asselin and Zaitlin, 1978). Studies of the peptide structure of this protein testify to the presence in its composition of all the peptides of the coat protein and of another protein of host origin (Collmer and Zaitlin, 1983; Collmer etal., 1983). The mechanism giving rise to such a protein is unknown. The complete nucleotide sequence has been reported for the RNA of TMV uulgare (Goelet etal., 1982); considerable parts of RNA of cowpea have been TMV strain C, and tomato strain OM very similar to uulgare sequenced by Japanese workers (Meshi etaL,1982a,b,c). Examination of the nucleotide sequences of TMV RNA (common strain) showed the presence of three open reading frames in the virus genome (Fig. 3). The first open reading frame corresponds to the protein of molecular weight 125,941 (130K protein) which terminates at the UAG triplet at residue 3417 from the 5‘ end, and its readthrough product is a protein of molecular weight 183,253 (165K protein). The right-hand portion of the cistron coding for the 130K and 165K protein overlaps by five codons with a reading frame, in the second phase, that codes for a protein of molecular weight 29,987 (30K protein). The gene coding for the 30K protein is terminated by the UAA triplet two nontranslated nucleotides before the initiator codon of the coat protein (Guilley etal., 1979; Goelet etal., 1982). The nucleotide sequences

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amber terminator UA6

;

mG ’

p 130

T

I

PI60

r

t

I

V

7 r

.

RNA molecule =translation initiation m’G = cap I = translation termination t = aminoacylating n = protein p = MW x 10-3 CP= coat protein =

I

P 87

I

P 83

I

.

I

. P46

,

.

’

’

’

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FIG.3. A tentative map of the TMV (common strain) genome. For explanation see text.

coding for the 30K protein of common and cowpea C, strains have been compared by Meshi etal. (1982a,b). It turned out that the 30K protein of the C, strain is 15 residues longer than that of the common strain. In contrast to the common strain, the 30K cistron of the C, strain overlaps with the coat protein gene and is terminated within its initial region. Therefore, the 30K gene of the C, strain overlaps by its left portion with the 165K gene and by its right portion with the coat protein gene. In addition to LMC and subgenomic RNA coding for the 30K protein, a family of 3’-coterminal and colinear messengers (probably subgenomic RNAs) have been found by Goelet and Karn (1982). These RNAs can be

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encapsidated (since they contain the origin of assembly) and code for the proteins of molecular weights in the range 20,000-90,000. A set of proteins can be translated in uitro from the encapsidated subgenomic RNAs, including those of molecular weights 30,000,29,000 and 23,000 which have overlapping COOH-terminal sequences (data of T. Hunter, R. J. Jackson, and D. Zimmern, referred to by Goelet et al., 1982). The origin and significance of the population of 3'-colinear subgenomic TMV RNAs is obscure. It should be noted that there is no direct evidence that all the members of this population are functionally essential and code for functionally active TMV-specific proteins. It cannot be excluded that at least partly these RNAs are produced by inefficient termination of the transcription of the genomic RNA into negative strands with subsequent synthesis of the positive strands to yield double-stranded (ds) RNA. It is noteworthy that a set of discrete dsRNAs presumably serving as the replicative form (RF) for subgenomic RNAs (sub-RF) have been isolated from TMV-infected cells (Zelcer and Zaitlin, 1981; Dawson and Dodds, 1982), including the sub-RF for the 30K protein gene. Interestingly, no sub-RF corresponding to LMC has been found. A reasonable suggestion is that with TMV and some other plant viruses (Smit and Jaspars, 1982; Nassuth et al., 1983) the subgenomic RNAs coding for the coat protein are not produced by autonomous replication through sub-RF formation. 2. Analysis of T M V Mutants

Certain advance in studying the structure of TMV genome was achieved through analysis of mutants. It must, however, be admitted that though quite a number of TMV mutants have been described, in the overwhelming majority of cases it is difficult if at all possible to establish the connection between the phenotypic manifestation of mutation(s) and the virus-specific function affected. For instance, mutants selected by particular symptoms they produce on appropriate indicator plants (e.g., by their ability to cause lesions on N.siluestris Speg and Comes) may actually have different functions impaired, i.e., differentiating between these mutants requires other more specific tests. The properties and peculiarities of behavior of TMV mutants with mutations in different genes have been discussed in a number of reviews (see, e.g., Atabekov, 1977). Here TMV mutations are analyzed with respect t o their possible influence on the T F of the virus. a. Coat Protein Mutants. We still cannot definitely state that the coat protein has any other function(s) besides the structural one; mutation in this gene seems to have no dramatic effect on the expression of other viral genes. It is well known that in the absence of an active coat protein RNA of many viruses can replicate to form infective daughter RNAs that are not

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protected from RNase action in the sap of the infected plant. Consequently, the infective principle is unstable without the coat protein (Jockusch, 1966a). Various forms have been described of “defective” mutants in the coat protein that are deficient in their ability to produce mature TMV particles. Siegel etal. (1962) isolated two unusual TMV mutants deficient in production of mature TMV particles. No virus-like nucleoprotein particles could be isolated from plants infected with these mutants; in the cells of such plants the viral RNA is not protected with the coat protein. These coat protein mutants were designated as PM1 and PM2. Defective mutants spread from cell to cell but usually remain within the inoculated leaves. The long-distance spreading of the infection from the lower inoculated leaves to the upper ones occurs extremely slowly (Siegelet aL, 1962;Sarkar, 1969;Kapitsa etaL,1969;Bhalla and Sehgal, 1973). The deep suppression of the long-distance transport is characteristic of all TMV mutants defective in the coat protein, which “tend to travel in vascular parenchyma, and do not enter the conducting elements or are inactivated there” (Siegel and Zaitlin, 1964). The causes of suppressed long-distance transport in the absence of the coat protein are discussed below (see Section IV). Recently a new interesting mutant (DT-1G) defective in the coat protein has been described which differs from all previously known by its complete inability to produce the coat protein (Sarkar and Smitamana, 1981a,b). Irrespective of the causes of the absence of the viral protein, the DT-1G mutant offers an attractive model for studying the influence of the coat protein on the transport function. It turned out that the DT-1G infection lacks symptoms and the infective principle is present in the green tissues of the infected leaf (Gfrom “green”). This prompts a conclusion that even mutation(s) causing the complete abolishment of the synthesis of the TMV structural protein do not preclude the short-distance transport of the virus, i.e., the coat protein is not indispensable for the expression of the TF. It cannot be excluded, however, that in this case the TF is also somewhat suppressed; this can be inferred only from the smaller lesions produced on leaves of Xanthi-nc tobacco by the DT-1G (as compared to vulgure) (Sarkar and Smitamana, 1981a). It is quite probable that the transport efficiency is lower simply because of the lack of mature virus particles which, albeit not obligate for the TF, nevertheless provide an additional possible form of near and long-distance transport of the infection. In the systemic spreading the virus may probably use different forms of particles to transfer the viral genome, which functionally complement each other. We should specially dwell on the effect of temperature-sensitive ( t s ) mutations in the coat protein gene on the expression of the TF.

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Jockusch (1966a) showed that various TMV ts mutants fall into two classes: 1. The tsmutants of class I are typically represented by the mutants Nil18 and flauumand possess the following characteristics. A t nonpermissive temperatures viral particles are formed in negligible amounts; the infectivity of the extracts at 30-33°C is low or entirely lacking. Under nonpermissive conditions, cells infected with tsImutants accumulate infectious viral RNA unprotected or partially protected by the protein, i.e., the situation is in principle similar to that reported for plants infected with defective mutants of the PM series (see above). The behavior of the ts1 mutants at high temperatures and of the constitutively defective PM mutants is also similar in respect of the T F their long-distance transport is suppressed but they retain the cell-to-cell transport, although it may also be partly inhibited. Hence provisionally both these types of mutants (PM series and tsI) can be regarded as defective in the coat protein synthesis but active in the short-distance transport and RNA replication. For several other plant viruses the coat protein gene has been shown to be not essential for viral RNA replication (for references, see Sarachy etaL, 1983). 2. TMV tsmutants of class I 1contain a mutation in a portion of the virus genome beyond the coat protein gene. Informaton concerning the properties and functions of nonstructural proteins encoded in the TMV genome is quite scanty. Class I 1 mutants are considered below.

b.Non-Coat-Protein Mutants.We do not know for certain how many other genes essential for the virus replication the TMV RNA contains besides the coat protein gene. Unfortunately, in those rare cases when the mutant can be assigned to the tsII class in Jockusch’s terminology, it is rather difficult to reliably identify the affected gene(s). Among the tsmutants deficient in the nonstructural protein function there are grounds for discerning at least two types of mutations whose phenotypic expression may affect the TF. i.Mutations which influence thesynthesis of genomic andlor subgenomic RNAs. According to the classical scheme, the parental plus-strand RNA molecule serves as a template for the synthesis of RNA chains through formation of dsRNA (RF) and partial dsRNA, or the replicative intermediate. Subsequently the minus-strand RNA direct the synthesis of complementary daughter RNA plus chains. It is assumed that a virus-specific enzyme, RNA-dependent RNA polymerase (or RNA replicase) is responsible for the replication of the virus genome. More exactly, viral RNA is replicated in the so-called replication complex consisting of viral RNA, virus-specific and/or cell-specific proteins. It can be concluded that an indispensable function in the replica-

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tion of plant virus RNA is performed by some nonstructural virus-coded protein(s). This conclusion is supported by the existence of mutants conditionally lethal in this function (Dawson and White, 1978,1979;Dawson, 1983). Unfortunately, no definitive evidence has been obtained on the structure and even the origin of the RNA replicase activity in the virus-infected plant (for references, see Dorssers etal., 1983). A number of indirect facts accumulated in recent years argues in favor of the host (and not the virusspecific) origin of the enzymic system providing for the synthesis of viral RNA in the infected cell. Firstly, some (albeit rather low) RNA-dependent RNA polymerase activity was found in the healthy cells of some plants (for review, see Ikegami and Fraenkel-Conrat, 1979;Dorssers etal., 1983; Fraenkel-Conrat, 1983), and preparations of RNA polymerase isolated from healthy and from TMV-infected cells proved similar in several respects (Romaine and Zaitlin, 1978). Second, RNA polymerase preparations isolated from plants infected with different viruses did not show template specificity (Semal and Hamilton, 1968; May etal., 1970; Zaitlin et al., 1973; Weening and Bol, 1975; Le Roy etal., 1977), i.e., showed no significant preference for the RNA of the infecting virus as template. Third, the products of polymerase reaction, when analyzed, were shown to be small and heterogeneous (for review, see Miller and Hall, 1983). On the other hand, a considerable body of evidence suggests that viruscoded product(s) participate in the replication of plant virus RNA (De Jager etal., 1977; Dawson and White, 1978, 1979; Hardy etal., 1979; Goldbach et al., 1980;Robinson et al., 1980; Nassuth et at., 1981;Bujarski et al., 1981; Nassuth and Bol, 1983; Sarachy etal., 1983). Dorssers etal. (1983) showed that two different RNA polymerase activities are operative in CPMV-infected cowpea, one associated with replicating viral RNA and the other representing the host-coded RNA-dependent RNA polymerase system. Very important data of Hall and co-workers (Bujarskiet al., 1982; Miller and Hall, 1983) show that RNA 1 of brome mosaic virus (BMV) codes for the template-specific active viral replicase. The question of the coding of the TMV-specific polymerase remains obscure, most probably because of the peculiarities of the system and the difficulties of detecting minor TMV-coded protein( s) in RNA replicase preparations isolated from TMV-infected plants. The facts available at present allow of three possibilities: (1)The TMV RNA replicase is a virusinduced protein, i.e., its synthesis is directed by the cell genome but activated upon viral infection; (2) the RNA replicase is a virus-specific product, encoded in the viral genome; (3) the RNA replicase system is of mixed origin and contains, besides the cell components (which is characteristic of many RNA replicase systems), the virus-specific protein(s) as well, probably as minor constituents.

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It is not clear whether any connection exists between the mutations affecting the replication of viral RNA and the virus TF. No information is available in the literature on the dependence of the transport rate on the rate of synthesis of virus components (RNA, coat protein) in the infected cell. The lack of such information complicates the interpretation of a number of facts; in particular, it would have substantially clarified the central question whether the transport is an active virus-coded function. An adherent of the passive transport, De Zoeten (1981) claimed that a membrane-associated dsRNA (the replication complex) is the form in which viral RNA can be transported from cell to cell, and speculated that the “transport of infective entity must be achieved in a passive manner” following “the movement of the normal cell constituents.” Assuming that the transport of the infective principle (as free viral RNA or in another form) is passive, there should be a dependence between the efficiency of the synthesis of viral RNA and the rate of the cell-to-cell transport of infection. On the other hand, it is quite obvious that in any case the ts mutations affecting the replication of genomic RNA would inevitably manifest themselves as temperature-sensitive in the TF; mutations suppressing the synthesis of genomic RNA and mutations in transport would be phenotypically similar. Therefore the question can be rephrased: are there real mutants in TF, or does the decline in the transport rate merely reflect the decreased synthesis rate of genomic TMV RNA? If the TF is performed independently from RNA replication (i.e., by the protein product of a special transport gene), then mutants must exist that are ts in transport by trin RNA replication. TMV mutants temperaturesensitive only in transport have indeed been described; the genomic RNA of such ts mutants is normally replicated but the virus transport is inhibited at nonpermissive temperature (see further). It may be assumed that genomic TNA replication and the synthesis of subgenomic TMV RNAs are two independent enough processes, i.e., the synthesis of subgenomic RNAs is controlled by a particular virus-specific protein. If this suggestion is correct, one should expect mutations in the TMV genome affecting the synthesis of different subgenomic TMV RNAs (LMC and I-class RNAs) but not of the genomic RNA. Would such mutations tell on the transport function? Since the production of the TMV coat protein is not obligate for the virus transport, a mutation suppressing the synthesis of the subgenomic LMC RNA is hardly likely to have appreciable influence on the TF. On the other hand, mutations affecting the synthesis of the I-class subgenomic RNAs can be thought to have basically different phenotypic manifestations. Earlier we have suggested (Atabekov and Morozov, 1979) that the 30K protein, the product of translation of the intermediate-class

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subgenomic RNAs, is the transport protein. If this is so, the transport function would be inhibited to an equal extent by (1)mutations causing the ts behavior of the transport gene product and (2) mutations suppressing the synthesis of subgenomic RNAs coding for this protein. From this it followsthat with the ts mutations exhibiting retarded transport there are at least three conceivable causes giving rise to this phenotype: (1)temperature sensitivity of the synthesis of genomic RNA, (2) temperature sensitivity of the synthesis of subgenomic RNA coding for the putative virus-specific transport protein, and (3) temperature sensitivity of the transport protein as such. The idea of the existence of a special transport gene independent of the RNA replicase gene(s) is compatible with the results of the studies on the expression of the multipartite genome of CPMV (see a review by Jaspars, 1974). The heavy (B) component of CPMV RNA was known to code for functions related to viral RNA replication (Goldbach et al., 1980). Recently Rezelman etal. (1982) showed that B-RNA is capable of infecting cells and replicating there; however, the infection is not transferred to adjacent cells since the TF is encoded in the other genomic component, the light CPMV RNA (M-RNA). Thus the replicase and the transport protein are coded for by B-RNA and M-RNA, respectively. It is of interest that in another bipartite virus, the tobacco rattle virus (TRV), both these functions (RNA replication and transport) are encoded in the heavy RNA. Inoculation of plants with the heavy TRV component results in RNA replication and systemic spreading of the infection which exists in an “unstable form” (Sanger and Brandenburg, 1961; Cadman, 1961). ii. Mutations in thetransport gene. At least three TMV mutants temperature-sensitive in the function of systemic spreading have been isolated Ni2519 (Jockusch, 1968), 11-27 (Peters and Murphy, 1975), and Lsl (Nishiguchi et aL, 1978). It seems reasonable to assume that acertaingene product nonfunctional at restrictive temperatures is responsible for this effect. Strain Lsl has been derived from the temperature-resistant tomato strain L (Nishiguchi et aL, 1978); its temperature sensitivity manifests itself as the block of cell-to-cell transport of infection at nonpermissive temperatures (32- 33“C). Under such conditions the virus replicates only in the primarily infected cells. It must be emphasized that Lsl is not ts in either RNA replication, coat protein synthesis, or virion assembly; the virus normally infects and reproduces in cultured protoplasts at 32’ C, the temperature nonpermissive for Lsl in the intact plant. Moreover, at restrictive temperature Lsl accumulates in infected cells in greater amounts than at permissive temperature (22”C),i.e., its genomic RNA is even more actively replicated at

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higher temperatures. Taking into account that the TF under such conditions is completely inhibited, one can suppose that the transport of the viral genome into neighboring cells does not directly depend on the amount of genomic RNA produced in the primarily infected cell but is controlled only by the transport gene(s). The rate of short-distance transport in tissues infected with a trstrain L was found to be the same at 22 and 32 C although at 32 C the virus accumulated in infected cells in substantially larger quantities than at 22 C (Nishiguchi et al., 1978). It is not excluded that the transport depends on RNA synthesis only at certain critical RNA concentrations in the infected cell. There may be a minimal threshold RNA concentration that must be attained for the transport to become possible. It remains utterly obscure, however, why after overcoming the threshold further increase in the viral RNA concentration within the cell does not accelerate the transport of strain L temperature-resistant in this function. If the lack of dependence between the rates of transport and RNA synthesis would be proved, the transport of infection from infected to healthy cells could be thought not to be a continuous process concurrent with RNA replication, but to take place probably in a single step at a moment when the total amount of RNA is relatively small; after this the virus transport from this particular cell stops though the synthesis of the viral RNA goes on. This is in accordance with the suggestion that the 30K protein is the most probable candidate for the transport protein (Atabekov and Morozov, 1979;Leonard and Zaitlin, 1982). Ooshika et al. (1983)and Watanabe et al. (1983)succeeded in detecting the 30K protein and its mRNA in TMV-infected protoplasts and studied their time course in viva The synthesis of the 30K protein was first detected in 3- 4 hours after inoculation (before infectivity appeared). However, the 30K protein and its mRNA were transiently synthesized in 2 to 3 hours after inoculation and gradually declined afterward. In contrast, the 130K,180K,and coat protein, as well as genomic RNA and LMC, were synthesized continuously. These data suggest first that the synthesis of subgenomic RNAs of different classes is controlled by different mechanisms and, second, that the 30K protein is unstable and degrades or becomes inaccessible for the extraction relatively soon after inoculation. If the 30K protein is indeed the transport protein the T F would cease to operate simultaneouslywith this event. The transport rate may be independent of the rate of genomic RNA replication for some other reasons. For instance, the transport of the viral RNA into adjoining cells at a certain stage of virus replication may compete with virion maturation: the “flux” of the newly synthesizedviral RNA changes its direction and is switched over from the transport to the assembly of the viral particles, which probably do not participate in transport.

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It can also be assumed that the transport form of infection (see further) contains a certain host or virus-specific protein necessary for its functioning. Then the TF is controlled not by the RNA synthesis rate but by the availability and amount of this hypothetical protein. Unfortunately, we are practically ignorant of the properties and functions of the transport protein. Leonard and Zaitlin (1982) carried out a comparative peptide analysis of the 30K proteins directed in a cell-free protein-synthesizing system by RNAs from two TMV strains: L (wild type) and ts-transport mutant Lsl; a small difference was found in the peptide maps of the 30K protein coded for by these viruses. The nucleotide sequences of the 30K protein cistrons of L ( t rand ) Lsl (ts in TF) strains have been recently compared by Ohno etal.(1983). The authors found a single base substitution which caused the replacement of a proline in the L strain 30K protein by serine in the Lsl 30K protein. Detection of a single amino acid substitution in the 30K protein of Lsl compared to that of the L strain is not enough to state that this protein is the product of the transport gene. This result cannot yet be linked to the T F for several reasons. First, the 30K protein coded for by Lsl RNA was not shown to be temperature-sensitive and, second, and most importantly, there is yet no direct proof that the 30K protein performs the transport function. If nevertheless the 30K protein does perform the TF, it must be noted that different TMV strains differ dramatically in the amino acid sequences of their 30K proteins. The amino acid compositions of the 30K proteins of several TMV strains (uulgare, OM, C,, L) have been deduced from the 1982a,b;Goelet etal., nucleotide sequence data on TMV RNA (Meshi etal., 1982; Takamatsu etal., 1983; Watanabe etal., 1983). The 30K proteins coded for by different TMV strains may strongly differ in the content of particular amino acids (Watanabe etal., 1983). For example, the 30K of OM TMV contains 11methionines in the total 267 amino acid residues; in contrast, the 30K of the C, strain has only three in the total 282 (Watanabe etal., 1983). 3.Complementation of tsTransport Function by trHelperVirus

Various forms of complementation between plant viruses have long been known: during mixed infection the partner viruses provide each other with the gene products which can rescue the defective virus (for review, see Dodds and Hamilton, 1976). Pertinent examples are the phenomena of satellitism of viruses (for review, see Atabekov, 1977)and the replication of viruses with fragmented genomes (Jaspars, 1974). The possibility of complementation between plant virus mutants was first demonstrated with a number of TMV mutants in this group (Shas-

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333

kolskaya etal., 1968;Atabekov etal., 1970a,b,c) and then reproduced (Sarkar, 1969) and confirmed (Kassanis and Bastow, 1971a,b; Kassanis and Conti, 1971). Upon mixed infection of plants with two TMV strains one of which produces a tsand the other a tr coat protein, their complementation can take place under conditions nonpermissive for the ts mutant alone. This gives rise to virions with a so-called masked genome (mixed virus particles in which the RNA of the tsmutant is encapsidated with the coat protein of the trhelper). Later we used in the complementation analysis the ts TMV mutant Ni2519 (Taliansky etal., 1982a) that has no mutational defect in the coat protein gene. This non-coat mutant was derived from the natural TMV strain A14 and described as ts (Jockusch, 1968; Bosch and Jockusch, 1972). A more detailed characteristic of this mutant was given by Taliansky etd.(1982a,b) and Kaplan etal. (1982). At nonpermissive temperature (33°C) the assembly of Ni2519 particles is blocked (Bosch and Jockusch, 1972). It has been shown that in uitro the Ni2519 RNA can be reconstituted with the coat protein, yet infectious virions are formed only at permissive temperature; at restrictive temperature the assembly gives rise to defective particles. The aberrant assembly was found to result from the ts behavior of the Ni2519 RNA molecule itself, its conformational instability at 33°C. Under such conditions the assembly of the virus particles is initiated not from a single initiation site (as with trstrains or with Ni2519 RNA a t permissive temperature); at 33°C an additional assembly initiation site is demasked in the Ni2519 RNA molecule directly in the coat protein gene (Kaplan etal., 1982; Taliansky etal., 1982a,b). Thus, Ni2519 is a representative of a new peculiar class of ts virus mutants. Taking into account this peculiarity of Ni2519, it was logical to suppose that its assembly function would not be complemented by any helper virus. Indeed, complementation of the assembly of Ni2519 was shown to be impossible (Taliansky etal., 1982a). However, Ni2519 is ts not only in the virion assembly. Two distinct virus-specific functions, namely virus assembly and spreading of infection from cell to cell (TF),are t s in Ni2519 (Bosch and Jockusch, 1972). It has been recently shown by Taliansky etal. (1982a,b) that the ts TF of Ni2519 can be complemented by a helper virus. The experimental system for the complementation of virus spreading was as follows. The N'-gene-carrying plants used developed local lesions in response to Ni2519 (dependent ts virus) but formed a systemic reaction upon infection with a common TMV strain uulgare (helper tr virus). The leaves were jointly infected with a mixture of the viruses, and the plants were subjected to the TST. Since Ni2519 alone is incapable of spreading systemically a t 33"C, the local

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lesions produced by Ni2519 could increase upon TST only by virtue of complementation by the helper virus which can spread at high temperatures. The appearance of lesions surrounded by a collapsed area testified to the complementation of the cell-to-cell transport of Ni2519 at 33°C. Obviously the Ni2519 infection spreads under these conditions in a form other than mature virions, as its assembly cannot be complemented by the helper virus (Taliansky etal., 1982a). In another work (Taliansky etal., 1982c) uae was made of the ts mutant Lsl which is temperature-sensitive only in TF but normally multiplies at nonpermissive temperature in primarily infected cells and protoplasts. Complementation of the cell-to-cell movement of Lsl in the presence of a trhelper was tested either by the TST of necrotic N'-gene-carrying plants (as above) or by immunofluorescence microscopy. The results in a general form are given in Table I. It is important that the role of the helper virus in transport can be played not only by a related TMV strain but also by a quite unrelated, foreign virus that can systemically infect the particular host. Thus potato virus X (PVX) could serve as a helper for TMV-Ls1 in the cell-to-cell movement. The next experimental system used for complementing the TF in Lsl TABLE I COMPLEMENTATION OF t s TRANSPORT FUNCTION BY tr HELPER VIRUS** Virus ts TMV

Transport of the ts T("C) virus from cell to cell

strains

Lsl

33

Ni2519

33

24 24

trhelper

TMV-UI

33

24 Potato virus X

+

ts tr Lsl UI Lsl potato virus X Ni2519 UI

+ +

+

33

24 33 33 33

+ + + + + +

+

Summarized results of Taliansky etal.(1982a,d). (+) and ( - ) correspond to effective and blocked transport, respectively; the trhelper viruses spread normally at both temperatures used.

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

335

was extremely simple; yet it served its purpose. It is well known that it is impossible to infect the upper leaves of a healthy plant when the virus is imbibed through the cut stem. The virus moves along the conducting tissues of the stem (probably through the xylem), accumulates there, but cannot move to the parenchyma and infect the leaf. It was assumed that the penetration of the virus from the conducting tissues into the leaf parenchyma is a transport event, which is blocked since the TF cannot be expressed without virus replication. Therefore the influence of preinfection of the upper leaves with a helper virus (which systemically spreads there) on the transport of the dependent virus from the conducting tissues to the leaf parenchyma was studied. Such preinfection before imbibing the dependent virus into the stem was found (Taliansksy etd,1982c,d) to promote its penetration into the mesophyll of the preinfected leaf and its spreading there. The systemic infection of the plant upper leaves with the helper virus appeared to “open the gates” for the transport of the dependent virus. The helper virus renders the leaf susceptible for systemic infection with the dependent virus that otherwise - when alone- would be confined to the conducting tissues (Table 11). As can be seen from Table 11, the TF of the dependent virus can be complemented in this system not only by strains of the same virus (TMV) TABLE I1 HELPER VIRUSPROMOTES THE TRANSPORT OF DEPENDENT VIRUS** Dependent virus imbibed through the stem

Helper virus: preinfection of upper leaves

TMV U2

TMV UI No helper Potato virus X No helper TMV Nil18 ( t s coat protein, trtransport) No helper TMV L (tr transport) No helper TMV LsI (ts transport)

TMV L TMV Lsl ( t s in transport) TMV Lsl TMV L

Transport and

T replication of ( C) dependent virus 24 24

33

+++ -

24 - 33

+

24 33

Summarized results of Taliansky etal. (1982a). and (-) correspond to effective and blocked transport function. a

* ( +)

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J. G. ATABEKOV AND YU. L. DOROKHOV

but also by an unrelated helper (PVX). Thus, the T F is not very specific since an unrelated virus can assist the spreading of the dependent virus over the leaf. It would be interesting to identify and compare the transport proteins coded for by TMV and PVX; it is not excluded that the transport proteins of different viruses (capable of systemic spreading in the same host species) have basically different properties. Nevertheless such proteins must have some common functional specialties determining their transport faculty. It is evident from Table I1 that TMV Lsl can be used as a dependent virus, and its transport at nonpermissive temperature can be complemented with TMV Nil18 (ts coat protein, trtransport) or with its parental strain L. The Lsl was used not only as a dependent virus but also as a helper at restrictive and permissive temperature. As can be seen (Table 11),Lsl can serve as the transport helper for TMV L only at low temperature (24°C) but is ineffective at 33°C. It is noteworthy that when the upper leaves are preinfected with Lsl (helper) at 24°C and then the upper portion of the plant is cut off and transferred to 33' C, placing the stem into the suspension of TMV L (dependent virus), the ability of Lsl to assist the transport is lost not immediately but within several hours after the transfer from 24 to 33°C (Taliansky etal., 1982~).It seems likely that at restrictive temperature the t s transport protein of Lsl is rendered nonfunctional and its activity is gradually lost. 4.Interaction betweenViruses Possibly LeadingtoTF Complementation

Numerous different forms of nongenetic interaction between plant viruses under conditions of mixed infection have been repeatedly reviewed (see, e.g., Kassanis, 1963; Dodds and Hamilton, 1976; Matthews, 1981). The multiplication rate and/or distribution of one virus are often enhanced in the presence of another. A classical example of the synergism between plant viruses is the stimulation of PVX replication in double infection with different unrelated viruses potato virus Y (PVY), TMV, tobacco etch virus, cucumber mosaic virus (CMV). The increase in PVX concentration is attended by more severe symptoms (Rochow and Ross, 1954, 1955; Stoffer and Ross, 1961a,b). This phenomenon might be ascribed to the increased rate of PVX cell-to-cell transport in the presence of a helper virus; however, it has been found that (1)the rate of PVX transport remains practically the same in single and double infection, and (2) PVX is stimulated in the presence of TMV upon mixed infection of isolated protoplasts (M. E. Taliansky etal., unpublished data). And still, the interaction between PVX and PVY seems to involve complementation of the TF as well. According to Close (1962),at 31°C PVX replication was restricted to the inoculated leaves, i.e., the long-distance transport was

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

337

inhibited. However, PVX spread systemically a t 31°C in plants doubly infected with PVX and PVY. It is of interest that the transport of PVX in plants kept at 31°C could also be assisted by TMV and some strains of tobacco ringspot virus and CMV (Close, 1962). The experiments of Rochow and Ross (1955) and Close (1962) support the suggestion that the phenomenon described by them represents the complementation of TF; the transport of PVX from parenchyma into phloem (which is inhibited in single infection, especially at high temperature) can be nonspecifically complemented by unrelated viruses that are active in transport at this temperature. Some examples of synergism between strains reviewed by Kassanis (1968) are probably based on the effects of TF complementation. Thus the necrotic strain of tomato spotted wilt virus (TSWV) acquired the ability to spread systemically over the potato plants in the presence of the ringspot TSWV strain (Norris, 1951) which appeared to act as a helper virus complementing the TF. One more example of interaction between viruses that can be regarded as TF complementation was reported by Benda (1957) who used two TMV strains for mixed inoculation of N. syluestris plants. The common TMV spread systemically while the yellow aucuba (YA) remained confined to the inoculated leaf, producing local lesions. The plants infected with both strains developed yellow spots produced by YA in the presence of the common strain. It seems reasonable to think that the common TMV helped the YA strain in the TF, providing for its systemic spreading. There are some variants of barley yellow dwarf virus (BYDV) confined to the phloem in oats. However, double infection with two different BYDV variants predisposed the xylem to infection (Gill and Chong, 1981). This unexpected phenomenon shows that interaction between two viruses each of which by itself is inable to penetrate the xylem can lead to the appearance of such ability, possibly as a result of the combined action of their transport proteins. 111. RESISTANCE OF PLANTS TO VIRUSES AS A PROBLEM OF TRANSPORT OF THE VIRUSGENOME FROM INFECTED TO HEALTHY CELLS

A. Extremeand Facultative Resistance of Plants toViruses The biochemical mechanisms determining the success or failure of the viral infection in the plant cell are not clear. Solution of this question is hampered by the lack of information on the functions performed by the virus and by the host plant in virus replication. It is quite probable that

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the mechanisms controllingthe resistance of plants to viruses may operate at any level of virus replication, i.e., nonperformance of any function(s) may render the plant resistant to the virus. Normal development of infectionproceeds through a sequence of events: 1. First phases of infection: virus penetration, adsorption on the cell receptors, and deproteinization. 2. Replication of viral RNA and its translation into virus-specific proteins. 3. Systemic spreading of the infection from the primarily infected to healthy cells (transport). 4. Assembly of mature virions.

The first phases of interaction with the cell, which play such a prominent role in the restraint of infection and host-range control of animal viruses, turn out to be of lesser importance during plant infection by plant viruses (see reviews by Atabekov, 1975; De Zoeten, 1981). It can be stated that in most cases plant resistance to the particular virus is caused not by the blockage of the first phases of infection in the host cells but by other factors. Plant cells probably also have cell receptors specific for certain viruses (Novikovand Atabekov, 1970), however, the virus -receptor interaction does not seem to be so decisive for the development of the viral infection as with most animal viruses. It has more than once been demonstrated that the resistance of plants to viruses usually holds at the level of the whole plant but is lost at the single-cell level (for review, see Van Loon, 1983). Only in a few cases the resistance of a plant species to a particular virus is maintained in isolated protoplasts. Van Loon (1983) argued that “in general, individual plant cells are capable of replicating essentially any virus.’’ In spite of its apparent extremeness,this concept seems to be true for many host-virus combinations. The term “extreme resistance’’ may be conveniently adopted for those probably infrequent cases when the plant and isolated cells (protoplasts) prove resistant both to the whole virus and to its free RNA (ie., when the resistance is determined not by the first phases of virus replication). Accordingly,when the resistance is exhibited only at the level of the intact plant (or tissues) while the individual cell is susceptible to the virus an appropriate term would be “facultative resistance.” The same term can also be reasonably applied to those infrequent situations when the plant resistant to the native virus can be infected with a preparation of free viral RNA (see a review by Atabekov, 1975).

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339

Thus, the phenomenon of extreme resistance consists in the fact that the virus replication turns out to be in principle impossible in the cells of resistant plants. Facultative resistance implies that virus replication in the cells is not basically forbidden, though in the whole plant the infection is blocked by (1)a barrier for the first phases of infection, which can be overcome by infecting the resistant plants with free viral RNA; (2) impossibility of the transport of infection from the primarily infected cells where the virus can multiply (in this case the protoplasts isolated from the resistant plant are infected by the virus); and (3) the antiviral defense reactions operating in plant tissues but not in protoplasts or individual cells. Extreme resistance can be exemplified by the immunity of N . tabacurn plants and protoplasts to the wild-type strain of bromegrass mosaic virus (BMV) (Motoyoshi et al., 1974). It must be noted that even in this case some amount of BMV-susceptible protoplasts can be revealed (Sakai et al., 1983). Similar resistance is exhibited by the tomato line Tm-1 toward TMV L (strain of type 0)(Motoyoshi and Oshima, 1977) and by the Arlington line of Vigna sinensis unguiculata Walp. toward the SB strain of CPMV (Beier et al., 1977, 1979). Virus replication turns out to be impossible or profoundly suppressed in the cells and protoplasts of the plants having extreme resistance. Interestingly, extreme resistance may cover not all strains of a given virus. Thus, N . tabacum plants and protoplasts proved susceptible for another BMV strain, V5 (Motoyoshi et al., 1974),the TMV strain CH2 (a strain of type 1)can overcome the resistance of TM-1, and the plants or protoplasts of the V. sinensis unguiculata line resistant to CPMV SB are infected by CPMV strain DG (Beier et al., 1979). Extreme resistance appears to be underlied by the impossibility of carrying out the late phases of virus reproduction after the completion of the early phases. Most probably the barrier is put up at the level of replication of genomic or subgenomic viral RNAs. The obstacle can hardly be in translation since the results of numerous experiments testify to the lack of selectivity in mRNA translation by eukaryotic ribosomes (see, e.g., a review by Atabekov and Morozov, 1979). It was found that accumulation of infective TMV RNA was extremely inhibited in the presence of the Tm-1 gene in tomato, suggesting that this gene influenced the viral RNA replication (Motoyoshi, 1982). In the manifestation of the extreme resistance in protoplasts a prominent role is played by some cell substances suppressing viral replication. Thus it was observed that BMV, which is incapable of multiplying in radish and turnip mesophyll protoplasts, acquires this ability upon exposure of the protoplasts to ultraviolet light or actinomycin D (Maekawa et al., 1981).

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Separate consideration should be given to those cases when the infected cell (and protoplast) isolated from a virus-resistant host nevertheless proves capable of replicating the virus, i.e., when immunity to the virus exists in tissues but not in individual cells. Quite a number of examples of this type have been described 1.TMV can infect cowpea protoplasts but not the whole plants (Huber et al., 1977). 2. BMV strain ATCC66 can infect protoplasts of Raphanus sativus L. while the plants are BMV-resistant (Furusawa and Okuno, 1978). 3. CMV can infect protoplasts of cowpea, the plant normally resistant to this virus (Kioke etal., 1977; Gonda and Symons, 1979; Maule etal., 1980). 4. Several lines of V. sinensis unguiculata were found to be resistant to CPMV-SB; upon inoculation of the resistant plants with the virus at a concentration of 250 pg/ml no symptoms develop and the virus is virtually not accumulated. The resistance is lost at the level of protoplasts: the protoplasts of all resistant lines (except Arlington) could be infected with CPMV-SB (Beier etal., 1977, 1979). 5. Tomato line TM-2 plants are resistant to TMV but their protoplasts are quite susceptible (Motoyoshi and Oshima, 1977).

All the above cases can arbitrarily be classed as examples of facultative plant resistance based on the block of systemic spreading (transport) of the virus from the primarily infected to the surrounding healthy cells in the resistant plant. It must, however, be noted that per se the ability of the virus to replicate in protoplasts isolated from the tissues where it cannot accumulate does not always mean that the host resistance is based on the block of just the virus-specific TF. The resistance in this virus-host system may be due to the inability of the virus to replicate in the host cells organized into leaf tissue (e.g., because these cells contain a certain substance impeding virus multiplication). During isolation of protoplasts the cell wall is removed and the plasma membrane undergoes fundamental changes, accompanied by partial exchange of the cell contents with the medium and probably loss of some cytoplasmic components. It is admissible that all these events alter the composition of the cytoplasm, enabling the virus to replicate in the protoplasts though this was impossible in the tissue. Rigorous proof of the fact that one is dealing with the resistance actually based on blocked infection transport for each particular virus - host system requires demonstration that the virus replicating in protoplasts can also accumulate in primarily infected cells of the intact facultatively resistant (1978) in the work plant. Such proof was presented by Nishiguchi etal.

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341

with the TMV ts mutant Lsl, and by Sulzinski and Zaitlin (1982)who used TMV to infect resistant plants of V.sinensis End1 and Gossipium hirsutum L. In both cases TMV replication was shown to take place only in the primarily infected cells, i.e., the plants behaved as facultatively resistant (see also Section 111,D).

B. Role of Transport Function in Virus Host Range Control The role of virus-coded TF in the host range control of a plant virus was studied by Taliansky et al. (1982d) using the experimental system described above: the dependent virus was imbibed into the conducting tissues and moved into the mesophyll cells of the upper leaves preinfected with the helper virus (Taliansky et al., 1982~). It was assumed that a certain plant species may be resistant to a given virus (be a nonhost for it) simply because the virus genetic material cannot move from cell to cell over this plant. As a result, the virus remains and multiplies only in the primarily infected cells. The plant looks like a nonhost only owing to blocked TF. Taliansky et al. (1982d) demonstrated that in several cases this type of resistance can be overcome, and the nonhost plant surrenders to infection in the presence of a helper -another virus that can normally spread in this plant. It can be seen from Table I11 that the BMV Russian strain acquires the ability to spread over the leaves of tomato (the plant facultatively resistant to BMV) in the presence of a helper virus-tomato strain of TMV. Similarly, BMV can infect beans (which are also resistant to BMV) when assisted by another helper TMV strain (bean strain, or dolichos enation mosaic virus, DEMV) which is able to systemically infect bean plants (Table 111). Dodds and Hamilton (1972) and Hamilton and Nichols (1977) demonstrated the complementation between barley stripe mosaic virus (BSMV) and TMV in barley plants (Hordeurn vulgare L. var Black Hulless) doubly infected with these viruses. A similar effect was observed when BMV was used in mixed infection with TMV (Hamilton and Nichols, 1977). As a result of this interaction, TMV systemically spread over barley plants coinfected with BSMV or BMV. Taliansky et al. (1982d) presumed that this phenomenon was based on the complementation of TMV TF by BSMV in barley where the transport of TMV was restricted. As can be seen from Table 111,BSMV served as a helper virus promoting the systemic spreading of TMV vulgare over wheat plants. It is important that similar results were obtained when the TMV mutant Lsl (ts in TF) was used as a dependent virus. Lsl was shown to be capable of systemically spreading at

342

J. G. ATABEKOV AND YU. L. DOROKHOV TABLE I11

HELPER VIRUSPROMOTES THE TRANSPORT OF DEPENDENT VIRUSOVER NONHOST PLANTS"* ~

Plant resistant to dependent virus

Dependent virus imbibed through the stem

Tomato Bean Wheat

Bromegrass mosaic virus Bromegrass mosaic virus TMV UI

Wheat Tomato Tm-2 (resistant to TMV)

Helper virus preinfection of upper leaves

Transport and T replication of ("C) dependent virus 24 24 24 33

TMV LsI ( t s in transport)

TMV L TMV bean strain Barley stripe mosaic virus Barley stripe mosaic virus

TMV L

Potato virus X

24

33

+

+ + + + +

Summarized results of Taliansky etal.(1982d). correspond to effective and blocked transport function.

* (+) and (-)

nonpermissive temperature (33"C) in the presence of a temperature-resistance helper (BSMV). These observations indicated that it was the TF that was complemented by BSMV under such conditions. Similar results were obtained when tomatoes of the Tm-2 line were used in such experiments. Table I11 shows that Tm-2 tomatoes can be systemically infected with TMV in the presence of another unrelated virus, PVX, used as a helper. These results led to the conclusion that a "nonhost" (facultatively resistant) plant, whose resistance to the virus was due only to the blockage of the TF, can be infected by this virus owing to complementation of transport by the unrelated helper virus. One could ask whether the dependent virus which in the presence of a helper overcomes the transport blockage and moves from the conducting system into parenchyma then continuously needs the assistance of the helper virus for spreading over the parenchyma or, having once moved from the vascular system into parenchyma with the help of the assisting virus, it can then spread alone, without the helper. To clarify this question a complementation experiment was performed where Lsl TMV was used as asystemically spreading helper virus and BMV as a dependent one. Three days after BMV was imbibed into the cut stem of Lsl-infected tomatoes kept at 24"C, the plants were transferred to 32°C. As can be seen in Table IV, the spreading of BMV within the tomato leaf parenchyma started during the 3 days of incubation at 24°C. In controls continuously incubated at 24"C the virus moved continuously. However, BMV transport within the tomato leaf was arrested as soon as the T F of the helper virus (Lsl) was switched off at 32°C (M. E. Taliansky etaL, unpublished).

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

343

TABLE IV T F SHOULD BE CONSTANTLY COMPLEMENTED FOR SPREADING OF BMV OVER TOMATO LEAF PARENCHYMA

Effectiveness of transport: percent of BMV-infected mesophyll protoplasts isolated from TMV-infected tomato leaf (days after incubation)

T

Helper virus used

("C)

3

5 ~

Lsl(ts) Control' Lsl Control L (tr) Control

24 24 24-,32* 24-32 24 24 24-32 24-32

L

Control ~

10 0

8 0 13 0 9 0

7 1 0 ~

18 0 11

0 27 0 21 0

29 0 10 0 35 0 36 0

~~

52 0 11 0 58 0 53 0

~~~~~

No helper used. Three days after BMV was imbibed into the cut stem of TMV (Lsl or L)-infected tomatoes (at 24°C) the plants were transferred to 32°C and kept for the time indicated.

Another phenomenon that can provisorily be regarded as a manifestation of plant facultative resistance is the tropism of viruses to particular cell types. Multiplication of many viruses is known to be restricted to phloem cells (see above). Apparently in these cases we again encounter the suppression of the infection transport, but here the barrier arises at the stage of the transfer of infection from the phloem to the parenchyma cells. That this barrier has at its foundation the infection transport block is favored by the results of Kubo and Takanami (1979) for the tobacco necrotic dwarf virus (TNDV). Replication of TNDV is restricted to the phloem, but the authors have shown that this virus can infect tobacco mesophyll protoplasts in uitro. By analogy to the experiments on TF complementation described above, it seemed reasonable to use a systemically spreading virus as a helper compensating for the inability of a phloem-limited virus to be transported from the phloem to parenchyma. It was demonstrated by Atabekov etal. (1983) that in Datum stramonium L. plants mixedly infected with PVX

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J. G . ATABEKOV AND YU. L. DOROKHOV

(systemically spreading helper virus) and potato leafroll virus, PLRV (dependent phloem-limited virus) the latter moves from the phloem into parenchyma cells and multiplies there. As a result of TF complementation PLRV accumulates in doubly infected plants in amounts exceeding that in the controls (PLRV alone). Like results were obtained by Carr and Kim (1983a,b) with another phloem-limited virus, bean golden mosaic virus (BGMV) which was shown to spread systemically in beans coinfected with BGMV and TMV (bean strain).

C. Two Conceivable Means ofCell-to-Cell Transport: Modification of Plusmodesmata and Suppression ofthePlantDefense Reactions At least two different mechanisms can be envisaged as the general means of cell-to-cell transport of the virus genome. The first one, already mentioned, postulates that a plant cell is normally prepared for the replication of a virus introduced into it but “not prepared” to let the virus out, to let it pass to the neighboring healthy cells. A virus-coded TF should be performed to “open the gates.” The plasmodesmata of healthy cells playing the role of such gates are closed for the transport of virus infection and should be modified by a virus-specific transport proteins to ensure virus spreading. Another quite different mechanism can be offered as well to explain the phenomena of transport and its blockage and complementation. It is based on the generally accepted notion than upon the primary inoculation of a plant certain defense mechanism(s) may be triggered, rendering the neighboring noninoculated cells and tissues resistant to challenge inoculation. Apparently, this phenomenon should be due to systemic movement of certain antiviral factor(s) from inoculated to noninoculated cells (for review, see Kassanis, 1963; Sela, 1981; Fraser, 1982; Gianinazzi, 1982, 1983; Van Loon, 1982, 1983; Loebenstein etal., 1982). Thus, it can be speculated that there is no need to modify the plasmodesmata since they are always opened for transport, but the surrounding healthy cells may happen to be resistant to the infection. The virus-coded TF probably consists in overcoming this defense in the cells at or around the point of entry of the virus, making them susceptible to the virus infection. This function can be thought to be performed only in a certain host-virus combination, the transport protein coded by a given virus being effective in suppressing the defense reaction in surrounding cells only in a certain plant species (in which the virus spreads systemically). If it is so, the mechanism of T F complementation consists in overcoming the plant antiviral defense reactions by the transport protein of the helper virus, and not in modification of plasmodesmata.

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

345

The question arises why the virus replicates in the primarily infected cells. It can be speculated that in this case the defense reaction is induced only after the virus replication has begun, i.e., later than necessary for the cell to achieve a fully “antiviral” state. On the other hand, the blockage of the virus transport may be due not to the virus-induced but to some constitutive resistance to a particular virus in aparticular host. This constitutive defense mechanism may be inactive when isolated protoplasts or primarily inoculated cells are infected, but operates against secondary infection to cause complete inhibition of virus replication or some kind of abortive infection. Thus, the term “transport function” may mean not an active mechanism for transporting the virus genome from infected to healthy cells but a mechanism that serves to break the defense of healthy cells against virus replication.

D. The Subliminal Symptomless Infections Several forms of localized virus infection have been listed by Loebenstein etal. (1982) including different types of necrotic reactions, starch lesions produced by TMV in cucumber cotyledons, and the so-called subliminal symptomless infection. The last phenomenon is considered below, being the less studied and the one directly connected with the problem of the cell-to-cell transport of virus infection. The term “subliminal infection” was offered to designate the kind of virus - host interactions when no necrotization or any visible symptoms develop and extremely little infective virus progeny can be recovered from the inoculated leaves (Cheo, 1970; Cheo and Gerard, 1971). The phenomenon of subliminal infection basically differs from tolerance (in a tolerant plant the replication and transport of the virus are not prevented although the infection is symptomless) and from immunity (an immune plant is totally unable to support the virus replication in individual cells and tissues). In the subliminally infected host plant the virus infection can be localized by a mechanism similar to that operating upon localization at nonpermissive temperature of TMV mutant Lsl temperature-sensitive in the TF (see above). In these cases the localization mechanism does not operate through inhibiting virus multiplication: the virus can replicate but cannot move to the surrounding cells (Koike etal., 1976; Wieringa-Brant etal., 1978; Huber etal., 1981; Sulzinski and Zaitlin, 1982). It can be assumed that the virus-specific transport protein is nonfunctional in the subliminally infected host. The restriction of virus spreading also takes place in the hypersensitive host reaction upon production of ringspot-type local lesions or some other

346

J. G. ATABEKOV AND YU. L. DOROKHOV

symptoms. However, at least in some of these cases the localization reactions are temperature-sensitive and can be switched off at 30-33°C; as a result, the virus moves at these temperatures (see above). On the contrary, the cell-to-cell virus transport seems to be invariably blocked at either temperature in subliminally infected plants. Therefore, it is possible to discriminate between the localization of a virus owing to suppressed TF and that due to the inhibition of virus replication upon the development of the hypersensitive reaction. The evidence is accumulating that the phenomenon of subliminal infection represents a separate form of localizing resistance which is a common system of plant defense against viruses. Several examples of “facultative” resistance of plants to viruses have been mentioned above, including the resistance to TMV of barley plants and tomatoes homozygous for the TM-2 gene. The host plant mechanically inoculated by the phloem-limited viruses can be regarded as subliminally infected since some phloemlimited viruses were shown to multiply in the primarily inoculated epidermal cells. The phloem-limited viruses normally transferred by the vector are restricted to the phloem and cannot be transported into parenchyma cells, i.e., such host -virus combinations in a certain sense can also exemplify the subliminal form of virus infection. It can be assumed that more than one mechanism may be responsible for the phenomenon of subliminal infection in different host -virus combinations. It is important to know whether the resistance developed in the subliminally infected host is constitutive or induced by the virus; in other words, whether the blocking of the TF in the subliminally infected plant is an intrinsic capacity of a given plant species with a given virus, or it is induced by this virus as an active defense reaction against the virus infection. If the blockage of the TF is a virus-induced reaction of the host cell, there are at least two possibilities: (1) the defense reaction(s) involved in the localization of the virus in the primarily infected cells develop in noninfected neighboring cells in response to a signal from primarily infected cells; or (2) the defense reaction(s) develop only directly in the primarily infected cells owing to a certain modification of the latter. It was shown by Shalla etal. (1982) that the number of plasmodesmata between the cells in LsI-infected tissues is significantly smaller at nonpermissive than at normal temperature. One can suggest that a similar reduction of the number of plasmodesmata between the primarily infected and neighboring healthy cells occurs in the subliminally infected host. However, the reduction in the number of plasmodesmata can hardly explain the phenomenon of complete transport blockage. The observations of Nishiguchi etal. (1980) allow a suggestion that the cell-to-cell translo-

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

347

cation of Lsl at nonpermissive temperature is stopped completely although the reduction in the number of plasmodesmata is rather a quantitative than a qualitative event: no differences in the structure of plasmodesmata were observed upon blocking of the cell-to-cell virus trans1982). It is not excluded, however, that the reduction of port (Shalla etal., the number of plasmodesmata connecting the primarily infected cells with the neighboring healthy ones represents a universal defense reaction of a plant against the virus infection, and the function of a virus-specific transport protein consists in overcoming this reaction. It can be speculated that the transport protein coded for by a certain virus is functionally active not in any plant species, hence a subliminal infection develops in a host plant where the transport protein is inactive. Apparently, the transport-blocking mechanism operating in a subliminally infected plant (as well as in a plant infected with TMV ts transport mutant Lsl at nonpermissive temperature) is specific, i.e., it is directed only against the definite virus (e.g., against TMV in cowpea and cotton, or against Lsl TMV a t 33°C) although it does not prevent the infection of the same plant by other viruses and their systemic spreading. It would be of interest to know whether the physiological changes upon subliminal infection and blockage of virus transport are localized in the primarily infected cells, i.e., the neighboring cells are not involved in the process. To our knowledge there is no information about the replication of viral RNA in subliminally infected plants. Unexpectedly it turned out that in some cases when the transport of infection is apparently blocked, replication and transport of at least some species of viral RNA are not suppressed. Appreciable synthesis of short (MW 0.7 X lo6 to 1.2 X lo6) “subgenomic” RNAs with very little if any replication of genomic RNA was demonstrated in wheat infected with TMV or PVX. It is important that newly synthesized “subgenomic” RNAs are detected in the noninoculated parts of TMV-inoculated wheat leaves although no virus progeny and no coat protein (or subgenomic RNA coding for it) can be found in noninoculated tissue (Dorokhov etal., 1983a,b). Hence it can be concluded that not only the originally infected cells are involved in the subliminal infection in TMV-infected wheat plants; secondarily infected cells do not produce the virus progeny probably because abortive infection develops. Similar results were obtained when another experimental system of this kind was analyzed. Different authors (Caldwell, 1931; Matthews, 1981) reported that plants cannot be infected with TMV, TYMV, or their RNA through the cut stem. It might have been thought that the virus introduced into the plant vascular system through the cut stem and entering the upper leaves is confined there and remains

348

J. G . ATABEKOV AND YU. L. DOROKHOV

inert, i.e., the TMV genome is not expressed under these conditions. However, the observations of Dorokhov etal. (198313) indicate that TMV introduced into the plant through the cut stem induces in the upper leaves the synthesis of TMV-specific RNAs shorter than genomic TMV RNA. The synthesis of these RNAs was localized in conducting and possibly in parenchymal cells closely associated with the vessels. The synthesis of genomic RNA does not occur or is deeply suppressed under such conditions. In other words, TMV introduced through the cut stem induces abortive infection in the upper leaves, marked by the lack of infectivity growth probably resulting from the block on genomic RNA synthesis. Tobacco leaves inoculated with the TMV ts-transport mutant Lsl at 33"C represent the third experimental system demonstrating that the virus infection is abortive when its transport is blocked. Dorokhov etal. (1983b) showed that in all three experimental systems (TMV in wheat, TMV in vascular system, and Lsl TMV in tobacco at 33"C)the blockage of the transport function was accompanied by the development of an unusual abortive form of infection; synthesized and transported are TMV-specific RNAs shorter than the genomic one whose synthesis is suppressed. In these cases the transport of the normal virus infection was indeed blocked. Still, an abortive form of TMV infection developed and seemed to spread at least in two of the three cases listed. IV. THETRANSPORT FORM OF VIRALINFECTION

A. MatureVirions Do NotAppearto Participate inInfection Transport Though the identity of the transport form (i.e., structures carrying the virus genetic material during the moving of infection) was obscure, it was assumed that this part could be played by mature virions, free plus RNA, or the replicative complex associated with cell membranes (De Zoeten, 1981). Mature virions are observable in the plasmodesmata of infected tissues; hence they were supposed to participate in the cell-to-cell transport of infection (Weintraub etal., 1976). However, there are several lines of evidence that virus particles are not used as the transport form or play only an ancillary role in the virus transport. 1.It is clear that the mere presence of virions in the primarily infected cell (even though daughter virions may have accumulated there through active replication) does not suffice for infection transport to healthy cells. This is illustrated by the results of Nishiguchi etal. (1978) with the TMV ts-transport mutant Lsl at nonpermissive temperature.

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

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2. Infection transport is a relatively early function and takes place not after but before the formation of mature virions. This is proved by experiments on the determination of the rate of TMV movement from the leaf epidermis to the underlying mesophyll (Matthews, 1981). The epidermal cells acquire the ability to infect mesophyll3 hours before the appearance of mature viral particles in the epidermis. 3. Evidence against the role of mature virions as the obligate form of infection spreading was obtained in studies of some viruses defective in the coat protein and TMV tsmutants in the coat protein. It is also obvious that the coat protein as well is not indispensable for the transport of infection from the infected cell. 4. Finally, the transport role of mature virions is contradicted by the results obtained by Dawson etal. (1975) by differential temperature treatment (DTT). In the course of DTT the lower leaves of a mechanically TMV-inoculated plant are kept at a temperature optimal for virus multiplication (25°C) while the upper leaves are placed into a cold chamber and kept at a temperature (5°C) precluding virus replication. Under such conditions a certain potentially infective material (the transport from of the virus) penetrates the upper noninfected leaves and accumulates there in substantial amounts. However, it is important to note that the infective entity gets there not as mature virions but in some other form. This follows from the fact that the upper leaves in the DTT conditions (several days at 5°C) contain no infective viral particles. On the other hand, there is no doubt that genomic TMV RNA reaches the upper leaves during DTT, since subsequent incubation at normal temperature (25°C) leads to intensive virus replication in these leaves, and the infection is even noticeably synchronized (Dawson etal., 1975; Dorokhov etal., 1981).

B. Detection and Properties of Virus-Specific Informosome-Like Ribonucleoprotein (vRNP) Taking into account that in eukaryotic cells the messenger RNA is contained in informosomes -cytoplasmic RNP particles built of cell proteins in complex with mRNA (for review, see Spirin, 1969,1972; Preobrazhensky and Spirin, 1978)-the viral RNA could also be thought to be able to form such structures. A convenient object to study this question is the TMV coat-protein ts mutant Nil18 (see above). Despite the absence of mature particles the Nil 18 infection can systemically spread a t nonpermissive temperature, i.e., this model is quite convenient for studying the identity of the transport form of virus infection. Dorokhov etal.(1980a, 1983a) found that the cells of plants infected

350

J. G . ATABEKOV AND YU. L. DOROKHOV

with Nil18 at nonpermissive temperature contain new types of ribonucleoprotein particles differing from mature virus in structure and buoyant density. These RNP are composed of genomic and subgenomic TMV RNAs and TMV-specific and cell proteins. Henceforth these structures are referred to as virus-specific RNP, or vRNP. It has been demonstrated that vRNP are present in cells infected with different TMV strains (including vulgare at 25°C) and with PVX (Dorokhov etal., 1983a,b). Formation of vRNP can be thought to be characteristic of the replication of various plant viruses. The properties of vRNP distinguishing them from mature virions have been elucidated (Dorokhov etal., 1983a): 1. The buoyant density of vRNP is considerably higher and corresponds to that of plant and animal cell informosomes. In cesium chloride or cesium sulfate density gradients vRNP particles are found in the zone of buoyant density 1.36-1.45 g/cm3 (Fig. 4). 2. The RNA of vRNP, unlike that of mature virions, is susceptible to RNase. 3. In the electron microscope TMV vRNP appear as filamentous structures differing from the rodlike TMV virions (Dorokhov et al., 1983a). 4. The results of pulse-chase experiments showed that vRNP does not serve as a precursor for the mature virion (Dorokhov etal., 1983b).

C. Structure of vRNP In TMV vRNP, besides the genomic RNA, two types of RNA were found (Dorokhovet al., 1983a)which appear to be intermediate-class subgenomic RNA. This suggestion agrees first, with the results of hybridization of these RNAs with DNA of the recombinant plasmid pBR322-TMV containing nucleotide sequences complementary to genomic TMV RNA; second, RNA isolated from TMV vRNP stimulates in a rabbit reticulocyte cell-free protein-synthesizing system, among other TMV-specific proteins, the synthesis of the 30K protein (Dorokhov etd., 198313) which is known to be coded for by the I-class subgenomic TMV RNA (Beachy and Zaitlin, 1977). It should be noted that PVX vRNP contain genomic PVX RNA and a considerable amount of shorter (probably subgenomic) RNAs capable of coding for PVX-specific proteins in a cell-free system (Miroshnichenko etal., 1983). Polypeptide analysis revealed in TMV (vulgare) vRNP several proteins, the main components being 39K, 37K, 31K, and the coat protein (17.5K) (Fig. 5).

351

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION N i l 18 (ts-coat protein; tr-transport)

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FIG. 4. CsCl gradient diagrams of vRNP synthesized in Nill8-infected plants. (A,B) vRNP revealed after labeling with [3H]uridineat 25°C for6 and20 hours, respectively. (D,E) vRNP revealed after labelingwith [3H]uridineat 33°C for 6 and 20 hours, respectively. (C,F) Healthy leaf tissue labeling for 6 hours at 25 and 33"C, respectively. Buoyant densities are given above the zones.

The virus-specific nature of the 31k, 37K, and 39K polypeptides is evidenced by the following facts: (1)They are synthesized in the presence of actinomycin D when the host protein synthesis is suppressed. (2) They have overlapping amino acid sequences (Fig. 6 ) .This phenomenon is widely encountered in viruses: overlapping amino acid sequences are found in the TMV-specific proteins (Pelham, 1978) and several nonstructural proteins of other plant viruses (for review, see Davies and Hall, 1982). (3) It is important that the 31K, 37K, and 39K proteins are not observed in vRNP isolated from tobacco leaves infected with another virus, PVX. The PVX vRNP, besides the coat protein (23K), contain polypeptides 55K, 78K, 95K, 120K, and 145K (Dorokhov etal., 1983a). It is of interest that the 55K, 120K, and 145K polypeptides are found among the in vitro

352

J. G. ATABEKOV AND YU. L. DOROKHOV

A

B

x 1o3

-95.2 69

46

FIG. 5. Slab polyacrylamide gel electrophoresis of polypeptides of vRNP of TMV uulLane B, 14Cgare.Lane A represents the polypeptide pattern of vRNP of TMV uulgare. methylated marker proteins.

translation products of RNA isolated from PVX vRNP (Miroshnichenko et al., 1983). On the other hand, vRNP obtained from different plant tabacum, N .glutinosa, Lycopersicon esculentum) infected with species (N. TMV contain identical sets of polypeptides labeled in the presence of actinomycin D (Dorokhov et aL,1983b).

D. Functions of vRNP As already mentioned above, vRNP is not a precursor to the mature virion, and it can be supposed that one of its functions is to take part in the FIG.6. Peptide analysis of “C-labeled polypeptides of molecular weights 31,000 (A),

37,000 (B), and 39,000 ( C ) isolated from vRNP of TMV uulgare. (A,B,C) Autoradiograms of

peptide maps; (D,E,F) a scheme reflecting the positions of ninhydrin-stained spots of the TMV coat protein (not hatched) and radioactive spots of vRNP proteins (hatched).

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J. G. ATABEKOV AND YU. L. DOROKHOV

synthesis of virus-specific proteins. This point of view is supported by a number of facts. 1. In the cells vRNP are found not only in the free state but also in the composition of polyribosomes synthesizing virus-specific proteins (Dorokhov etal., 1983b). 2. As already noted, in the cell-free protein-sythesizing system from rabbit reticulocytes the RNA from TMV vRNP can direct the synthesis of TMV-specific proteins, among which the 30K protein coded for by the I-class subgenomic TMV RNA is distinguished. Fingerprinting of the 30K species translated from vRNP RNA and from short I-class RNAs isolated from virions showed an identical set of peptides (Dorokhov etal., 1983b).

Evidence for the probable involvement of vRNP in transport was obtained in the work with two TMV mutants ts in transport, Ni2519 and Lsl. These mutants were shown to be able to form vRNP only at permissive but not at restrictive temperature when the transport is inhibited (Dorokhov etal., 1980a, 198313). The ways of spreading of the TMV transport form and its location in the upper noninoculated tobacco leaves were studied with the use of a modified DTT system (Dorokhov etal., 1981). The object was Samsun NN tobacco which, as mentioned above, develops lesions at 25°C but systemically responds to TMV at 33°C. The lower leaves of plants were infected with TMV and kept at 33°C for systemic spreading of the infection while the upper leaves of the same plants were kept at 5°C. As a result, lesions developedd neither in the lower (33°C) nor in the upper (5°C) leaves, but the transport form of infection moved from the lower to the upper noninfected leaves of the experimental plant. After 7- 10 days the upper leaves were transferred to 25°C to switch on the necrotic reaction (Fig. 7). The results obtained revealed the way of spreading and made it possible to locate the transport form of TMV in the DTT system. It is shown that when the N gene is switched off the transport form of the infection moves from the lowest mechanically inoculated leaves first to the basal regions of the top noninfected leaves and then to their distal parts and to the basal regions of the leaves of the next lower circle, spreading through the vascular system. Direct proof of the participation of vRNP in the transport of viral infection was obtained by Dorokhov etal. (1980b, 1983b) with the DTT technique. A study was made of the nature of the infective principle getting into the upper noninfected leaves (5°C) from the lower TMV-inoculated leaves (25°C) of tobacco plants. Labeling with [3H]uridine and 14C-la-

355

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION 1

2

8 crn 10 crn

FIG.7. Distribution and localization of infection in the upper leaves of TMV-infected tobacco Samsun NN plants after DTT. The upper leaves of tobacco Samsun NN were placed in a cold (5°C)chamber after inoculation of the lower leaves with TMV. The lower leaves were maintained at 33°C. Afterbeing kept in the chamber for aweek, the top oftheplantwas cut off and maintained at 25°C. Two days later lesions appeared on the leaves; the pattern of their localization is shown. Numbers correspond to the numeration and size of the leaves.

beled amino acids through the stem in DTT conditions in the presence of the inhibitors of host RNA synthesis and further analysis of extracts from the upper leaves revealed vRNP (Fig. 8). The absence of such structures from controls gave grounds for concluding that vRNP synthesized in the lower (infected, 25"C) leaves are transported to the upper (noninoculated, 5°C) ones under DTT conditions, i.e., that vRNP plays the role of the potentially infective material (the transport form) in the systemic spreading of viral infection. The fact that vRNP particles contain subgenomic I-class RNAs may reflect the functional role of vRNP. It has been suggested by Atabekov and Morozov (1979) and by Leonard and Zaitlin (1982) that the 30K protein coded for by the I, subgenomic RNA in vitro (Beachy and Zaitlin, 1977) acts as the transport protein essential for the systemic spreading of TMV infection in plant tissue. It is admissible that after vRNP containing the I, subgenomic RNA have moved from TMV-infected to healthy cells, the RNA can be translated to yield the 30K transport protein. Further, it is not excluded that this takes place even before the genomic

356

J. G. ATABEKOV AND YU. L. DOROKHOV

4

-1.6 -1.5 -1.4 -1.3

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FIG.8. Detection of vRNP in the upper noninoculated tobacco leaves infected with TMV under the DTT conditions. Healthy plants and those infected with TMV were kept for 72 hours under DTT conditions, then the plants were cut and the lower part of the stem was placed into a solution with AMD for 4 hours and then into a solution with [3H]uridine(A,B) or 14C-labeledamino acid mixture (C,D) in the presence of AMD and rifampicin. Plants under DTT conditions were labeled through the stems for 24 hours. Extracts from the upper noninoculated leaves were examined for vRNP presence by density gradient fractionation in CsCl. (A) TMV-infected plant; (B) healthy plant labeled in the presence of AMD and rifampicin; (C) TMV-infected plant, ‘“-labeled amino acid; (D) healthy control, “C-labeled amino acid.

TMV RNA penetrates the cell; vRNP translation and 30K production could be essential for subsequent modification of the cell which may precede the penetration and/or replication of full-length genomic RNA. It was already mentioned that if a systemic host is infected with a TMV strain effective in the coat protein the infection is confined to the mechanically inoculated leaves, i.e., the unstable form can perform only the shortdistance transport but is not involved in the long-distance transport along the vascular system. The movement of the virus is restricted to the vascu-

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lar parenchyma. It was supposed that the long-distance transport of the unstable form is impossible because the viral RNA is more readily degraded in the vascular system than in the parenchymal cells (Siege1etal., 1962). The situation changes radically on assuming that the transport form is not free RNA but vRNP. It has been mentioned above that one of the main vRNP components is the coat protein (Fig. 5 ) which was found in vRNP formed by TMV vulgare and by TMV ts mutants at permissive temperature. On the other hand, the coat protein was shown to be absent from the vRNP of TMV class I ts mutants at nonpermissive temperature (Dorokhov etal., 1983b). It is important that the lack of the coat protein in vRNP is not accompanied by a loss of short-distance transport of these mutants, i.e., the presence of the coat protein is not crucial for the near transport. Conversely, there are grounds for believing that the coat protein in the transport form (vRNP) is needed to protect viral RNAs during the long-distance transport. To test this suggestion, experiments were run on the complementation of the long-distance transport of viral infection. To this end, plants were simultaneously infected with two TMV mutants: Lsl (ts in transport but trin the coat protein) and Nil18 ( t s in the coat protein and in long-distance transport but trin short-distance transport). It was expected that during mixed infection Lsl will make the tr coat protein which will be incorporated into the vRNP of the ts mutant Nil18 instead of its homologous coat protein at 33°C. As a result, the Nil18 RNA will be protected from RNase attack in mixed vRNP and will therefore be able to participate in long-distance transport. It should be borne in mind that under such conditions the complementation could be reciprocal: first, it was expected that the mixed Nil18 vRNP having acquired the Lsl coat protein would be involved in long-distance transport, and second, it was less possible but not excluded that Lsl itself could spread in the presence of Ni118. In agreement with this suggestion, Dorokhov etal. (1983b)found considerable amounts of 3H-labeled vRNP in the upper leaves of tobacco plants infected with both Nil18 and Lsl; no such material was seen in controls singly infected with Nil18 or Lsl (Fig. 9). It should be noted that the viral RNA was considerably more sensitive to RNase in the coat-protein-free vRNP of Nil18 than in vRNP of vulgare containing the coat protein. The coat protein can be supposed to indirectly serve for T F expression by protecting the viral RNA in the vRNP particles, thereby being essential for the long-distance transport. In this connection the mechanism of transport of viroid infection appears enigmatic. It is quite clear that the viroid RNA does not code for any viroid-specific proteins in the infected plant (for review, see Dickson, 1979), but nevertheless the infection spreads. Hence the transport of

358

J. G. ATABEKOV AND YU. L. DOROKHOV

- 12

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.

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FIG.9. Biochemical testing of complementation between TMV mutants Lsl and Ni118. (A-C) CsCl gradient diagrams of RNP synthesized in leaves mechanically inoculated with Lsl (A), Nil18 (B),Lsl Nil18 (C). (E-G) CsCl gradient diagrams of RNP synthesized in noninoculated upper leaves systemically infected by L s l (E),Nil18 (B),and Lsl Nil18 (G); D,H, healthy controls. (From Dorokhov et aL,1983b.)

+

+

viroids does not require any new proteins or may involve some viroid-induced proteins of host origin. The molecular form in which viroid RNA is transported (free RNA or some form of RNP) is also unknown.

V. CONCLUDING REMARKS Systemic spreading of viral infection over the plant, i.e., the transfer of the virus genome from infected to healthy cells, is regarded not as a passive process but as an active function (TF) performed by virus-coded protein (s). The TF can be complemented in mixed infection with a helper virus: a temperature-resistant helper assists systemic spreading of the virus temperature-sensitive in TF. Complementation can be achieved with some unrelated viruses, i.e., the TF is nonspecific. Resistance of nonhost plants to viruses can be due to the impossibility of

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359

the infection transport (subliminal infection) and the virus is confined to the primarily infected cells. In these cases the TF can be compensated by a heterologous helper so that in mixed infection the plant surrenders to both viruses. In other words, a TF-complementing helper expands the host range of some other viruses, making them infectious for normally resistant plants. The virus genetic material is transported through the infected plant in the form of special transport particles -virus-specific informosome-like ribonucleoproteins (vRNP) differing from mature virions. These vRNP contain viral genomic and subgenomic RNAs and virus-specific and cell proteins. Some hypothetical possibilities are considered as to why the virus genetic material may or may not be actively transported in a particular virus - host combination.

ACKNOWLEDGMENTS We wish to thank Drs. J. F. Bol, R. S. S. Fraser, S. Gianinazzi, T. C. Hall, G. Loebenstein, T. Meshi, M. Nichiguchi, I. Sela, S. Sarkar, A. Van Kammen, and L. C. Van Loon for sending us their reprints andpreprints. Thanks are due to Dr. A. V. Galkin for his help in translating this text into English.

REFERENCES Asselin, A., and Zaitlin, M. (1978). Virology 91,173. Atabekov, J. G. (1975). Annu. Rev. Phytopathol. 13,127. Atabekov, J. G. (1977). Compr. Virol. 1 1 , 143-216. Atabekov, J. G., and Morozov, Yu. S. (1979). Adv. Virus Res.25, 1. Atabekov, J. G., Novikov, V. K., Vishnichenko, V. K., and Javakhia, V. V. G. (1970a). Virology 4 1 , 108. Atabekov, J. G., Schaskolskaya, N. D., Atabekova, T. I., and Sacharovskaya, G. A. (1970b). Virology 4 1, 397. Atabekov, J. G., Novikov,V. K., Vishnichenko, V. K., andgaftanova, A. S. (1970~).Virology 4 1 , 519.

Atabekov, J. G., Drampyan, A. H., and Taliansky, M. E. (1983). In preparation. Beachy, R. N., and Zaitlin, M. (1977). Virology 81, 160. Beachy, R. N., Zaitlin, M., Bruening, G., and Israel, H. W. (1976). Virology 73, 498. Beier, H., Siler, D. J., Russell, M. L., and Bruening, G .(1977). Phytopathology 6 7 , 917. Beier, H., Bruening, G., Russell, M. L., and Tucker, C. L. (1979). Virology 96, 165. Benda, G. T. A. (1957). Virology 3 , 601. Bennett, C. W. (1956). Annu. Rev. Plant Physiol. 7, 143. Bhalla, R. B., and Sehgal, 0. P. (1973). Phytopathology 63,906. Bosch, F. X., and Jockusch, H. (1972). Mol. Gen. Genet. 116,95. Bruening, G., Beachy, R. N., Scalla, R., and Zaitlin, M. (1976). Virology 71,498. Bujarski, J. J., Hardy, S. F., Miller, W. A,, and Hall, T. C. (1982). Virology 1 1 9 , 465.

360

J. G. ATABEKOV AND YU. L. DOROKHOV

Cadman, C. H. (1961). Nature (London) 193,49. Caldwell, J. (1931). Ann. Appl. Biol. 1 8 , 19. Carr, R. J., and Kim, K. S. (1983a). Phytopathology 73,499. Carr, R. J., and Kim, K. S. (198313). J.Gen.Virol. 64, 2489-2492. Chamberlain, J. A., Catherall, P. L., and Jellings, A. J. (1977). J. Gen.Virol. 3 6 , 297. Cheo, P. C. (1970). Phytopathology 60,41. Cheo, P. C., and Gerard, J. S. (1971). Phytopathology 6 1 , 1010. Close, R. (1962). Referred to by Kassanis (1963). Cohen, J., and Loebenstein, G. (1975). Phytoputhology 6 5 , 3 2 . Collmer, C. W., and Zaitlin, M. (1983). Virology 126,449. Collmer, C. W., Vogt, V. M., and Zaitlin, M. (1983). Virology 126,429. Cronshaw, J. (1974). In “Dynamic Aspects of Plant Ultrastructure” (A. W. Robards, ed.), pp. 391-413. McGraw-Hill, New York. Da Graca, J. V., and Martin, M. M. (1975). Physiol. Plant Pathol. 7,287. Davies, J. W. (1979). In “Nucleic Acids in Plants” (T. C. Hall and J. W. Davies, eds.), Vol. 2, Chapter 10. CRC Press, Boca Raton, Florida. Davies, J. W., and Hall, R. (1982). J. Gen. Virol. 6 1 , l . Davison, E. M. (1969). Virology 37,694. Dawson, W. 0. (1983). Virology 1 2 5 , 314. Dawson, W. O., andDodds, J. A. (1982). Bwchem.Biophys. Res.Commun. 107,1230. Dawson. W . O., and White, J. L. (1978). Virology90, 209. Dawson, W. O., and White, J. L. (1979). Virology 93,104. Dawson, W. O., Schlegel, D. E., and Lung, M. C. Y. (1975). Virology 65,565. De Jager, C. P., Zabel, P., van der Beek, C. P., andVan Kammen, A. (1977). Virology76,164. De Zoeten, G. A. (1981). In “Plant Diseases and Vectors: Ecology and Epidemiology” (K. Maramorosch and K. F. Harris, eds.), p. 221. Academic Press, New York. Dickson, E. (1979). In “Nucleic Acids in Plants” (T. C. Hall and J. W. Davies, eds.), Vol. 2, pp. 153-193. CRC Press, Boca Raton, Florida. Dodds, J. A., and Hamilton, R. I. (1972). Virology 50,404. Dodds, J. A., and Hamilton, R. I. (1976). Adu. Virus Res.2 0 , 3 3 . Dorokhov, Yu. L., Alexandrova, N. M., Miroshnichenko, N. A., and Atabekov, J. G. (1980a). Biol. Nauki (Moscow) No. 6, p. 22. Dorokhov, Yu. L., Alexandrova, N. M., Miroshnickenko, N. A., and Atabekov, J. G. (1980b). Biol. Nauki (Moscow) No. 9, p. 23. Dorokhov, Yu. L., Miroshnichenko, N. A., Alexandrova, N. M., and Atabekov, J. G. (1981). Virology 108, 507. Dorokhov, Yu. L., Alexandrova, N. M., Miroshnichenko, N. A., and Atabekov, J. G. (1983a). Virology 127, 237. Dorokhov, Yu. L., Alexandrova, N. M., Miroshnichenko, N. A.,andAtabekov, J. G. (1983b). Virology (in press). Dorssers, L., Zabel, P., van der Meer, J., and Van Kammen, A. (1982). Virology 116,236. Dorssers, L., van der Meer, J., Van Kammen, A., and Zabel, P. (1983). Virology 125,155. Esau, K. (1956). Am. J. Bot. 4 3 , 739. Esau, K. (1960a). Virology 10, 73. Esau, K. (1960b). Virology 11, 317. Esau, K. (1972). New Phytol. 71,161. Esau, K. (1978). J. Ultrustruct.Res. 6 3 , 224. Esau, K., and Cronshaw, J. (1967). J. Cell Biol. 33,665. Esau, K., and Hoefert, L. L. (1972). Virology48, 724. Esau, K., Cronshaw, J., and Hoefert, L. L. (1967). J.CellBiol. 3 2 , 71.

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

361

Fraenkel-Conrat, H. (1983). Proc. Natl. Acad.Sci. U.S.A. 80, 422. Franssen, H., Goldbach, R., Broekhuysen, M., Moerman, M., andVan Kammen, A. (1982) J. Gen.Virol. 41, 8. Fraser, R. S. S. (1982). ActaHortic. 127, 101. Fraser, R. S. S. (1983). Physiol. PlantPathol. 22, 109. Fraser, R. S. S., and Clay, C. M. (1983). Neth.J. PlantPathol. (in press). Fraser, R. S. S., and Loughlin, S. A. R. (1980). J . Gen.Virol. 48,87. Fraser, R. S. S., and Loughlin, S. A. R. (1982). Physiol. PlantPathol. 20,109. Furusawa, J., and Okuno, T. (1978). J. Gen.Virol. 40, 489. Gianinazzi, S. (1982). In “Active Defence Mechanisms in Plants” fR. K. S. Wood, ed.), pp. 275 - 298. Plenum, New York. Gianinazzi, S. (1983). In “Plant-Microbe Interactions, Molecular and Genetic Perspectives” (E. W. Nester and T. Kosuge, eds.), Vol. 1. Macmillan, New York (in press). Gianinazzi, S., and Kassanis, B. (1974). J. Gen.Virol. 23, 1. Gianinazzi, S., Vallee, J. G., and Martin, C. (1969). C.R. Hebd.Seances Acad.Sci. 282,800. Gianinazzi, S.,Martin, C., andVallee, J. C. (1970). C. R.Hebd.SeancesAcad. Sci. 270,2383. Gibbs, A,, and Harrison, B. (1976). “Plant Virology. The Principles.” Edward Arnold, London. Gill, C. C., and Chong, J. (1981). Virology 114, 405. Goelet, P., and Karn, J. (1982). J.Mot. Biol. 164, 541. Goelet, P., Lomonosoff, G. P., Butler, P. J. P., Akam, M. E., Gait, M. J., and Karn, J. (1982). Proc. Natl. Acad.Sci. U.S.A. 79, 5818. Goldbach, R., and Krijt, Y. (1982). J. Virol. 43, 1151. Goldbach, R., Rezelman, G., and Van Kammen, A. (1980). Nature(London) 286,297. Goldbach, R., Frenssen, H., Rezelman, G., Van Kammen, A., Verver, J., and Wezenbeek, P. (1983). ColdSpring HarborSymp.Quant. Biol. (in press). Gonda, T. J., and Symons, R. T. (1979). J.Gen.Virol. 45, 723. Guilley, H., Jonard, G., Kukla, B., and Richards, K. E. (1979). Nucleic AcidsRes.6, 1287. Hamilton, R. I., and Nichols, C. (1977). Phytopathology 67,484. Hardy, S. F., German, T. L., Loesch-Fries, S. L., and Hall, T. C. (1979). Proc. Natl. Acad.Sci. U.S.A. 76,4956. Hirth, L., and Richards, K. E. (1981). Adv.VirusRes.26, 145. Hiruki, C., Shukla, P., and Rao, D. V. (1976). Phytopathology 66,594. Huber, R., Rezelman, G., Hibi, T., and Van Kammen, A. (1977). J. Gen.Virol. 34, 315. Huber, R., Hontelez, J., and Van Kammen, A. (1981). J. Gen.Virol. 5 5 , 241. Hunter, T. R., Hunt, T., Knowland, J., and Zimmern, D. (1976). Nature(London)260,759. Ikegami, M., and Fraenkel-Conrat, H. (1979). J. Biol. Chem.264,149. Imazumi, I., and Kubo, S. (19BOa). Ann.Phytopathol. Soc.Jpn.46, 54. Imazumi, I., andKubo, S. (1980b). Ann.Phytopathol. Soc. Jpn.46, 422. Jackson, A. O., Zaitlin, M., Siegel, A., and Francki, R. I. B. (1972). Virology 48,655. Jaspars, E. M. J. (1974). Adu.Virus Res.19, 37. Jockusch, H. (1966a). 2. Vererbungsl. 98, 320. Jockusch, H. (1966b). 2. Vererbungsl. 98,344. Jockusch, H. (1966~). Phytopathol. 2.55, 185. Jockusch, H. (1968). Virology 35,94. Joshi, S., Pleij, C. W., Haenni, A. L., Chapeville, F., andBosch, L. (1983). Virology 127,100. Kapitsa, 0. S., Vostrova, N. G., and Andreeva, E. N. (1969). Vopr.Virusol. 5,623. Kaplan, I. B., Kozlov, Yu. V., Pshennikova, E. S., Taliansky, M. E., and Atabekov, J. G. (1982). Virology 118,317. Res.10,219. Kassanis, B. (1963). Adv.Virus

362

J. G. ATABEKOV AND YU. L. DOROKHOV

Kassanis, B. (1968). Adu. Virus Res.13,147. Kassanis, B. (1981). Phytoputhol. 2.1 0 2 , 277. Kassanis, B., and Bastow, C. (1971a). J. Gen. Virol. 1 0 , 95. Kassanis, B., and Bastow, C. (1971b). J.Gen.Virol. 1 1 , 171. Kassanis, B., and Conti, M. (1971). J. Gen. Virol. 1 3 , 361. Kassanis, B., Gianinazzi, S., and White, R. F. (1974). J . Gen. Virol. 2 3 , 11. Kim, K. S., and Fulton, J. P. (1973). J. Ultrastruct. Res. 45,328. Kitajima, E. W., and Lauritis, J. A. (1969). Virology 3 7 , 681. Knowland, J., Hunter, T., Hunt, T., and Zimmern, D. (1975). Colloq. -Zmt. Nutl. Sunte Rech.Med. 47,211. Koike, M., Hibi, T., and Yora, K. (1976). Ann. Phytoputhol. SOC.Jpn.42, 105. Koike, M., Hibi, T., and Yora, K. (1977). Virology 8 3 , 413. Kubo, S. (1981). “SMI/AAB Descriptions of Plant Viruses,” No. 234. Kubo, S., and Takanami, Y. (1979). J. Gen.Virol. 4 2 , 387. Leonard, D. A., and Zaitlin, M. (1982). Virology 117,416. Le Roy, C., Stussi-Garaud, C., and Hirth, L. (1977). Virology 8 2 , 4 8 . Loebenstein, G., Spiegel, S., and Gera, A. (1982). 1n“Active Defense Mechanisms in Plants” (R. K. S. Wood, ed.), pp. 211-230. Plenum, New York. Maekawa, E., Furusawa, I., and Okuno, T. (1981). J. Gen.Virol. 5 3 , 353. Matthews, R. E. F. (1980). Compr. Virol. 16,297-359. Matthews, R. E. F. (1981). “Plant Virology,” 2nd ed., Academic Press, New York. Maule, A. J., Boulton, M. I., and Wood, K. R. (1980). J . Gen. Virol. 6 1 , 27. May, J. T., Gilliland, J. M., and Symons, R. H. (1970). Virology 4 1 , 653. Meshi, T., Ohno, T., and Okada, Y. (1982a). Nuckic Acids Res.10,611. Meshi, T., Ohno, T., and Okada, Y. (1982b). J. Biochern. (Tokyo) 9 1 , 1441. Meshi, T., Takanami, N., Ohno, T., and Okada, Y. (1982~). Virology 118,64. Miller, W. A., and Hall, T. C. (1983). Virology 1 2 5 , 236. Miroshnichenko, N. A., Dorokhov, Yu. L., Alexandrova, N. M., and Atabekov, J. G. (1983). In preparation. Motoyoshi, F. (1982). Proc. Int. Congr.Tissue Cell Cult. 5th,1982 pp. 659-660. Motoyoshi, F., and Oshima, N. (1977). J. Gen.Virol. 34,499. Motoyoshi, F., and Oshima, N. (1979). J. Gen.Virol. 44,801. Motoyoshi, F., Bancroft, J. B., and Watts, J. W. (1974). J. Gen.Virol. 25, 31. Nassuth, A., and Bol, J. F. (1983). Virology 1 2 4 , 75. Nassuth, A., Alblas, F., and Bol, J. F. (1981). J. Gen.Virol. 63,207. Nassuth, A., ten Bruggencate, G., and Bol, J. F. (1983). Virology 1 2 6 , 75. Nishiguchi, M., Motoyoshi, F., and Oshima, N. (1978). J. Gen.Virol. 3 9 , 5 3 . Nishiguchi, M., Motoyoshi, F., and Oshima, N. (1980). J. Gen.Virol. 46,497. Norris, D. 0. (1951). Aust. J. Agric. Res.2 , 221. Novikov, V. K., and Atabekov, J. G. (1970). Virology 4 1 , 101. Ohno,T., Takamatsu, T., Meshi, T., Okada, J., Nishiguchi, M., andKiho, Y. (1983). Virology 131,255.

Ooshika, I., Watanabe, Y., Meshi, T., Okada, Y., Igano, K., Inouye, K., and Yoshida, N. (1983). Virology 132, 71. Pelham, H. R. B. (1978). Nature (London) 272,469. Pelham, H. R. B., and Jackson, R. J . (1976). Eur.J. Biochem. 6 7 , 247. Peters, D. L., and Murphy, T. M. (1975). Virology 6 5 , 595. Preobrazhensky, A., and Spirin, A. (1978). Prog. Nucleic Acid Res.Mol. Biol.2 1 , 1. Rappaport, I., and Wildman, S.G. (1957). Virology 4 , 265. Rezelman, G., Franssen, H. J., Goldbach, R. W., Ie, T. S., and Van Kammen, A. (1982). J. Gen.Virol. 60, 335.

PLANT VIRUS-SPECIFIC TRANSPORT FUNCTION

363

Robards, A. W. (1975). Annu. Rev. Plant Physiol. 26, 13. Roberts, D. A. (1982). Virology 122,207. Robinson, D. J., Barker, H., Harrison, B. D., andMayo, M. A. (1980). J .Gen. Virol. 51,317. Rochow, W. F., and Ross, A. F. (1954). Phytoputhology 44, 504. Rochow, W. F.,and Ross, A. F. (1955). Virology 1, 10. Romaine, C. P., and Zaitlin, M. (1978). Virology 86,241. Rottier, R. J. M., Rezelman, C., and Van Kammen, A. (1979). Virology 92,299. Sakai, F.,Dawson, J. R. O., and Watts, J. W.(1983). J. Gen. Virol. 64, 1347. Samuel, G.(1931). Ann. Appl. Biol. 18, 494. Sanger, H.L., and Brandenburg, E. (1961). Naturwissenschajten 48,391. Sarachy, A. N., Nassuth, A., Roosien, J., Van Vloten-Doting, L., and Bol, J. F. (1983). Virology 125,64. Sarkar, S. (1969). Mol. Gen. Genet. 105, 87. Sarkar, S.,and Smitamana, P. (1981a). Naturwissenschajten 68, 145. Sarkar, S.,and Smitamana, P. (1981b). Mol. Gen. Genet. 184,158. Scalla, R., Romaine, C. P., Asselin, A.,Rigaud, J., and Zaitlin, M. (1978). Virology 91,182. Schneider, I. R. (1965). Adv. Virus Res.11,163. Schneider, I. R., and Worley, J. F. (1959a). Virology 8, 230. Schneider, I. R.,and Worley, J. F. (1959b). Virology 8,243. Sehgal, 0.P.(1973). Mol.Gen. Genet. 121, 15. Sela, I. (1981). Adu. Virus Res. 26,201. Semal, J., and Hamilton, R. (1968). Virology 36,293. Shalla, T. A., Peterson, L. J., and Zaitlin, M. (1982). Virology 60,355. Shaskolskaya,N. D., Atabekov, J. G., Sacharovekaya, G. N.,andJavachia,V. G. (1968). Biol. Nauki USSR 8,101. Siegel, A. (1960). Virology 11, 156. Siegel, A., and Zaitlin, M. (1964). Annu. Reu.Phytopathol. 2,179. Siegel, A., Zaitlin, M., and Sehgal, 0. P. (1962). Proc. Nutl. Acad. Sci. U.S.A. 48,1845. Siegel, A., Zaitlin, M., and Duda, C. T. (1973). Virology 53, 75. Siegel, A., Hari, V., Montgomery, I., and Kolacz, K. (1976). Virology 73, 363. Smit, C. H., and Jaspars, E. M. J. (1982). Virology 117,271. Spirin, A. S.(1969). Eur.J. Biochern. 10, 20. Spirin, A. S. (1972). I n “Mechanism of Protein Biosynthesis and its Regulation (L. Boscb, ed.), pp. 515-537. North-Holland Publ. Amsterdam. Stoffer, R. F., and Ross, A. F. (1961a). Phytopathology 51,5. Stoffer, R. F.,and Ross, A. F. (1961b). Phytopathology 51, 740. Sulzinski, M.A., and Zaitlin, M.(1982). Virology 121,lZ. Takamatsu, N., Ohno, T., Meshi, T., and Okada, Y. (1983). Nuckic Acids Res.11,3767. Taliansky, M.E.,Atabekova, T. I., Kaplan, I. B., Morozov, S. Yu., Maeyshenko, S. I., and Atabekov, J. G. (1982a). Virology 118,301. Taliansky, M. E., Kaplan, I. B., Yarvekulg, L. V., Atabekova, T. I., Agranovsky, A. A., and Atabekov, J. G.(1982b). Virology 116,309. Taliansky, M. E., Malyshenko, S. I., Pshennikova, E, S., Kaplan, I. B., Ulanova, E. F., and Atabekov, J. G.(1982~).Virology 122,318. Taliansky, M. E., Malyshenko, S. I., Pshennikova, E. S., and Atabekov, J. G. (1982d). Virology 122,327. Van Loon, L.S. (1976). J. Gen. Virol. 30,375. Van Loon, L. C. (1982). I n “Active Defense Mechanisms in Plants” (R. K. S. Wood, ed.), p. 247. Plenum, New York. Van Loon, L. S. (1983). I n “The Dynamics of Host Defense” (J. A. Bailey, ed.). Academic Press, New York (in press).

364

J. G. ATABEKOV AND YU. L. DOROKHOV

Van Vloten-Doting, L., and Neelman, L. (1982). Encycl. Plant Physiol., New Ser.16. Watanabe, Y., Emori, Y., Ooshika, I., Meshi, T., Ohno, T., and Okada, Y. (1983). Virology 133,18. Weening, C. J., and Bol, J. F. (1975). Virology63, 77. Weintraub, M.,Ragetli, H. W. J., and Leung, E. (1976). J. Ultrastruct. Res. 66,351. Whitfeld, P.R.,and Higgins, T. J. V. (1976). Virology 71,471. Wieringa-Brants, D. H.(1981). J.Gen.Virol. 64, 209. Wieringa-Brants, D. H., Timmer, F. A., and Rouweler, H. C. (1978). Neth. J.Plant Puthol. 84,239. Zaitlin, M., Duda, C. T., and Petti, M. A. (1973). Virology 53,300. Zaitlin, M., Beachy, R. N., Bruening, G., Romaine, C. P., and Scalla, R. (1976). I n “Animal Virology” (D. Baltimore, A. S. Huang, andF. C. Fox, eds.),pp. 567-581. Academic Press, New York. Zelcer, A., and Zaitlin, M. (1981). Virology 113,417. Zhuravlev, Yu.N.(1979). “Phytoviruses in Plants and Model Systems.” Nauka, Moscow.

AUTHOR INDEX Numbers in italics refer to pages on which the complete references are listed.

A Abbott, J. D., 304,305,307 Abelson, J., 250,258 Aberle, H., 256, 258 Ackermann, H. W., 267,286, 287,288,289, 290,291,293,307

Adam, G., 69,91, 152,165, 227,261 Adams, A. N., 132,166, 246,258 Adams, D. H., 45,48 Adams, M. H., 267,307 Adams, W. R. J., 238,261 Adhikari, P. C., 271, 302,307,308 Adhya, S., 300,301,305,308 Aebig, J., 134,135,142, 149, 151,167 Agranovsky, A. A., 318,333,363 Ahmad-Zadeh, C., 187,194 Ahmed, R., 78, 81,83, 85,90 Air, G. M., 103, 104, 110, 111, 112, 127, 130 Akam, M. E., 323,325,332,361 Alain, R., 193,193 Albert, D. M., 11,53 Alblas, F., 228, 231, 234, 235, 254,258, 261, 328,362

Alemohammad, M. M., 298,311 Alexandrova, N. M., 314,347,348,349, 350,351, 352,354,357,358,360 Almeida, J. D., 169, 170, 188, 191, 193, 194 Almond, J. W., 112,126, 128 A1 Moudallal, Z., 102, 124, 133, 135, 137, 138, 139,142, 144,146,147,148, 149, 154,155, 156,158,164,165, 166 Alper, T., 2, 29, 30, 32, 33, 45, 48, 53 Alpers, M. P., 7, 8, 10, 11, 13, 14, 15, 48, 50,

51,55 Altschuh, D., 132, 144, 145, 147, 154, 155, 166, 168 Alvarez, G., 289,307 Amako, K., 279,294,295,296,297,302,312 Amyx, H. L., 10, 11, 14, 21, 25, 50, 51, 52 Anacker, R. L., 271,311 Anderegg, J., 116, 127 Anderer, F. A., 132,160, 166 Anderson, L. A. P., 265,307 Anderson, N. G., 163, 166 Anderson, R. D., 45,56

Anderson, T. F., 169,193 Andral, L., 102, 124 Andreeva, E. N., 319,326,361 Andrews, P., 34, 48 Angelo, J., 7, 8, 11,53 AnnB, J., 257,258 Antczak, D. F., 96,103,124,128 Anthony, D. W., 197,211 Aoki, S., 67, 93, 215, 216, 217, 219, 221, 236,237,258,262

Aoki, Y.,304,311 Appleton, J. A., 103, 106, 122, 124, 127 Appleyard, G., 198,213 Arcioni, S., 243,258 Arif, B. M., 196,200,202,204,205,206,211 Armagier, A., 197,212 Aronson, A. I., 204,212 Arutynnov, Y. I., 292,309 Asher, D. M., 7,8, 10,11, 12, 13, 51, 53 Asheshov, I., 265,267, 276,290,307 Asheshov, I. N., 265,267,276,290,307 Askonas, B. A., 117,126 Asselin, A., 322, 323,359, 363 Asuyama, H., 228,259 Atabekov, J. G., 314,318, 322,325, 329, 331, 332, 333, 334, 335, 336, 338, 339, 341, 343, 347, 348, 349, 350, 351, 352, 354,355,357,358,359,360,361,362,363 Atabekova, T. I., 318,333,334,335,363 Atassi, M. Z., 123, 124 Aubert, M., 119,125 Audurier, A,, 267, 307

B Babiuk, L. A., 115,128 Bacchetti, S., 101, 114, 115, 121, 122,124 Bacon, L. D., 101, 113, 127 Bacote, A,, 14,51 Bairagi, A. K., 288,310 Baird, S. M., 113, 114, 129 Bajet, N. B., 228, 241, 258, 259 Baker, F., 19,51 Balachandran, N., 101, 114, 115, 121, 122, 124

Balfour, H. H., 101, 128 365

366

AUTHOR INDEX

Balganesh, M., 288,307 Balganesh, T. S., 305,310 Balian, G., 32, 49 Baltz, H. R., 257, 258 Banatvala, J. E., 170,193 Bancroft, J. B., 230, 231,234,236,237,247, 260,339,362 Banerjee, A. K., 60,63,64,66,90,92,108,125 Barbara, D. J., 150,166 Baringer, J. R., 9, 11, 19,26,27, 33, 43,48, 51, 55, 121,125 Barker, H., 217, 227,228,231,254,258, 259,260, 328,363 Barksdale, L., 306,307 Barlass, M., 217,219,220, 221,222,232,258 Barnet, B., 106, 129 Barnett, A., 227,242,258, 260 Barnett, 0. W., 248,259 Baross, J. A., 267,307 Barrett, P. N., 120, 121, 124 Bartkowski, J., 197,212 Barua, D., 264, 283,287, 290, 5'. 3, 299,307, 308 Baserga, R., 106,128 Bastian, F. O., 43, 46,48 Bastow, C., 333,362 Basu, J., 300, 301, 305,308 Basu, S., 266,289, 290,292,307, 310,311 Bauer, R. J., 255,262 Bautz, E., 32,50 Bautz-Freese, E., 32,50 Bayley, P. M., 117,129 Bazin, H., 103, 124 Beachy, R. N., 67,93, 216,248,249,250, 252, 253, 256,258, 261, 262, 322, 323, 350,355,359,364 Beale, H. P., 132, 166 Bean, W. J., 96, 110, 126, 128 Bech-Hansen, C. W., 225,259 Beck, E., 2,8, 9, 10, 11, 21, 22, 49, 51 Beetfink, F., 223,224,225,261 Beier, H., 218, 219, 220, 221, 228, 231, 237, 258,339,340,359 Beiiersbergen, J. C. M., 134, 135,167 Bellamy, A. R., 66, 91 Bellini, W. J., 115, 118, 124 Benda, G. T. A., 337,359 Benedetti, E. L., 42, 49 Benenson, A.S., 277,310 Benjamin, D. C., 110, 130 Bennett, C. W., 315,319,359

Benton, C. V., 104,112,124,126 Berek, C., 146, 168 Berenstein, E. H., 138, 139, 142, 166, 168 Berger, R., 103,126 Bergoin, M., 196, 198,200,203,207, 209, 21 1,212 Berkeley, R. C. W., 300,310 Berliner, M., 256, 258 Bernhard, W., 110,111,127 Bernoulli, C., 7, 8, 10, 12, 13, 53 Berns, K. I., 205,211 Bers, N., 196, 212 Bert, J., 19, 51 Berte, C., 99, 108, 110,124 Berthiaume, L., 193, 193, 267,307 Berton, M. T., 103,104,112,120,130 Best, J. M., 170, 193 Beth, E., 171, 176, 190, 192, 194 Beverley, P. C., 121,125 Bhalla, R. B., 319, 326,359 Bhaskaran, K., 299,307 Bhattacharya, F. K., 264,298,308,312 Bhattacharya, P., 292,311 Bignami, A., 43,49 Bildstein, C., 5, 26, 29,34,42,55 Bilimoria, S. L., 202, 204,211 Bilkey, P. C., 225,258 Bils, R. F., 58, 59, 90 Binington, H. B., 42,56 Bird, F. T., 196,200,207,208,210,211 Bister, F., 271,312 Black, D. R., 58, 59, 61, 63, 64, 66, 90, 91, 103,126 Black, L. M., 57, 58,59, 60, 61,62,63, 64, 66, 67, 69, 72, 73, 74, 75, 76, 78, 79, 80, 81, 83, 85,86, 87, 88, 90, 91, 92 Blancou, J., 102,119,124, 125 Blann, A. D., 96,124 Blattner, F. R., 88,93 Bloemendal, H., 42,49 Bloomer, A. C., 157, 166 Blythman, H. E., 122,124 Bobkova, 0. V., 20,50 Bobowick, A. R., 11,53 Boccardo, G., 187,194 Boeye, A., 116, 124, 129 Bogers, R. J., 219,258 Bohn, W., 96,124 Bol, J. F., 228, 231, 234, 235, 254,258, 261, 325,327,328,362, 363, 364 Bolton, A. E., 39,49

367

AUTHOR INDEX Bolton, D. C., 3, 4, 27, 38, 39,41,42, 45,48, 49, 53, 55 Bon, S., 42, 56 Bootman, J., 112,128 Borgert, K., 31,49 Bornstein, P., 32,49 Borsa, J., 64, 91 Bosch, F. X., 333,359 Bosch, L., 323, 361 Both, G. W., 69,91 Boucher, D. W., 102,125 Boue, A., 102,125 Boulton, M. I., 227, 232, 237, 239,260, 340, 362 Bouras, C., 19,51 BovB, J. M., 227, 242, 243, 254, 255,261 Bowman, K. A., 3, 4, 9, 16, 17, 19, 27, 29, 32, 34, 35, 38, 42, 45, 48, 48, 55 Boxall, E., 170, 194 Boyle, C. C., 9, 18, 51 Bozarth, R. F., 171,174,189,192,194, 205, 206,212 Bradley, D. E., 290, 303, 308 Brakke, M. K., 58,73, 87,90,91 Branch, A., 250,258 Brandenburg, E., 330,363 Brandis, H., 265,308 Brandriss, M. W., 102, 121,128, 129 Brandt, W. E., 102,107,121, 224, 126 Braun, A. C., 88,91 Braun, D. G., 101,128 Braun, D. K., 97,124 Brennan, V. E., 79,91 Brenner, S., 169, 193, 283,308 Bres, P., 102, 128 Briand, J. P., 102, 124, 132, 133, 135, 137, 138, 139, 142, 144, 146,147, 148, 149, 154, 155, 156, 158, 164,165, 166, 168 Bricogne, G., 157, 166 Brioen, P., 116, 124, 129 Britt, W. J., 113, 114, 124 Brlansky, R. H., 171, 172,173,193 Brochard, M., 242,243,261 Broekhuysen, M., 316,361 Brown, D. M., 32,54 Brown, E. B., 117, 129 Brown, E. G., 78,79,85,91 Brown, L. E., 103, 107, 112,127 Brown, P., 26,49 Brown, R., 271,311 Brownlee, G. G., 111, 125

Bruce, M. E., 21,49, 50, 56 Bruck, C., 99, 108, 110, 111,124 Bruening, G., 218, 219, 220, 221, 228, 231, 237,258, 322, 323, 339, 340,359, 364

Bruenn, J. A., 79,91 Bryner, J. H., 271,310 Brzosko, W. J., 194 Bucana, C., 26,56 Buchan, A., 104,127 Buchatskyi, L. P., 197,211 Buchmeier, M. J., 106, 113, 125, 128, 129 Buckmaster, A., 104, 127 Buckner, C. D., 101,129 Bujarski, J. J., 328,359 Bulla, L. A,, Jr., 204,212 Burckard, J., 171, 175, 180, 194 Burger, D., 9, 51 Burgess, J., 230, 236, 237, 247,258, 260 Burke, J. M., 196,211 Burkholder, P. R., 87,92, 92 Burnette, W. N., 138,149, 167 Burny, A., 99, 108, 110, 111, 124 Burrows, W., 264, 293, 307 Burstin, S. J., 107, 119, 124 Burtonboy, G., 103,124 Butcher, G .W., 138,166 Butcher, R. N., 137,139,144,167 Butler, P. J. G., 115, 124, 323, 325, 332,361 Butzow, J. J., 31, 49 Buyukmihci, N., 9,49 C

Cabau, N., 102,125 Cadman, C. H., 330,360 Caldwell, J., 318, 347,360 Calisher, C. E., 103, 105, 107, 126 Calkins, E., 42,49 Callard, R. E., 121, 125 Cancilla, P. A., 43, 46,48 Cann, A. J., 112,128 Carlberg, I., 243,258 Carlson, R. I., 100, 101,130 Carmichael, L. E., 103, 128 Carp, R. A., 9,52 Carp, R. I., 43,54 Carr, R. J., 344,360 Carroll, R. B., 118, 124 Carson, D. A., 113, 114,129 Carter, M. J., 102, 104, 107,109, 110, 113, 114,115, 118,124, 125, 129

368

AUTHOR INDEX

Carter, W. G., 42,49 Casey, G. A., 102,125 Casjens, S. R., 35, 50 Caspar, D. L. C., 160,167 Caspary, E. A., 45,48 Casper, R., 150,166 Cassells, A. C., 217, 219, 220, 221, 222, 232, 233,244,245,258 Castro, A., 104, 125 Catherall, P. L., 316,360 Caton, A. J., 111, 125 Catt, K. J., 170, 193 Celis, J. E., 289,307 Cepko, C. L., 117,125 Chakrabarty, A. N., 305,310 Chamberlain, J. A., 316,360 Champness, J. N., 157,166 Chanas, A. C., 107,112,121,125 Chancock, R. M., 170,194 Chanda, P. K., 268,269,273, 278,279,280, 281,282,283,284,289,308, 310 Chandler, R. L., 2, 20,49, 52 Chandra, P., 281,308 Chang, K., 134,135,167 Chang, M. W., 295,308 Chao, J., 170, 193 Chapeville, F.,323,361 Charlton, K. M., 102,125 Charpilienne, A., 64, 91, 97, 129 Chase, R., 118,129 Chatterjee, S. K., 267, 276, 290,307 Chatterjee, S. N., 268, 269, 270, 271, 272, 273, 274, 275, 277, 278, 279, 280, 281, 282, 283, 284, 287, 289, 290, 297, 302, 307,308,310,311,312 Chaudhuri, K., 268,270,272,273,277,278, 280,282,283,288,289,308,310 Chelle, P. L., 45, 49 Chen, S. N., 112, 113,128 Cheo, P. C., 345,360 Chernesky, M., 101,124, 171,176,190,192, 194 Chesebro, B., 113, 114,124 Cheung, R., 301,304,309 Cheyne, I. M., 289,309 Chilton, M. D., 88,91, 93 Chiu, R. J., 58, 61, 69, 73,91 Cho, H. J., 36,45,49 Chong, J., 337, 361 Choppin, P. W., 113,126

Chou, S. M., 22,49 Chun, D., 295,308 Chung, J. K., 295,308 Cianciolo, G., 106, 125 Claflin, L., 136,142,166 Clark, M. F.,132, 150,166, 246,258 Clark, R., 108, 111, 125 Clark, W. W., 7,8, 13, 51 Clarke, L. D., 112, 128 Clarke, M. C., 2, 20, 29, 30, 32, 33,45, 48, 49,53 Clay, C. M., 361 Clegg, J. C., 121, 125 Cleveland, P. H., 101,129 Clift, R. A., 101, 129 Close, R., 336, 337,360, 362 Cochran, S. P., 3, 4, 5 , 15, 16, 17, 25, 26, 27, 29, 32, 33, 34, 35, 38, 42, 45, 48,55 Cocker, F.M., 232,233,244,258 Cocking, E. C., 67, 91, 215, 216, 219,221, 225, 233,243,247,256,258,261 Codd, A. A., 188,194 Cohen, A. S., 42,45,49 Cohen, G. H., 109, 111,118,125, 187,194 Cohen, J., 64, 91, 97,129, 176, 190, 193, 242,260,320,360 Cole, G. A,, 121, 129 Collins, A. R., 106, 125 Collins, J. K., 113, 114, 124 Collis, S. C., 21, 49 Collmer, C. W., 323, 360 Colman, P. M., 103,111,120, 125, 130 Comoglio, P. M., 118,126 Conrad, K., 85,91 Conscience, J.-F.,144,166 Consigli, R. A., 204,212 Constabel, F., 220, 225,259 Conti, M., 333, 362 Cooper, D., 236,237,262 Corey, L., 96, 101, 102, 104, 128 CBte, P. J., Jr., 97, 102, 125 Couillin, P., 102, 125 Coulon, P., 119,125 Coutts, R. H. A,, 227,233,247,258 Cove, D. J., 256,259 Covey, L., 106, 129 Cox, N. A., 20,54 Cox, R. A., 42,49 Crainic, R., 102,125 Cramer, R., 33,53

AUTHOR INDEX Cramp, W. A., 2, 29, 30, 32, 48 Crawford, A. M., 210,211 Crawford, D. H., 121, 125 Crawford, L., 123,125 Crawford, L. V., 123,126 Cremer, N., 101,114, 117, 128 Creutzfeldt, H. G., 5,49 Croce, C. M., 97, 126 Croizier, G., 200,211 Cronshaw, J., 317, 318,360 Crowther, D., 31,49 Cuille, J., 45, 49 Cunningham, J. C., 196,210,211 Cunnington, P. G., 20,49 Currier, T. C.,88, 93

369

decamp, M., 7,8, 13, 51 DeGrere, H., 88,91 De Harven, E., 171,176,190,192,194 d'Herelle, F., 265,308 De Jager, C. P., 328,360 Delbriick, M., 272,308, 309 DeLeo, A. B., 106,128 Delferriere, N., 103, 124 Dellaporta, S. L., 240, 246,259 de Milton, R. C., 157, 158,167 De Monte, A. J., 265, 276, 290, 311 Depicker, A., 88,93 Derks, A. F. L. M., 134,135, 167 Dernick, R., 117,125 Derrick, K. S., 170, 171, 172, 173, 187, 188, 193

D Da Graca, J. V., 360 Dahmus, M. E., 106, 125 Dale, P., 256,258 Dales, S., 198, 200, 203,211, 212 Dalrymple, J . M., 102, 107,121, 126, 129 Damico, J., 256,258 Dangl, J. L., 144,166 Daniel, P. M., 2,8, 9, 10, 11, 21, 22,49, 51 Daniels, R. S., 117, 125 Dapolito, G. M., 102, 125 Darlington, C. D., 45,49 Das, J., 283,284, 287,288,290,307,308 Dastidar, S. G., 300, 301, 305,308, 310 Datta, A., 301, 305,308 Davauchelle, G., 196, 200,211 Davey, M. R., 243, 256,258 David-Ferreira, J. F., 43,49 David-Ferreira, K. L., 43,49 Davies, J. W., 322, 351,360 Davies, T., 122, 125 Davis, J. M., 144, 166 Davis, L. J., 113, 126 Davis, W., 163, 166 Davison, E. M., 316, 360 Dawson, J. R. O., 227, 232,236,237,239, 259,262, 339,363

Dawson, W. O., 248,259, 325,328, 349,360 Day, J. W., 106, 117, 129 De, B. K., 108,125 De Beuckeleer, M., 88,93 Deblaere, R., 88,91 Debouck, P., 170,194

DesRosiers, M. H., 19, 56 Devauchelle, G., 198, 207,211 Devlin, V., 101, 128 De Zoeten, G. A., 314, 329, 338,348,360 Diaco, R., 134, 147,149, 151, 152,166 Dickerson, P. E., 232,236,237,239,259 Dickinson, A. G., 4, 5, 7, 8,22,24,25,49, 50 Dickson, E., 251,262,357,360 Diener, T. O.,3, 29, 45, 47, 48, 50 Dietzgen, R. G., 117, 125, 132, 133, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144, 146, 147, 148, 149, 152, 156, 157, 158,159,160,163,164,166 Dietzschold, B., 119, 125 Digoutte, J. P., 102, 103, 125, 128 Dijkstra, J., 363 Dilworth, S. M., 97, 125 Di Marco, A., 281,308 Dittmar, D., 104, 125 Divry, P., 22,50 Dix, R. D., 121, 125 Doane, F. W., 170,193 Dodds, J. A., 255,260,325,332,336,341,360 Doermann, A. H., 272,308,309 Doherty, P. C., 121, 125 Doi, H., 11, 56 Dolin, R., 170, 194 Domdey, H., 29,55 Don, G., 101, 130 Dondero, D. V., 101, 104,114, 115, 128 Dopheide, T. A. A,, 110, 111, 127 Dorokhov, Y. L., 314, 347, 348, 349, 350, 351,352,354,357,358,360 Dorssers, L., 328,360

370

AUTHOR INDEX

Dos Santos, A. V. P., 243,258 Douglas, A. R., 117, 125 Douillard, J. Y., 114,166 Downey, D. E., 3,4, 16, 17, 27,55 Downie, J. C., 187, 194 Drampyan, A. H., 343,359 Dreesman, G. R., 96,129 Drexler, H., 268, 273, 277, 279, 282, 284,

Engvall, E., 159, 166, 170, 193 Epstein, D. A,, 204,211 Ericson, R. L., 205,212 Eriksson, T., 221,224,225,235,243,258,262 Esau, K., 315, 316,317, 318,319,360 Esiri, M. M., 22, 56 Evans, D. M., 112,128

295, 296,297,312

Drost, G., 106,122, 128 Drozhevkina, M. S., 292,309 Ducrest, P., 292, 299,311 Duda, C. T., 322,328,363, 364 Duffus, J. E., 151,167 Dulbecco, R., 137, 166 Dunez, J., 171, 175, 190, 194 Dunia, I., 42,49 Dunnebacke, T. H., 59,91 Durand, D. P., 134,135, 147, 149, 151, 152, 162,166, 167 Durbin, R., 118, 127 Durham, A. C. H., 115,127 Durr, A., 250,259 Duthoit, J. L., 196, 197,200,211,212 Dutta, N. K., 292,312 Dvorak, M., 131,166

E Earnshaw, W. C., 35,50 Ebihara, S., 11, 52 Ebringer, L., 304,312 Edmunds, C., 227,232,237,239,260 Egami, F., 300,310 Eichorn, G. L., 31,49 Einschermann, P., 197,211 Eisenberg, R. J., 109,111, 118,125, 187,194 Eisenstark, A., 290, 291, 292,307, 311 Eklund, C. M., 2, 7, 8, 9, 13, 15, 18, 26, 27, 33,45,48,50, 51, 55

Elder, J. H., 110, 128 Ellis, D. S., 107, 112, 121, 125 Ellis, E. L., 272, 309 Elmore, J., 271,310 Emerson, E. E., 42, 50 Emini, E. A., 110,116,125 Emori, Y., 323, 331, 332,364 Engel, J., 42,54 Engler, D. E., 219, 221,259 Engler, G . ,88, 93

F Falkow, S., 298,310 Farkas, G. L., 220,261 Farkas-Himsley, H., 299, 300, 301, 302, 303,304,305,306,309,310

Faulkner, P., 163, 167, 205, 213 Faustmann, O., 250,261 Fazekas de St. Groth, S., 137, 138,139, 145,146,166

Feary, T. W., 267,309 Feder, J., 145,166 Federici, B. A., 197,211 Feeley, J. C., 264, 309 Fefer, A., 101, 129 Feinstone, S. M., 170, 193 Feorino, P. M., 102, 127 Ferenczy, L., 257,259 Ferguson, M., 102,112,116,126, 128 Fernie, B. F., 97, 125 Ferracini, R., 118, 126 Feynerol, C., 97, 129 Field, A. M., 169, 192, 193 Field, E. J., 21, 43, 45,48, 50, 55 Field, H. J., 18, 52, 121, 127 Fields, B. N., 78,79, 81,83, 85,90, 91, 107, 119,120, 124, 126, 129

Filipowicz, W., 42, 50, 250,260 Filshie, B. K., 196, 197, 198,211 Finberg, R., 120,126 Fink, G. R., 79,93 Finkelstein, R. A., 277,309 Fisher, E., Jr., 267, 309 Fisher, T. N., 267,309 Flack, J. H., 227,260 Flamand, A., 102,119, 125, 130 Fleming, E. N., 230,258 Fleurdelys, B., 113,126 Flewett, T. H., 170, 194 Flores, J., 103, 126 Flu, P. C., 265,309

371

A U T H O R INDEX

Ford, E. C., 97,125 Forghani, B., 101,126 Forni, L., 146,168 Fosset, J., 196, 212 Fowke, L. C., 220,225,259 Fox, P. C., 138,139, 142,168 Fox, V. L., 102,129 Foxe, M. J., 256,259 Fraenkel-Conrat, H., 131,168, 328,361 Fraley, R. T., 240, 246,259 Frame, B., 101,124 Francis, R. T., 46, 51 Francki, R. I. B., 150,168,218,259,322,361 Franke, J., 134,167 Frankel, M. E., 96, 110, 126 Franklin, E. C., 42,55 Franklin, H. A,, 137, 167 Franklin, R. M., 32,50 Franssen, H. J., 252,261, 316, 330,362 Franssen, J. D., 99, 108,110, 124 Franzblan, C., 42,52 Fraser, H., 7, 8, 18, 21, 24, 25, 49, 50, 56 Fraser, R. S. S., 314, 320, 344,361 FrBdBricq, P., 299, 300, 301,309 Freeman, G., 137,166 Freeman, J., 256, 258 Freeman, V. J., 306,309 Freese, E., 32, 50 Friedrich-Freksa, H., 132, 168 Fries, P., 133, 135, 136, 137, 141, 142, 143, 144, 145, 146,148, 149, 153, 162, 163, 164,165,166, 167 Frisch-Niggemeyer, W., 237,259 Frisman, D. M., 113,114,129 Fritsch, C., 228,259 Fryd, D. S., 101, 128 Fujinami, R. S., 101, 120, 126 Fujise, N., 304,311 Fukami, H., 264,312 Fukatsu, R., 21, 50 Fukunaga, Y., 224,225,239,240,241,259 Fukushi, H., 103,126 Fuller, A., 104, 127 Fulton, J. P., 316, 362 Fulton, R. W., 150, 166, 168 Furniss, A. L., 266, 286, 287, 288, 289, 291, 293,294,307,310 Furth, M. E., 113, 126 Furthmayr, H., 42,54 Furuichi, Y., 59, 60, 64, 91, 93

Furusawa, I., 226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 241, 254, 259, 260, 261, 262, 339,362 Furusawa, J., 340,361

G Gait, M. J., 323, 325, 332,361 Gafford, L. G., 205,212 Gajdusek, D. C., 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 25, 26, 43, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 GalfrB, G., 137, 138, 143, 145, 146, 147, 166 Galland, G. G., 100, 104, 127, 128 Gallo, D., 101, 102, 114, 117, 126, 128, 129 Gallut, J., 265, 284, 292, 293, 299, 307, 309, 311,312 Galston, A. W., 238,261 Gamborg, 0. L., 220, 225,259 Gandar, J., 242, 243,261 Garcia, D., 101, 124 Gardash’yan, A. M., 20,50 Garden, E., 301,302,310 Gardiner, A. C., 20,50 Gardner, A. B., 264,309 Gardner, P. S., 104, 129 Garfin, D. E., 26,21,33,48,55 Gary, G. W., Jr., 103,126 Garzon, S., 197, 198,200, 207,212 Gatenby, A. A., 245, 258 Gaur, V. P., 281,308 Geelen, J. L., 26,56 Gegenheimer, P., 250,258 Gentile, A. C., 87,91 Gentry, G. A., 205,212 Gentry, M. K., 102, 107, 120, 124, 126 Georg-Fries, B., 101, 128 Gera, A., 220, 221, 242, 246, 252,260, 262, 314,320, 344, 345,362 Gerard, J. S., 345, 360 Gerdes, J. C., 295,309 Gerhard, W., 96,97, 109, 110,111,121, 125, 126, 127, 130, 147, 153, 163, 164, 165,168 Gericke, D., 281,308 GBrin, J. L., 97, 102, 125 German, T. L., 328,361 Gerner, R. E., 137,167 Gerstmann, J., 5, 50 Geshelin, P., 205,211

372

AUTHOR INDEX

Ghabrial, S. A., 289,309 Gianinazzi, S., 314, 344,361, 362 Giannoni, G., 35, 50 Gibbons, R. A,, 26,36,37,45,50, 52 Gibbs, A., 315,361 Gibbs, C. J., Jr., 7,8, 10, 11, 12, 13, 14, 20, 21, 22, 25, 26, 43, 45,49, 50, 51, 52, 53, 54, 55, 56 Gilbert, S. F., 116, 127 Gilbert, W., 79, 83, 84,92 Gilchrist, J. E., 115, 128 Gilden, R. V., 104,124 Gill, C. C., 337, 361 Gillies, R. R., 305, 309 Gillies, S., 83, 91 Gilliland, J. M., 328, 362 Giraldo, G., 171, 176, 190, 192, 194 Giraudon, P., 102, 114,126, 130 Gish, D. T., 131, 168 Gisiger, V., 42, 56 Glimelius, K., 224, 225, 243,258, 262 Gloeckler, R., 171, 180, 194 Glucksberg, H., 101,129 Goding, J. W., 137,138,142,146,166 Goebel, W. F., 270,309 Goelet, P., 323, 324, 325, 332,362 Gold, P., 203,211 Goldbach, R. W., 252,261, 316,330,362 Goldstein, L. C., 96,101, 102, 104,126, 128 Gomatos, P. J., 59,91 Gomez, C. Z., 264,295,297,312 Gonda, M. A., 112,126 Gonda, T. J., 228,259, 340,361 Gonzalez-Scarano, F., 103, 105,107,126 Goodman, H. M., 88,93 Goodman, R. M., 228,241,258,259 Goodwin, R. H., 196,197,198,211 Gorde, J.-M., 19,51 Gorde-Durand, J . M., 19,51 Gordon, J., 97, 129 Gordon, M. P., 88,91, 93 Gordon, W. S., 2, 7,8, 22,23, 51 Gorgacz, E. J., 7,8,11,19, 53 Gorski, D., 116, 129 Gotoh, F., 11, 52 Gotz, A., 281,308 Gotz, P., 197, 205,211 Goudsmit, J., 11,51 Gough, K. H., 171,177,178,190,194 Gould, E. A., 107,112,121,125

Grabski, C., 256,259 Graham, A. F., 64, 81,90, 91, 92, 93 Graham, E. A., 118,126 Granados, R. R., 58,59, 72,88,91, 93, 196, 197,200,204,205,207,208,209,21 I, 212 Grandien, M., 107, 112, 128 Grange, J. M., 306,309 Gras, J., 235, 245, 246, 256,259, 261 Gratia, A., 298, 299, 309 Gray, A., 46,51 Green, E. M., 26,49 Green, M. I., 85,91 Green, N. M., 42, 51 Greenberg, H., 103,126 Greenburg, J. F., 209,212 Greenfield, P., 87, 92 Greer, C. H., 250,258 GrBgoire, N., 19,51 Greig, J. R., 3, 51 Grieves, J., 116, 127 Griffin, B. E., 97,125 Griffith, J . S., 45,51 Grogan, R. G., 219,221,259 Gros, O., 122,124 Gros, P., 122, 124 Gross, H. J., 29, 42, 50, 55, 250,260 Gross, L., 47, 48, 51 Grossman, G., 109,128 Groth, D. F., 3,4, 16, 17, 21, 25, 27, 29, 30, 32, 33, 34, 35, 38, 45, 48, 55 Groth, D. G., 5,26,27, 29,34, 42,55 Gruber, M., 42,55 Gueft, B., 42,50 Gugerli, P., 133, 135, 136, 137, 141, 142, 143, 144, 145, 146, 148, 149, 153, 162, 163,164,165,166 Guha, D. K., 266,290,292,311 Guha Roy, U. K., 266,290,292,311 Guice, M. B., 276, 278, 280, 282,309 Guillemin, M. C., 97, 129 Guilley, H., 323,361 Gulyas, A., 236, 238,261 Gupta, S. K., 265, 276,290,311 Gurney, E. G., 118,124 Gustafson, D. P., 43,53

H Haas, B., 250,262 Haber, S., 241,259

AUTHOR INDEX Hackett, C. J., 117,126 Hackman, R., 101,126 Hadley, D., 25, 55 Hadlow, W. J., 2, 7, 8,9, 10, 11, 13, 15, 18, 25, 26, 27, 33, 45, 48, 50, 51, 55

Haemmerling, U., 107,110, 111,128 Haenni, A. L., 323,361 Haig, D. A., 2,20,29,30,32,33,45,48,49,53 Haines, H., 104,125 Hakomori, S., 42, 49 Halk, E. L., 134, 135, 136, 137,138, 139, 141, 142, 143, 144, 146, 148, 149, 150, 151,161, 164,167 Halk, E. T., 134, 135, 164, 167 Hall, C. E., 58, 59, 90 Hall, I. M., 210,211 Hall, R., 322, 351,360 Hall, T. C., 227,260, 328,359,361,362 Hall, W. W., 113, 126 Halliday, J. W., 101, 130 Hamilton, L. D., 35,53 Hamilton, R. I., 332, 336, 341,360, 361 Hammerling, G. J., 137, 138, 139, 142, 144, 146, 148,167 Hammerling, U., 137, 139, 142, 144, 146, 148,167 Hammond, J., 227,258 Hampar,B., 101, 104, 109, 111, 114, 115, 125, 129, 130 Hampson, A. W., 103,109, 127, 128 Han, G. K., 277,309 Hancock, D., 104,127 Handsfield, H. H., 96, 102,128 Hanson, C. V., 31,51 Hanson, R. P., 5, 7,8, 9,26,29,34,46,53, 56 Hardy, S. F., 328,359, 361 Hare, J. D., 255,260 Hari, V., 226,262, 322, 363 Harkrider, J. R., 210,211 Harlow, E., 123,125, 126 Harmann, D. F., 146, 168 Harnish, D., 101, 114, 115, 124 Harrap, K. A., 206,211 Harris, J. O., 7,8, 10, 12, 13,53 Harris, W. J., 198,213 Harrison, B. D., 171, 173, 174, 189, 190, 194, 217, 227, 228, 231, 258, 259, 260, 328,363 Hart, M. N., 43,46,48 Hartman, D., 154,166

373

Hartmann, J. X., 248, 249,261 Hartsough, G. R., 9,51 Hashib, T., 256,259 Haskill, J. S., 106, 125 Haskins, W. T., 271,311 Haslam, E. A,, 289,309 Haspel, M. V., 110,119,122, 128 Hathaway, A. E., 170,194 Hauptmann, R., 112,128 Hay, A. J., 66,91 Hazard, E. I., 197,211 Hazendonk, A. G., 106, 122,128 Hazendonk, T. G., 102, 128 Hearst, J. E., 31, 33, 48, 51, 52, 53 Hedges, A. J., 300,310, 312 Heilman, C. J., 114, 130 Heinz, F. X., 103, 126 Helman, T., 145, 167 Henchal, E. A., 102,107,126 Henco, K., 29,55 Hengartner, H., 146,168 Henry, J. E., 197,211 Herbst, H., 101,128 Herion, P., 99, 108, 110, 124 Hermsen, T., 42,49 Hernalsteens, S. P., 88,91 Herzenberg, L. A., 144, 166 Hewish, D. R., 134,135, 167 Heyn, R. F., 236,259 Hibi, T., 217, 228, 230, 243, 259, 260, 340, 345,361, 362

Higgins, T. J. V., 322,364 Hikita, K., 21, 56 Hill, E. K., 135, 162,167 Hill, J. H., 134, 135, 136, 137, 138, 139, 142, 146, 147, 148, 149, 151, 152, 153, 161, 162,166, 167 Hills, G. J., 58, 91 Hinshaw, V. S., 96, 103, 104,110,112, 117, 120, 126, 130 Hinsman, E. J., 43,53 Hirth, L., 250, 259, 322, 328, 361, 362 Hiruki, C., 232,261, 317, 361 Hirumi, H., 72,91, 93 Hoare, M. N., 13, 54 Hoefert, L. L., 316,360 Hoeffler, W. K., 123,127 Hoffman, M., 101,114,117,128 Hoffman, T., 144,166 Hogan, R. N., 4, 9, 11, 21, 23, 51,52

374

AUTHOR INDEX

Hohmann, A. W., 163,167 Hohn, T., 243,261 Holland, J. J., 118, 126 Holloway, B. W., 304,309 Holmes, K. K., 96, 102, 128 Holmes, K. T., 103,109,127, 128 Holsters, M., 88, 93 Honda, Y., 228,230,233,254,261 Honnen, W. J., 107,110,111,128 Hontelez, J., 228, 231, 232, 234, 235, 242, 244,259, 345,361 Hooks, J., 43, 53 Hooper, C. W., 35,53 Hoorgenraad, J., 145,167 Hoorgenraad, N., 145,167 Hornabrook, R. W., 10,51 Horne, R. W., 169,193, 283,308 Horodniceanu, F., 102, 125 Hoshino, Y., 103,126 Hotchin, J., 19,51 Hotta, Y., 236, 260 Hourrigan, J., 7, 8, 13, 51 Howard, J. C., 138,166 Howard, R. J., 101,128 Howe, S. C., 138,166 Howell, S. H., 227,259 Howlett, G. J., 109, 227 Howse, G. W., 210,211 Hsieh, C., 208, 212 Hsu, C. H., 154,167 Hsu, H. T., 69,91, 134, 135, 142, 149, 151, 164,167 Huber, I., 222, 245, 246, 254, 255, 256,259, 261 Huber, R., 228, 231,232,234,235, 242, 244, 259, 340, 345,361 Huger, A. M., 197, 205,211 Hugh, R., 264,265,309 Huismans, H., 64,93 Hull, R., 226, 227, 228, 230, 232, 233, 234, 235,240,247,254,259,260,261 Humphrey, D. D., 102,127 Hunt, T., 322,361,362 Hunter, G. D., 2, 20,26, 29, 36, 37, 45, 46, 49, 50, 52, 54, 56 Hunter, J., 106, 125 Hunter, T., 322,325,362 Hunter, T. R., 322,361 Hunter, W. M., 39,49 Huq, M. I., 277, 310

Hurpin, B., 196,212 Hutcheson, A. M., 227, 231,259, 260

I Icenogle, J., 116, 127 Ie, T. S., 252,261, 316, 330,362 Igano, K., 323,331,362 Igarashi, A., 83,93 Iida, K., 279, 294, 296, 297, 312 Ikeda, K., 300,310 Ikegami, M., 241,259, 328,361 Imaizumi, s.,254,262 Irnazumi, I., 318,361 Inouye, K., 323,331,362 Iqbal, K., 22, 43, 54, 56 Ishii, S., 219,261 Israel, H. W., 322, 323,359 Isaacs, S. T., 31, 33,52, 53 Isakson, P., 122, 127 Ishii, K., 11,56 Ishii, T., 11, 52 Isselbacher, K. J., 100, 101, 129, 130 Iwanani, S., 264,312

J Jackers, A., 116,124 Jackson, A. O., 322,361 Jackson, D. C., 109,111,127 Jackson, R. J., 322,325,362 Jackson, T. A., 9, 18,51 Jacob, F., 299,309 Jakob, A., 5,52 Jakobs, M., 223, 224, 225,261 Jameson, B. A., 110,116,125 Jamieson, J. D., 169,194 Jansen, F. K., 122,124 Jarvis, N. P., 224,225,229,231,232,235,259 Jaspars, E. M. J., 325, 330, 332,361, 363 Javachia, V. G., 332,333,363 Javakhia, V. V. G., 333,359 Jayawardene, A., 299,300,301,303,304,309 Jebbett, J. N., 13,54 Jellings, A. J., 316, 360 Jenkins, G. I., 256, 259 Jensen, R. G., 218,259 Jerne, N. K., 165,167 Jesaitis, M. A,, 270, 309

AUTHOR INDEX Jockusch, H., 320,326, 327,330, 333,359, 361 John, A,, 102, 128 Johnson, E. D., 121,129 Johnson, F. L., 101,129 Johnson, K. P., 112,113, 228 Johnson, S. R., 295, 296,309 Johnstone, G. R., 134, 135, 267 Joklik, W. K., 60, 64, 81, 91, 92, 93, 205,212 Jonard, G., 323,361 Jones, C. L., 118,126 Jones, K. M., 45,54 Jones, L. A., 267,307 Jones, R. A., 248,259 Joshi, S., 323,361 Jotten, K. W., 265,310 Joyce, G., 21,43,50 Junga, U., 253, 254,259 Jutila, J. W., 197, 21 1

K Kado, C .I., 88, 92 Kaftanova, A. S., 333,359 Kageyama, M., 300,310 Kahl, G., 85, 91 Kajita, S., 228, 254,261 Kakehi, T., 241,259 Kalica, A. R., 170, 194 Kalmakoff, J., 58,59, 61,64,91, 92, 210,211 Kanazawa, I., 12,52 Kao, K. N., 220,259 Kaper, J. M. K., 150,168 Kapikian, A. Z., 170, 293, 194 Kapitsa, 0.S., 319, 326, 361 Kaplan, I. B., 318, 333, 334, 335, 336, 341, 361,363 Kapoor, A. K., 121,127 Kapular, A. M., 64,91 Karn, J., 323,324,325,332,361 Karstad, L., 9 , 5 1 Kasatiya, S. S., 286, 287, 288, 289, 291, 293, 307 Kascsak, R., 43,54 Kasper, K. C., 2, 4, 20, 21, 23, 34, 38, 48, 52, 55 Kassanis, B., 216, 230, 235, 244,259, 314, 333,336, 337, 344,361, 362 Kasuga, S.,197,212 Katagiri, K., 196, 197, 212

375

Kates, J., 204, 212 Katterman, F., 79, 93 Katz, D., 170, 171, 181, 182, 183, 186, 189, 191,192, 194 Katzman, R., 13, 52 Kawanami, S., 11,54 Kawata, T., 266,284, 291,310 Kearney, J . F., 144, 167 Kedinger, C., 106, 125 Keeler, R. F., 271,310 Keene, T. D., 60,92 Keith, H. D., 35, 50 Kelen, A. E., 170, 194 Kellenberger, E., 272, 312 Keller, W. A,, 220,259 Kelly, J . F., 146, 168 Kelly, S. M., 85,91 Kendal, A. P., 100, 104, 127, 128 Kennedy, R. C., 2,7,8,9,11,13,18,45,50,51 Kennett, R. H., 96, 127 Kerlan, C., 171, 175, 190,194 Kew, 0. M., 102,127 Khan, S., 265, 267, 276, 290,307 Khare, G .P., 255,262 Khie, T. S.,277,309 Kida, H., 103, 107, 112, 126, 127 Kiho, Y., 332,362 Kikkawa, H., 224,225,226,229,230,232, 235,260 Kikkawa, Y., 42,50 Kikuchi, Y., 250,260 Killington, R. A., 101, 114, 115, 224 Kim, I. C., 42, 52 Kim, J., 7, 8, 11,53 Kim, K. S., 316,344,360, 362 Kimberlin, R. H., 3, 5, 7, 8, 9, 18, 20, 21, 25,26,21,29,36,37,45,46,49,52,53,54 Kimmins, W. C., 248,262 Kimura, H., 42,52 Kimura, I., 59, 73, 91, 92 Kimura-Kuroda, J., 107, 110, 227 King, J . M., 232, 236, 237,239,259, 262 Kingsbury, D. T., 4, 11, 12, 21, 23, 25, 33, 48, 51, 52, 55 Kinney, R. M., 102, 106, 117,128, 229 Kintner, C., 113,127 Kirschbaum, W. R., 10,52 Kitagawa, Y., 11, 52 Kitajima, E. W., 316, 362 Kjeldsberg, E., 171, 189, 193, 194

376

AUTHOR INDEX

Klassen, T., 43, 48 Klatzo, I., 10,52 Klein, J., 21, 52 Kleinschmidt, A. K., 59,91 Klenk, H. D., 104,127 Klingsporn, A., 7,8, 13, 51 Klug, A,, 115, 127, 157, 160, 166, 167 Knapp, J. S., 96,102, 128 Knapp, W., 103,126 Knight, C. A., 63, 64, 66, 90, 131, 168 Knobler, R. L., 106, 125 Knowland, J., 322,361, 362 Koda, Y., 85, 86, 91 Koenig, I., 243, 261 Koenig, R., 144,167, 171,174,189,192,194 Koga, M., 11, 12, 25,56 Koga, T., 266,283,284,291,310 Kohler, G., 95, 127, 132, 146, 167, 168 Kohn, A., 170,171,182,183,191,192,194 Kohn, B., 85,91 Koike, M., 228, 260, 340, 345,362 Kolacz, K., 322,363 Konarska, M., 42,50 Kono, M., 304,311 Koos, R., 83,93 Koprowski, H., 97,102,119, 123, 125, 126, 127, 130

Kormendy, A., 299,309 Koschel, K., 31, 49, 120, 124 Kotecha, S., 42,49 Koto, A., 11,52 Kouri, Y., 101, 124 Kozlov, Y. V., 333,361 Kozlowski, P. B., 9,52 Kraehenbuyl, J. P., 169, 180,194 Kraiselburd, E., 101, 124 Krasovich, M., 43, 53 Krauth, W., 42,52 Krens, F. A., 88, 91 Kreth, H. W., 112, 129 Krieg, A., 197, 205,211 Krijt, Y., 316, 361 Kristensson, K., 112, 113,127 Krol, P. M., 300,301,303,304,310 Krolick, K. A., 122, 127 Kropinski, A. M. B., 267,310 Kruger, K., 88,93 Krystal, G., 81,93 Kubler, A.-M., 144,166 Kubo, S., 217, 228, 231, 232, 253, 254,260, 262, 318,343,361, 362

Kukla, B., 323,361 Kunz, C., 103,126 Kuo, C. C., 96,102,128 Kuroiwa, Y., 11, 12, 25,54, 56 Kurstak, C., 169, 194 Kurstak, E., 169, 194, 197, 198,200,207,212 Kushida, T., 197,212 Kuzuhara, S., 12,52

L Lafon, M., 119, 125 Lahiri, M. N., 265, 267, 276, 290,307, 311 Lamar, C. H., 43,53 Lamb, R. A,, 113,126 Lampert, P., 43,53 Lampert, P. W., 113,129 Lancaster, W. D., 5,29, 46,53 Lane, D. P., 108,111,123,125, 127 Lane, L. C., 150, 168 Lang, D., 301,302,310 Langridge, W. H. R., 202, 204, 205, 206, 209,212

Laporte, J., 64,91 Larkin, P. J., 222,260 Larsen, G. R., 110,116,125 Latarjet, R., 10, 12, 29, 30, 32, 33, 45, 51, 53 Lauritis, J. A., 316, 362 Lausch, R. N., 121,129 Laver, W. G., 103, 104, 110, 111, 112, 117, 120,125, 127, 130, 187,194

Lavery, C., 101,124 Lavi, S., 69,91 Lawson, R. H., 134,135, 142, 149, 151,164, 167

Lax, A. J., 36,53 Lazar, G .B., 243,261 Leach, R. H., 43,53 Lederberg, E. M., 272,310 Lederberg, J., 272, 310 Lee, C. L., 86,90 Lee, F. K., 186,191,194 Lee, J. V., 266,286, 287, 288,289, 291, 293, 294,307,310

Lee, L. F., 101, 113,127 Leemans, L., 88,91 Leestma, J., 113, 127 Lefrancois, L., 109, 110, 120, 127 LeMinor, A., 292,311 Lemmers, M., 88,93 Lennette, E. H., 31,51

377

AUTHOR INDEX Leonard, D. A., 323,331,332,355,362 Leppard, K., 123,125 Leppla, S. H., 59, 92 Lerner, K. G., 101,129 Le Roy, C., 328,362 Lesemann, D. E., 171,173,174,178, 179, 181,187,189,192,194

Lung, M. C. Y., 349,360 Luoni, G., 281, 308 Lurquin, P. F., 236,260 Lutton, G. G., 210,212 Lutz, H., 118, 127 Lwoff, A,, 299,309 Lyles, D. S., 109, 110, 120, 127

Lesnaw, J. A., 64,66,92 Lesney, M. S., 226,229, 230, 231, 232, 233, 234,235,260 Lesser, R. L., 11,53 Letchworth, G. J., 103, 106, 122, 124, 127 Leung, E., 317,348,364 Levine, A. J., 113, 118, 129 Levinsohn, S., 204,212 Lew, S. T., 295,308 Lewandowski, L. J., 58, 59, 61, 62, 63, 64, 91, 92 Lewin, P., 45,53 Lewis, A. J., 110, 116, 125 Li, J. K.-K., 60,92 Lieberman, H. M., 101,129 Liesegang, B., 144, 167 Lindberg, A. A., 271,310 Lipa, J. J., 197, 212 Lister, R. M., 134, 147, 149, 151, 152, 166, 227,258, 289,309 Liston, J., 267,307 Littlefield, J. W., 139, 167 Liu, H. Y., 73,92 Liu, S.-T., 88, 92 Liu, X., 101, 113, 127 Loebenstein, G., 176,190, 193, 220,221, 242, 246, 252, 260, 262, 314, 320, 344, 345,360, 362 Loeffler, S., 102, 104, 107, 109, 110, 113, 118,124, 125,129 Loesch-Fries, S. L., 328,361 Lomniczi, B., 66, 91 Lomonosoff, G. P., 323,325,332,361 Long, D., 109, 111, 118, 125 Longa, G., 45,46,56 Lopes, D., 96,97, 126 Lossinsky, A. S., 21,54, 56 Lostrom, M. E., 138, 149, 167

Loughlin, S. A. R., 362 Lubeck, M., 109,127 Luderitz, O., 271, 312 Luftig, R. B., 115, 129 Luisoni, E., 170, 171, 172, 173. 175, 176, 187,189, 190,191,194

M McAuliffe, V., 103, 126 McCarthy, W. J., 200, 204, 205,212 McClintock, P. R., 110,119, 122,128 McCown, B., 221,262 McCown, J. M., 102, 107,120,124, 126 McCrae, M. A., 60,92 McCrea, J. F., 205,212 McCullough, K. C., 117, 127, 137, 139, 144, 167

McDonald, T. O., 301, 302,310 McDougall, J., 101, 126 M’Fadyean, J., 45,54 McFarland, H. F., 118,120,124,129 McFarlin, D. E., 115, 118, 120, 124, 129 McGeachie, J., 304,310 M’Gowan, J. P., 45,54 Mach, J., 304,312 Machold, O., 42,56 McIntyre, J. L., 255,260 MacKay, A., 102, 106, 107,128, 129 McKinley, M. P., 3, 4, 5, 16, 17, 26, 27, 29, 31, 32, 33, 34, 35, 36, 38, 39, 41, 42, 45, 48, 49, 50, 53, 55 McLaren, A. D., 30,53 McLaughlin, B., 193,193 McLean, C. S., 121,127 McLeod, D. A., 170,194 MacLeod, R., 58, 59, 60, 61, 62, 63, 64, 66, 69, 78, 92 McMillan, H. M., 146, 168 McMillan, P., 115, 129 Madalinski, K., 194 Maekawa, E., 339,362 Maekawa, K., 254,260 Magrath, D. I., 102, 126, 228 Maiti, M., 268, 269, 270, 271, 272, 273, 274, 275,277, 278, 279, 280, 282, 283, 284, 288,289,308,310,311,312 Maizel, J. V., 289, 310 Majdic, O.,103, 126 Makins, J. F., 239, 240, 241,260

378

AUTHOR INDEX

Malmgren, B., 271,311 Malone, R.H., 265,308 Malone, T.G., 5,26, 29, 34, 46, 53 Malyshenko, S. I., 334, 335, 336, 341, 363 Mandak, V., 243,261 Mandel, B., 159,167 Mandema, E., 42,55 Mannen, K., 102,124 Manning, E. J., 36, 53 Mannweiler, K., 96, 124 Manuelidis, E. E., 7, 8, 11, 19, 25,53 Manuelidis, L., 7,8, 11,19, 25,53 Maramorosch, K., 72, 73,91, 92, 93 Marchuk, L. M., 293, 310 Marcinak, R. A.,101, 130 Marker, S. C., 101,128 Markham, R.,58,59,90,91 Marquez, E.D., 135, 142, 152, 167 Marsh, R.F., 3,5, 7,8, 9, 25,26, 29, 34,46, 49, 53, 56

Martin, C., 361 Martin, J. D., 22,49 Martin, M. M., 360 Martin, R.R.,135, 137, 152, 167 Martin, S. A.,64,92 Martin, S. R.,117, 129 Martinez, H.M.,3, 4, 16,17, 25, 27, 34, 55 Masiarz, F. R.,3,5, 16, 17, 26, 27,29, 31, 32, 33, 34, 35, 36, 42, 45, 48,53, 55

Massey, R.J., 107, 109, 110, 112, 126, 127 Massoulie, J., 42,56 Masters, C. L., 7, 8, 10, 11, 12, 13, 14, 22, 51, 53, 54, 55

Mathot, S., 99, 108, 110, 124 Matsuhisa, T.,81, 92 Matsui, C., 224, 225, 226, 228, 229, 230, 232,233,235,240,241,254,259,260,261

Matsuyama, H., 11,52 Matthews, J. H., 121,128 Matthews, R.E. F., 195,212, 244,260, 266, 290,291,310, 314,315,318, 319,336, 347,349,362 Matthews, W. B., 10,43, 51, 53 Maule, A. J., 227,232,237,239,260,340,362 Maxam, A. M., 79,83,84,92 May, J. T.,328,362 Mayo, J. A., 267,307 Mayo, M. A.,228,229, 231,233, 254,259, 260, 328,363 Mayr-Harting, A., 300,310

Mayshenko, S. I., 318, 333,334,335,363 Mbiguino, A.,286, 287, 288, 289, 291,293, 307

Medappa, K. C., 9,53 Meegan, J. M., 103,128 Meikle, V., 7, 8,50 Meikle, V. M., 4, 22,49, 50 Mekalanos, J. J., 298,310 Melchers, G., 236, 237, 238, 239, 241,261 Melnick, J. L., 31, 49 Mercer, W. E., 106, 128 Mertes, G., 219, 224,225,229, 231,233, 236, 237, 238, 239, 242, 243, 245, 246, 248,249,252,253,256,260 Merz, D. C., 102, 129 Merz, G. S., 22,56 Merz, P. A.,22,43,54, 56 Meshi, T., 323, 324, 331, 332,362, 363, 364 Meyer, Y., 220,260 Meyers, J. D., 101,126 Meynadier, C., 196,212 Meynadier, G., 196,197,212 Miggiano, V., 139, 168 Mihalyi, E., 29, 42, 54 Miles, E. W., 38, 54 Mille, B., 171, 175, 190, 194 Miller, M. F., 171, 177, 189, 191, 194 Miller, R.A., 220,259 Miller, W.A.,328,359, 362 Millson, G. C., 2, 7, 8, 20, 22, 24, 26, 29, 36, 37, 46,52, 53, 54, 56 Milne, R. G., 170, 171, 172, 173, 174, 175, 176,178,179,187,189,190,191,193,194 Milner, K. C., 271,311 Milner, R.J., 210,212 Milstein, C., 95, 127, 132,137, 138, 143, 145,146,147,166, 167 Milton, S. C. F., 157, 167 Mingioli, G. S., 118, 124 Minor, P. D., 102,106,107,112,116,126, 128, 129 Minson, A. C., 104,127 Miroshnichenko, N. A., 314, 347,348,349, 350,351, 352, 353,357,358,360, 362 Misra, V., 115,128 Mitchell, D. J., 79,93 Mitchell, D. M., 121, 125 Mitra, S., 289, 305,310 Mitsuhashi, J., 72, 93 Miura, K., 60,93

379

AUTHOR INDEX Miura, N., 107, 228 Mock, N. I., 3, 27, 29, 32, 35,45, 55 Moerman, M., 252,259, 316,361 Molendijk, L., 88,91 Monath, T. P., 102, 121, 228, 129 Monsarrat, P., 197,212 Monsur, K. A., 273,277,293,310, 311 Montagnier, L., 33,53 Montgomery, I., 322,363 Moore, G. E., 137,167 Moore, S., 38, 56 Moretz, R. C., 9,21,52, 54 Morgan, M., 64,91 Mori, R., 12, 56 Morita, R. Y., 267, 307 Morozov, S. Y., 318,333, 334, 335,363 Morris, J. A. Q.,43,49 Morris, T. J., 33, 56 Morris, Krsinich, B. A. M., 226, 227, 230, 232, 233, 234,235, 247, 254,260

Morrison, J., 265,310 Morrow, C. H., 11,51 Mortensson-Egnund, K., 171, 180, 189, 193, 194

Mortez, R. C., 21, 56 Moseley, S. L., 298,310 Moss, R., 204,212 Motomura, S., 11,54 Motoyoshi, F., 219, 226, 227, 228,230, 231, 232, 234, 236,237, 238,247, 248, 249, 254,258, 260, 262, 321,330, 331, 339, 340,346, 348,362 Mouches, C., 242,243, 254, 255,261 Moulton, J. E., 20,54 Mountford, C. E., 103, 109,127, 128 Mountford, R. C., 112, 128 Mozes, R., 253,260 Mridha, K., 277,311 Muel, B., 45, 53 Mueller-Lantzsch, N., 101, 128 Muggeridge, M. I., 121, 125 Muhlbach, H. P., 216, 241, 242, 250, 256, 260, 261, 262 Mukerjee, S., 264, 265, 266, 267, 276, 277, 278, 279, 283, 290, 292, 293, 294, 296, 297,305,307,308, 309,311, 312 Murachi, T., 42,52 Murakishi, H. H., 224, 225, 226, 229, 230, 231, 232, 233, 234, 235, 248, 249, 250, 252, 253, 257,258, 259, 260, 261, 262

Murant, A. F., 152, 167, 228,259 Murashige, T., 224,262 Murphy, J. R., 298,310 Murphy, T. M., 330,362 Murray, J. M., 111, 127 Muthukrishnan, S., 60, 64,92, 92 Myoraku, C. K., 101,126 N

Nagata, N., 224, 225, 239, 240,241,259 Nagata, T., 219,224,225,226, 229,230, 232, 235, 240,260, 261

Nahmias, A. J., 186, 191,194 Nahmias, D. G. E., 186, 191,194 Naito, T., 304, 311 Nakanishi, T., 12,52 Nakayama, Y., 279, 294, 296,297,312 Narang, H. K., 43,45,50,54, 188,194 Narayanaswami, A., 264,308 Nartississov, N. V., 20, 50 Nash, A. A., 121, 127 Nassuth, A., 228,254, 255,261, 325,327, 328,362,363

Natali, A., 103, 120, 128 Nathanson, N., 103,105, 107,126 Naughton, M., 208, 209, 221 Naylor, A. W., 87,91 Nazerian, K., 101, 113, 127 Neelman, L., 322,364 Negrutiu, J., 223, 224, 225,261 Neiman, P. E., 101, 229 Nelson, B. P., 197,211 Nelson, D., 106, 128 Nelville, A. M., 121, 129 Neorgy, K. N., 295,311 Neser, C. F., 204,212 Nester, E. W., 88, 93 Nestorowicz, A., 109, 127 Neville, D. M., 122, 130 Newman, F. S., 276, 278, 280, 282, 286, 287,288,289,291,292,293,307,309,311

Newsome, P. M., 20,49 Nezri, C., 19, 52 Nibert, M. L., 78, 79, 85, 91 Nichols, C.,341,361 Nickell, L. G., 87, 88, 91, 92 Nicolaieff, A., 171, 174, 175, 176, 179, 182, 183, 186, 189, 191, 194

Nicolle, P., 284, 292, 299,309, 311. 312

380

AUTHOR INDEX

Nigara, H., 21,56 Niman, H. L., 110,128 Nishiguchi, M., 321, 330, 331, 332, 340, 346,348,362 Nobechi, K., 265,311 Nonoyama, M., 81,92 Noonan, A., 170,193 Norrby, E., 112,113, 127, 128, 187,194 Norrild, B., 97, 104, 114, 115, 124, 128 Norris, D. O., 337,362 Notkins, A. L., 110, 119, 122, 128 Novikov, V. K., 333,338,359 Nowinski, R. C., 96,101,102, 104,108, 109,126, 128, 129, 138,149,167 Nozaki, Y., 34,54 Nozth, A. C. T., 34,54 Nuss, D. L., 62, 63, 64, 66, 67, 69, 71, 72, 75, 77, 78, 79, 81, 83, 91, 92 Nutter, R., 88,93 Nytrai, A., 236, 238,261 0

Oakes, J. E., 121,129 Obdrzalek, V., 304,312 Obert, G., 171, 176,179, 189, 194 O’Brien, F. H., 11,53 O’Donnell, P. V., 107, 110, 111,128 Odermatt, E., 42,54 Ogawa, M., 256,261 Ogg, J. E., 298,311 Ohba, M., 197,212 Ohno, T., 323,324,331,332,362,363,364 Ohta, M., 11, 12, 25, 54, 56 Okada, J., 332,362 Okada, K., 240,261 Okada, Y., 107, 128, 323, 324,331,332,362, 363,364 Okayasu, H., 11,52 Okuno, T., 226,227,228,229,230,231, 232, 234, 235, 236, 237, 238, 254, 259, 260,261, 339,340,361,362 Olah, T., 220, 236, 238,261 Old, L. J., 106, 128 Oldstone, M. B. A., 113,120,126, 128 Ooshika, I., 323, 331,362, 364 Orenski, S. W., 72,93 Orvell, C., 107, 112, 113, 127, 128 Oshikata, H., 298,312

Oshima, N., 227,231,232, 236,237,238, 248, 249,260,321,330, 331,332, 339, 340,346, 348,362 Osterhaus, A. D., 102,106, 122,128 O’Sullivan, W. J., 103, 109, 127, 128 Ota, M., 102, I29 Otohuji, T., 266, 277,293,312 Otsuki, Y., 67, 93, 215, 216, 217, 219, 221, 226, 227, 228, 230, 231, 233, 244, 245, 254,261,262 Ott, U., 42, 54 Outram, G. W., 4,5, 22,50, 54 Oxford, J. S., 103, 120,128 Oxman, M. N., 101,129

P Padden, F. J., Jr., 35,50 Pagel, P., 305,309 Palfi, G., 236, 238,261 Palmer, A. C., 20,54 Palmer, E., 103,126 PBlsson, P. A., 13,54 Panitch, H., 20, 52 Paolett, E., 204,212 Paolucci, F., 122, 124 Papahadjopoulos, D., 240,246,259,262 Parkas, G. L., 236,238,261 Parker, C., 295,299,302,31 I Parkinson, D., 137, 139, 144,167 Parrish, C. R., 103, 128 Parry, H. B., 2,8,9,21,43,45,49,54 Partanen, C. R., 243,256,261 Pasricha, C. L., 265,276, 290,311 Paszowski, J., 243,261 Patel, G., 104, 129 Patlak, C. S., 19, 56 Patnaik, G., 219,261 Pattison, I. H., 2, 7, 8, 13, 20, 22, 24, 45, 54 Pau, B., 122,124 Paul, H. L., 171,178,179,181,189,194 Payment, P., 193, 193 Payne, C. C., 206,21 I Pearce, N., 256,258 Pearson, C. A., 219,220,221,262 Pearson, G. R., 118, I29 Pearson, T., 167 Pedersen, N. C., 118, I27 Peebles, C., 250,258

381

AUTHOR INDEX Peiris, J. S., 121, 128 Pelcher, L. E., 248, 249, 261 Pelham, H. R. B., 322,351,362 Pennington, J. E., 144, 166 Pensaert, M. B., 170, 194 Pereira, L., 97, 101, 104, 109, 111, 114, 115, 117,118, 121,124, 225, 128 Perez, N., 289,307 Peries, J., 97, 129 Perlmann, P., 159, 166, 170,193 Perry, K. L., 88,92 Peters, C. J., 103, 128 Peters, D. L., 330,362 Peterson, A. J., 62,63, 64,66, 67,69, 71, 72, 75, 81, 83, 92 Peterson, E., 101, 104,128 Peterson, L. J., 316, 346, 347, 363 Peterson, P. K., 101, 128 Petti, M. A,, 328,364 Pettigren, K. D., 19, 56 Pfau, C. J., 205,212 Phelan, J., 121, 127 Phillips, D. J., 100, 104, 227, 128 Phillips, J. H., 32, 54 Pim, D. C., 123,126 Pinter, A., 107, 110, 111,128 Pleij, C. W., 323,361 Pogo, B. G. T., 203,204,212 Pojnar, E., 215,258 Pollard, E., 280,311 Pollitzer, R., 265, 290,311 Ponce de Leon, M., 118,125, 187, 194 Porter, D. D., 20,54 Porter, H. G., 20,54 Porterfield, J. S., 121,128 Portetelle, D., 99, 108, 110, 111, 124 Portner, A., 96, 107, 110,128 Postle, K., 88, 93 Potrykus, I., 243,261 Poudayl, L., 298,312 Powell, C. A., 135, 142, 152, 267 Powell, H., 106, 225 Powell, L. W., 101, 130 Power, J. B., 221,261 Prabhakar, B. S., 110, 119,122, 128 Prakash, J., 256,259 Pras, M., 42,55 Prat, M., 118, 126 Precious, B., 104, 129

Premecz, G., 220, 236, 238,261 Preobrazhenshy, A., 349,362 Prescott, L. M., 301,305,308 Prives, C., 106, 129 Prusiner, S. B., 2, 3, 4, 5,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 35, 38, 39,41,42,43,45,48,49,50,52,52,53,55 Pshennikova, E. S., 333, 334,335, 336, 341, 361, 363 Purcell, R. H., 170, 193 Purdy, H. A., 131, 267

Q Qualtiere, L. F., 118, 129 QuBtier, F., 88, 91 Quiniou, J., 292, 299,311 Quiot, J. M., 209,222

R Rabin, H., 114,130 Race, R. E., 11, 13, 15, 18, 26, 27,33,51,55 Radbruch, A., 144,267 Ragetli, H. W. J., 317, 348,364 Raine, C. S., 21, 43,50, 55 Raison, R. L., 103, 109, 127, 128 Rajewski, K., 144, 167 Ramm, K., 29,55 Rammohan, K. W., 120,129 Randall, C. C., 205,212 Rao, D. V., 232,262, 317,361 Rapoport, H., 31,52 Rappaport, I., 314, 319,362 Rappaport, L., 85,92 Raveed, D., 45, 46,56 Rawls, W. E., 101, 114, 115, 121, 122,124 Raychaudhuri, C., 270,271,308,311 Raymer, W. B., 29,50 Reading, C. L., 163,167 Reaume, M., 280,311 Rector, J. T., 121,129 Reddi, K. K., 237,259 Reddy, D. V. R., 57, 58, 59, 60, 61, 62,63, 64, 66, 69, 73, 74, 75, 76, 78, 79,80, 81, 83, 85, 91, 92 Redmond, W. B., 306,309 Reeve, P., 112, 128

382

AUTHOR INDEX

Reeves, P. R., 299,305,311 Reimer, C. B., 100, 104,127, 128 Reinboldt, J., 154,166 Reivich, M., 19, 56 Renaudin, J., 227, 242, 243, 254, 255,261 Rennie, J. C., 7, 8, 56 Reynolds, D. J., 170,194 Reynolds, J. A., 34,54, 55 Rezelman, G., 217, 228, 230, 243, 252, 254, 255,259, 261, 316, 328, 330, 340,361, 362,363 Rhodes, D. P., 60,63, 64, 66,92 Ribi, E., 271,311 Rich, A., 34, 54, 59, 93 Richards, K. E., 322,323,361 Richardson, S. H., 268, 273, 277, 279, 282, 283, 295,296, 297, 299,302,311, 312 Richman, D. D., 101,129 Richow, W. F., 134, 142, 149, 151,167 Riesner, D., 29, 55 Riesterer, C., 227,261 Rifkind, R. A., 169,194 Riggs, J. L., 31, 51 Riguad, J., 322, 363 Rimon, A,, 42,55 Ritchie, A. E., 271, 310 Rizvi, S. H., 273,311 Rizza, J. J., 88, 93 Robards, A. W., 315,362 Robert, P., 196, 197,212 Roberts, D. A,, 320,363 Roberts, D. W., 196, 200, 202, 203, 204, 205,206,212 Roberts, I. M., 170, 171, 194, 229, 231,260 Roberts, M. I., 171,173, 174, 189, 190,194 Roberts, R. J., 196, 197,211 Roberts, W. D., 196, 198, 200, 205, 211 Robertson, H. D., 250,251,258,262 Robins, R. K., 255,262 Robinson, D. J., 231,260, 328,363 Rodriquez, M., 113,129 Roehrig, J. T., 106, 116, 117, 121, 128, 129 Rohwer, R. G., 45,55 Roizman, B., 97, 104, 114, 115, 124, 128 Rollin, P., 119, 125 Rollo, F., 240,261 Romaine, C. P., 322, 323,328,363, 364 Rombaut, B., 116,124, 129 Romig, W. R., 295,296,299,302,309,311 Ronald, W. P., 135, 148, 154, 168

Roos, R., 10,55 Roosien, J., 327, 328,363 Rorvik, M., 9,49 Rose, J. A., 29, 33,55 Rose, R. J., 219,261 Rosenblum, E. N., 204,212 Roseto, A,, 97,129 Ross, A. F., 336, 337,363 Roth, M. R., 219,220, 221,262 Rott, R., 104, 127 Rottier, P. J. M., 254, 255, 261 Rouhandeh, H., 205,213 Rouweler, H. C., 345,364 Roy, C., 277,311 Royston, I., 113, 114, 129 Rubin, B. A,, 187,194 Rubinstein, A. S., 171, 177, 189,191, 194 Rubenstein, D., 170, 193 Rudbach, J. A., 271,311 Rudra, B. C., 266, 292,311 Rueckert, R., 116, 127 Ruinen, L., 42,55 Rupert, E. A., 248,259 Rupley, J. A,, 42, 55 Russell, M. L., 339,340,359 Russell, P. K., 121, 124 Russell, W. C., 104,129 Russo, M., 226, 227, 230, 232, 233, 234, 235, 247,254,260

Rutter, G., 96, 124 Ruzicska, P., 220, 261 S

Sacharovskaya, G. A., 333,359 Sacharovskaya, G. N., 332,333,363 Sachs, M., 85,92 Saiki, R. K., 88, 91, 93 Sakai, F., 67, 92, 232, 236, 237, 239, 256, 259,261, 339,363

Sakazaki, R., 264,311,312 Sakurada, O., 19,56 Salamon, G., 19, 51 Salas, E., 289,307 Salazar, A., 12,55 Samuel, G., 320,363 Sander, E., 117,125, 132,133,135,136, 137, 138, 139, 141, 142, 143,144,146, 147, 148, 149, 152, 156, 157, 158, 159,

AUTHOR INDEX 160, 163, 165, 166, 224, 225, 229, 231, 233,235,242,245,246,254,256,260,261 Sanders, C. J., 196, 211 Sanger, H. L., 241,242, 250,256,260,261, 262 Sanyal, S. C., 292,312 Sanyal, S. N., 295,311 Sarachy, A. N., 327,328,363 Sarkar, S., 67, 92, 216, 218, 234, 236, 237, 238, 239, 241, 243,261, 326,333,363 Sarnow, P., 113,118,129 Sasaki, H., 12, 52 Satake, M., 115,129 Sato, Y., 11, 12, 21, 25,56 Sawhney, R. K., 238,261 Scalla, R., 200, 203, 212, 322, 323, 359, 363, 364 Schafer, W., 133,168 Schaffer, F. L., 29, 33,56, 59,91 Schardl, C. L., 88,92 Schaskolskaya, N. D., 333,359 Schechter, N. M., 34,54 Scheible, P. O., 60,92 Scheidegger, D., 137, 138, 139, 146, 166 Scheinker, I., 5, 50 Schell, J., 88, 93 Scheller, A., 106, 129 Scherch, A. R., 81,92 Scherrer, R., 64, 91, 97, 129 Schild, G. C., 102, 103, 106, 107, 112, 116, 120,126, 128, 129 Schild, G. S., 112, 126 Schilde-Rentschler, L., 219,261 Schilperoort, P. A., 236,259 Schlegel, D. E., 248,259, 349,360 Schlesinger, J. J., 102, 121, 128, 129 Schlesinger, M., 270, 312 Schlesinger, R. W., 83,93 Schlumberger, H. D., 160, 166 Schmaljohn, A. L., 121,129 Schmidt, N. J., 101, 102, 126, I29 Schmidt, 0. W., 101, 104,128 Schneider, I. R., 315, 317, 318, 363 Schneider, L. G., 102, I29 Schnolzer, M., 250, 262 Schochetman, G., 107, 109, 110, 127 Scholten, J. H., 42, 55 Scholtz, C. L., 46, 51 Schramm, G., 132, I68 Schreier, M., 146, 168

383

Schultz, A. M., 112,126 Schuster, H., 32, 56 Schwenk, F. W., 219,220,221,262 Schwerdt, C. E., 29,33,56 Scolnick, E. M., 113, 126 SBchaud, J., 272,312 Segiun, M. C., 102, 126 Sehgal, 0. P., 154, 167, 168, 319, 326,357, 359,363 Seitelberger, F., 12, 22,56 Sela, I., 247, 252, 253, 262, 314, 344,363 Semal, J., 328,363 Semancik, J. S., 5,9,26,29,33,34,46,53,56 Semler, B. L., 118, 126 Sen, R., 264,312 Senne, D. A., 96,126 Server, A. C., 102,129 Seyfried, P. L., 299, 300, 301, 303,309 Shabtai, S., 220, 221, 242, 246,260, 262 Shafritz, D. A., 101, 129 Shah, K. V., 104,124 Shahar, A., 170,171,182,183,191,192,194 Shalla, T. A,, 316, 346, 347, 363 Shannon, R., 300,304,305,307,312 Sharma, J. M., 101, 113,127 Sharp, I., 104, 229 Sharp, P. A., 117, I25 Shaskolskaya, N. D., 332, 333,363 Shatkin, A. J., 42, 50, 59, 60, 64, 69, 91, 92 Sheldon, R., 204,212 Shen, C. J., 31, 52 Shenk, T. E., 83,93 Shepard, J. F., 217, 218,221,262 Shepherd, R. J., 152, 165 Shesberadaran, H., 112, 113,128 Shih, J. W., 97, 102, 125 Shikata, E., 72, 92, 93 Shimada, T., 264,312 Shimamura, K., 12,52 Shimodori, S., 277,279, 293, 294,295, 296, 297, 299,302,312 Shimotohno, K., 60, 93 Shinohara, M., 19, 56 Shinshi, H., 243, 261 Shirahama, T., 42, 45, 49, 52 Shoemaker, H., 100, 101, 130 Shope, R. E., 103,105,107,126, 128 Showalter, S. D., 101, 104, 115, 129 Shrestha, M. B., 298,311 Shugar, D., 30, 53

384

AUTHOR INDEX

Shukla, D. D., 134, 135, 167, 171,177, 178, 190,194 Shukla, P., 317,361 Siakotos, A. N., 26,45, 46,56 Siddell, S. G., 113, 129 Siddiqui, K. A. I., 298,312 Sidwell, R. W., 255,262 Siegel, A., 226,262, 319, 322, 326, 357, 361, 363 Sigurdsson, B., 6,56 Sijens, R. J., 116, 124 Sikora, E., 19,51 Sikora, K., 121,129 Sil, J., 292, 305,308, 312 Siler, D. J., 339, 340,359 Silva, J., 106, 125 Silver, G. D., 115, 118, 124 Simminovitch, L., 299,309 Simmons, R. L., 101,128 Simon, L. N., 255,262 Singh, N., 103,126 Sinha, R. C., 72,93 Sinkovics, J. G., 96, 129 Sipe, J. D., 64, 92 Siraganian, R. P., 138, 139,142,166, 168 Skehel, J. J., 64, 66,91, 93, 103, 111, 117, 125, 129, 130 Skinner, M., 45,49 Slovin, S. F., 113, 114, 129 Smarda, J., 304,312 Smeltzer, D. A., 25, 52 Smit, C. H., 325,363 Smitamana, P., 326,363 Smith, C. L., 119,125 Smith, G. E., 202,212 Smith, K., 20,54 Smith, M., 221,262 Smith, R. E., 64,93 Smith, W., 45,56 Snyder, R. M., 110,130 Snyderman, R., 106,125 Sokoloff, L., 19, 56 Somers, J. M., 79,93 Somerville, R. A., 43, 46, 54, 56 Sorenson, G. D., 42,56 Spandidos, D. A., 81,93 Spencer, D. F., 248,262 Spendlove, R. S., 59,91 Spiegel, S., 314, 320, 344, 345,362 Spiesmacher, E., 250,262

Spirin, A., 349,361, 362 Spitz, M., 102, 106, 107, 112, 116, 126, 128, 129 Spriggs, D. R., 107, 119, 120,124, 126, 129 Stace-Smith, R., 135, 137,152,167 Staden, R., 157, 166 Staehelin, T., 97, 129, 139, 146, 168 Stkibli, C., 139, 168 Stamford, S., 107,112,121, 125 Stamrn, W. E., 96,102,128 Stamp, J. T., 45,56 Standt, L., 96, 126 Stanley, W. M., 131,168, 169, 193 Stanway, G., 112,128 Stark, G. R., 38,56 Stein, W. H., 38, 56 Stent, G. S., 267,312 Stephenson, J. R., 102, 104, 107,109, 112, 113,118,129 Stites, D. P., 2, 4, 20, 21, 23,48, 52 Stocker, J., 146,168 Stockman, S., 2, 8,56 Stoffer, R. F., 336,363 Stollar, V., 83, 91, 93 Stoltz, D. B., 197, 200, 207, 208,212 Stone, M. R., 108,109,129, 138, 149, 167 Stong, L., 96, 102, 128 Storb, R., 101,129 Stott, E. J., 170, 193, 194 Straussler, E., 5,50 Straussman, Y.,170, 171, 181, 182, 183, 191,192,194 Street, H. E., 224,262 Streeter, D. G., 255,262 Streissle, G., 58, 59,88,93 Stussi-Garaud, C., 328,362 Sudgen, B., 113,127 Suetsugu, M., 11,56 Sugimura, Y.,227,262 Sullivan, C. A., 113,118,129 Sulzinski, M. A., 315, 316, 341,345, 363 Summers, M. D., 197,200,202,207,208,212 Sur, P., 270, 271,272, 277,310, 312 Suss, K. H., 42, 56 Sutter, G. R., 196,212 Suzuki, M., 236, 237,238,262 Sward, R. J., 134,135,167 Symons, R. H., 228,259, 328,362 Symons, R. T., 340,361 Szoka, F., 240,262

385

AUTHOR INDEX Sztachelska-Budkowska, A., 194 Szybalski, W., 205,212

T Taber, R., 240,262 Tahara, S. M., 108,125 Takacs, B., 146,168 Takamatsu, N., 363 Takamatsu, T., 332, 362 Takanami, N., 323,324,332,362 Takanami, Y., 228,232,253,254,260,262, 343,362

Takebe, I., 67, 92, 93, 215, 216, 217, 219, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 235, 236, 237, 238, 239, 240, 241, 244, 245, 254, 256,258, 259, 260, 261, 262 Takeya, K., 265,266,277,279,286,287, 288, 289, 291, 292, 293, 294, 295, 296, 297,298,299,302,307,311,312 Taliansky, M. E., 318, 333, 334, 335, 336, 341, 342,343,359, 361, 363 Tam, M. R., 96,102,128, 138,149,167 Tamm, I., 59,91 Tamura, K., 264,312 Tanada, Y., 64,92 Tanford, C., 34,54, 56 Tateishi, J., 11, 12, 21, 25,56 Taiiber, H., 31,49 Tchubianishvili, C., 196,213 ten Bruggencate, G., 325,362 ter Meulen, V., 102, 104, 107, 109,110, 113, 118,124, 125, 129 Terry, R. D., 22, 56 Tessman, I., 32, 56 Theilen, G. H., 118, 127 Thiry, L., 31,51 Thomas, A. A., 116,124 Thomas, E. D., 101,126, 129 Thomas, J. A., 204,211 Thomas, P. S., 79,93 Thomashow, M. F., 88,93 Thorbeck, R., 281,308 Thornhill, T. S., 170,194 Thorpe, R., 106,107,129 Thresh, J. M., 150, 166 Timme, T. L., 298,311 Timmer, F. A., 345,364 Timpl, R., 42,54

Titmuss, D. H. J., 198,213 Tjian, R., 108, 111, 125 Togashi, T., 112, 113, 128 Tokiwa, H., 266,277,293,312 Tolbert, W. R., 145, 166 Tomita, K., 59, 93 Tompkins, W. A. F., 301,304,309 Torrance, L., 162,168 Totten, R. E., 217, 218, 221,262 Towbin, H., 97, 129 Toyoshima, S., 266,284, 291,310 Traub, R. D., 26,56 Traynor, B. L., 58,61,62,63,92 Tremaine, J. H., 135, 147, 148, 149,154,168 Trepanier, P., 193, 193 Trim, A. R. H., 232,236, 237,239,259 Trucco, M., 146, 168 Tsang, J., 238,261 Tsoukas, C. D., 113, 114, 129 Tsugita, A., 131, 168 Tucker, C. L., 339,340,359 Tudor, I., 256,258 Tuechy, H., 88,93 Tung, J. S., 107,110,111,128 Turner, R. H., 230,259 Tweeten, K. A., 204,212 Tyc, K., 250,260 Tyssen, P., 169,194 Tzen, J. C., 79, 93 U

Uchida, T., 107,128 Uchimiya, H., 224,262 Uhr, J. W., 122,127 Ulanova, E. F., 334,335, 336,341,363 Underwood, P. A,, 103,120,130, 146,168 Upadhya, M. D., 236,237,238,239,241,261 Ushiyama, R., 227,262

V Vago, C., 195, 196, 197, 198, 200, 207, 209, 211,212

Vago, D., 196,212 Valdesuso, J., 103, 126 Vallee, J. G., 361 Van Bruggen, E. F. J., 42,55 Van den Elsacker, S., 88, 93 van den Hurk, S., 115, 128 van der Beek, C. P., 328,360

386

AUTHOR INDEX

Van der Geest, J. M. C., 254,261 van der Meer, J., 328,360 van Deusen, R. A., 138,142,145,168 Van Dijk, A. A., 64,93 Van Kammen, A., 217,228, 230,231,232, 234, 235, 242,243, 244, 250, 252,253, 254, 255,259, 262, 316,328, 330, 340, 345,360,361,362,364 Van Larbeke, N., 88,93 Van Loon, L. C., 250,253,262, 314,320, 322,338,344,363,364 Van Montagu, M., 88,91,93 van Regenmortel, M. H. V., 132, 133, 135, 137, 138, 139, 142, 144, 145, 146, 147, 148, 149, 152, 153, 154, 155, 156, 157, 158, 164, 165, 166, 168, 170, 171, 174, 175, 176, 179, 180, 182, 183, 186, 189, 191, 194 van Steenis, B., 102, 106,122,128 van Vloten-Doting, L., 150, 168, 322, 327, 328,363, 364 Van Waerebeke, D., 197,212 van Wezel, A. L., 102, 106,122,128 van Wyke, K. L., 108,130 Varghese, J. N., 103, 111, 120, 125, 130 Varghese, M., 101, 130 Varma, M. G., 121,125 Vatter, A. E., 58, 73, 91 Vaughan, J. H., 113,114,129 Venkatraman, R. V., 264,309 Vernon, S. K., 187,194 Verver, J., 316, 361 Verwoerd, D. W., 64,93 Veyrunes, J. C., 200,203,211 Vidal, H., 122, 124 Vidaver, A. K., 267,307 Videtta, E. S., 122, 127 Vieu, J. F., 284, 286, 287, 288, 289, 291, 293,307, 312 Vigny, M., 42, 56 Villeriez, C., 122, 127 Vishnichenko, V. K., 333,359 Vodkin, M., 79,93 Vogt, V. M., 323,360 Volk, W. A., 110,130 Volkman, L. E., 208,212 von Arnold, S., 221, 235, 262 von Wechmar, M. B., 157,167 Vostrova, N. G., 319,326,361 Vrijsen, R., 116, 124, 129 Vroman, B., 118,129

W Wabba, A. H., 299,305,312 Wagner, R. R., 110,130 Walker, C. A., 7, 8, 18, 25, 27, 52 Walker, D. L., 72,93 Wallin, A., 224, 225,262 Wands, J. R., 100,101,129, 130 Ward, C. W., 110,111,120,127,130 Warren, R. A. J., 267,310 Watanabe, Y., 81,92,323,331,332,362,364 Waterfield, M. D., 117, 129 Waterson, A. P., 169, 170, 188, 191, 193 Watson, B., 88, 93 Watson, J. D., 4, 21, 23,52 Watson, W. A., 13,54 Watts, J. W., 219, 227, 230, 231, 232, 234, 236, 237, 239, 247,259, 260, 262, 339, 362, 363 Waxham, M. N., 102,129 Weathers, L. G., 33, 56 Webster, R. G., 96, 103, 104, 107, 108, 109, 110,111,112,117,120,126, 127, 130 Wecker, E., 31, 32,49, 50 Weeming, C. J., 328,364 Weidel, W., 270,312 Weinmaster, G., 115,128 Weintraub, M., 317, 348,364 Weiser, J., 196, 197,212, 213 Weissbach, A., 247,262 Wen, G. Y., 22,56 Werner, D., 42,52 Weston, L., 268, 273, 277, 279, 282, 284, 295,296, 297,312 Westphal, O., 271,312 Westwood, J. C. N., 198,213 Wezenbeek, P., 316,361 Whetstone, C. A., 138, 142, 145, 168 Whitcomb, R. F., 59, 73,91 White, D. D., 289,309 White, D. O., 111, 127 White, J. A., 154, 167 White, J. L., 248,262, 328,360 White, J. M., 117, 129 White, P. B., 265,312 White, R. F., 230,235,244,255,259,262,362 Whitfield, P. R., 322,364 Whitford, H. W., 9, 18,51 Wieringa-Brants, D. H., 316, 345,364 Wiktor, T. J., 102,119, 125, 130 Wilcock, G. K., 22, 56

387

AUTHOR INDEX Wild, T. F., 102, 114,126, 130 Wildman, S. G., 314, 319,362 Wildy, P., 121, 127 Wiley, D. C., 103, 111, 117, 125, 129, 130 Wilkins, M. H. F., 35,53 Will, R., 43, 53 Willcocks,M. M., 109,110,113,118,124, 125 Williams, A. R., 149, 168 Williams, E. S., 9, 21, 56 Williams, K., 136, 142, 166 Williamson, N. M., 123, 126 Willmitzer, L., 88, 91, 93 Wilson, D. R., 45, 56 Wilson, H. M., 224,262 Wilson, H. R., 35,53 Wilson, I. A., 103, 111, 117, 129, 130 Wilson, T., 240,262 Wimmer, E., 110, 116,125 Wisniewski, H., 22, 56 Wisniewski, 11. M., 9, 21, 22, 43, 52, 54, 56 Witkowski, J. T., 255,262 Witter, R. L., 101, 127 Wittman, H.-G., 32, 56 Wolcyrz, S., 73, 91 Wolinsky, J. S., 102, 129 Wollman, E., 299,309 Woltersdorf, M., 281,308 Wong, J., 43, 48 Wood, H. A,, 59,93 Wood, K. R., 227,232,237,239,258,260, 340,362 Wood, N. L., 256,258 Woodie, J. D., 101, 128 Woods, R. D., 230, 235,244,259 Worley, J. F., 318, 363 Wormsely, S. B., 113, 114, 129 Wrigley, N. G., 187,194 Wu, F. S., 248, 249, 257,262 Wullems, G. J., 88, 91 Wunner, W. H., 119,125 Wyatt, R., 103, 126

Y Yadav, N., 88,91, 93 Yagishita, S., 22, 56 Yakushiji, Y., 304,311 Yamada, M., 256,259 Yamamoto, M., 241,262 Yamamoto, N., 281,312

Yamaoka, N., 241,262 Yamashita, Y., 11,54 Yamato, S., 42, 52 Yanagawa, R., 103,126 Yanagida, M., 187, 194 Yanagihara, R. T., 11,51 Yarvekulg, L. V., 318,333,363 Yasui, K., 107, 110, 127 Yates, J. P., 102, 128 Yau, T., 205,213 Yewdell, J. W., 96, 110, 111, 125, 126, 130, 147, 153, 163, 164, 165, 168 Yi-hua, Q.,102,126 Yolken, R. H., 164, 165, 168 Yora, K., 228,259, 260, 340,345,362 Yoshida, N., 323, 331,362 Youle, R. J., 122, I30 Young, J., 131, 168 Young, S., 9, 21, 56

2 Zabel, P., 328, 360 Zaccara, A., 281,308 Zachary, A., 291,307 Zaenen, I., 88, 93 Zaitlin, M., 67, 93, 216, 218, 251, 256, 259, 260, 262, 315, 316, 322, 323, 325, 326, 328, 331, 332, 341, 345, 346, 347, 350, 355, 357,359, 360, 361, 362, 363, 364 Zambryski, P., 88,93 Zanders, E. D., 121,125 Zavada, J., 108, 110,111,124 Zelcer, A., 251, 262, 325, 364 Zhuravlev, Y. N., 315,364 Zigas, V., 5, 10, 12, 50, 52 Zimmerman, U., 138, 168 Zimmern, D., 322,325,361,362 Zinnaka, Y., 279,294,295,296,297,298,312 Zizka, Z., 196,213 Zlotnik, I., 2, 7, 8, 56 Zucker-Franklin, D., 42, 55 Zunino, F., 281,308 Zummer, M., 205,213 Zurawski, U. R., 100,101, 130 zur Hausen, H., 101,128 Zwartouw, H. T., 198,213 Zweerink, H. J., 64, 92 Zweig, M., 101, 104, 109, 111, 114, 115, 125, 129, 130

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SUBJECT INDEX A

Acetylcholinesterase, 42 Actinomycin C, effect on plant virus replication, 254-255 Actinomycin D, effects on BMV replication, 339 on plant virus replication, 254- 255 on tobacco mosaic virus vRNP polypeptides, 351 Adenocarcinoma, 304 Adenovirus 5, 113, 188, 193 hexon polymerization, 117 immunosorbent electron microscopy, 187 taxonomic polypeptides, 104 Adenovirus-antibody immunocomplexes, 186 Agallia constricta van Duzee, 73 Agalliopsis novella, 73 Agar double diffusion test, 148, 151 Agrobacterium tumefaciens, 88,256 Alfalfa mosaic virus (AMV) immunization of mice, 136 MCA-determined antigenic properties, 150 monoclonal antibodies, 134 positive hybridomas, 143 protoplast infection, 227-228, 231, 235 replication, inhibition, 254- 255 Alzheimer’s disease, 11, 13, 47 senile plaques, 22 Amyloid, 42,45 plaques, in prion diseases, 21 -22. See also specific disease Amyotrophic lateral sclerosis, 12, 47 Antigens, quantitation, 97-99 Apes Creutzfeldt-Jakob disease in, 7, 8,10,11 kuru in, 7, 8, 10 Apple mosaic virus (ApMV) diagnosis, with monoclonal antibody, 161 immunization of mice, 136 monoclonal antibody-determined antigenic properties, 150-151 monoclonal antibodies, 134 Arabidopsis thaliana, protoplast isolation, 224

Arabis mosaic virus (ArMV) immunization of mice, 136 monoclonal antibody-determined antigenic properties, 152-153 monoclonal antibodies, 134 positive hybridomas, 140- 142, 144 Aspergillusjaponicus, macerating enzyme, 219 Astrovirus, 188, 193 Avena sativa, leaf protoplasts, virus infection, 227 Avidin, 42

B Bacillus, 266, 294 Bacteriocine, 299 Bacteriocins, 301, 303. See also Vibriocins as diagnostic tool for sensitive cancer cells, 304, 306 high-molecular-weight type, 303 therapeutic use, 304,306 Bacteriophage, 32 Bacteriophage T4, 187 Baculoviruses, 204-205,209 Choristoneura biennis, 204 -205 DNA, 205 properties, 205 -206 genomes base composition, 205 size, 205-206 nucleic acid, 205-206 Barley facultative resistance, 346 virus complementation in, 341-342 Barley stripe mosaic virus (BSMV) as helper virus, 341-342 infection, detection, 245 protoplast infection, 227 Barley yellow dwarf virus (BYDV) MAV, monoclonal antibody-determined antigenic properties, 151- 152 monoclonal antibody-determined antigenic properties, 151-152 monoclonal antibodies, 134 PAV, monoclonal antibody-determined antigenic properties, 151- 152 389

390

SUBJECT INDEX

protoplast infection, 227 RPV. monoclonal antibody-determined antigenic properties, 151-152 transport function complementation, 337 Bean golden mosaic virus (BGMV) complementation with helper virus, 344 DNA, protoplast infection with, 241 protoplast infection, 228 Bean pod mottle virus (BPMV), protoplast infection, 229, 232, 234, 235 Beet western yellow virus, 152 Beet yellows virus, 318 Berberine chloride, 282 Betula, protoplasts, 220 BK virus, immunosorbent electron microscopy, 176- 177 Bluetongue virus, 64, 122 activation, 64 VP2, detection, 106 B lymphocytes, hybrid clone, 132 Bovine leukemia virus gp 51, structural analysis, 111 protein, functional areas, 107- 108 Brassica rapa, leaf protoplasts, virus infection, 221,234, 240 sinensis, protoplasts, virus infection, 227 Brome mosaic virus (BMV) complementation experiment, with tobacco mosaic virus, 342- 343 facultative resistance to, 340 helper virus promoting transport over nonhosts, 341-342 protoplast infection, 226-228, 231, 232, 234 replication, inhibition, 254 resistance to, 339 RNA, 328 protoplast infection with, 236-239 Bunyaviruses, study, monoclonal antibodies in, 103

C Calicivirus, 193 Cannibalism and kuru, 13 -15 and scrapie, 14-15

Carborundum powder, leaf treatment, 218-219 Carnation etched ring virus (CERV), monoclonal antibodies, 134 Carnation mottle virus (CarMV), immunosorbent electron microscopy, 176 Carnation vein mottle virus (CVMV), immunosorbent electron microscopy, 187 Cat, Creutzfeldt-Jakob disease in, 7 , 8 Cauliflower mosaic virus (CaMV) DNA, protoplast infection with, 241 immunosorbent electron microscopy, 174-175 protoplast infection, 227,232 protoplasts infected with, culture, 243 Cellulysin, 219, 225 Chenopodium hybridum, leaf protoplasts, virus infection, 227 Chimpanzee, Creutzfeldt-Jakob disease in, 11 Chloroform, 282-283 Chloroplast pigment protein, 42 Chlorotic leaf spot virus (CLSV), immunosorbent electron microscopy, 175 Cholera, 265 bacteriophages, 265. Seealso Choleraphage; Vibriophage Choleraphage, 276. Seealso Vibriophage 4 149,272,286 mode of action on classical and El Tor biotypes, 277-278 Chronic wasting disease, 9 amyloid plaques, 21 natural host, 6 Citrus exocortis virus (CEV), protoplast infection with, 241 Citrus leaf rugose virus (CLRV), monoclonal antibodies, 134 Clorix, 218 Clumping, 170, 173 Colicin, 301, 303-305 Colicine, 298 Complement fixation (CF), 99 Corn stunt mycoplasma, immunosorbent electron microscopy, 172 Corynebacterium diphtheriae, 306 Counterimmunoelectrophoresis, of rotavirus in human stools, 180

391

SUBJECT INDEX Cowpea (Vigna sinensis) facultative resistance, 340, 341 protoplasts isolation, 217-219 virus infection, 228 resistance t o viral infection, 251 -252 Cowpea chlorotic mottle virus (CCMV) protoplast infection, 227, 232, 234 stimulation by poly-D-lysine, 230- 231 RNA, protoplast infection with, 236- 237,239 Cowpea mosaic virus (CPMV) facultative resistance to, 340 proteolytic activity, 316 protoplast infection, 228-230, 231, 235 replication, inhibition, 254-255 resistance to, 339 RNA, 330 protoplast infection with, 237 RNA polymerase, 328 translocation protein, 252 Coxsackie virus, 122 B4, variants, 119 Creutzfeldt-Jakob disease, 3, 5-6, 10- 12, 15, 24, 43 adaptation process, 25 agent possible spiroplasma, 43 resistance to radiation, 12 sizes, 33 distribution, 1 2 duration of illness, 8 epidemiology, 13 fibrils found with, 43 incubation period, 7, 2 1 genes controlling, 23 natural host, 6 pathogenesis, 18 lack of immune response in, 19-21 plaques, 11,21-22,45 Crown-gall disease, 88 Cryptotopes, 147, 151, 157-158 antibodies, 145 Cucumber green mottle mosaic virus (CGMMV), protoplast infection, 227 Cucumber mosaic virus (CMV), 114 in double infection with potato virus X, 336 facultative resistance to, 340

immunosorbent electron microscopy, 176, 187, 190 protoplast infection, 227-228, 232 replication, inhibition, 254 RNA, protoplast infection with, 239 Cucumber pale fruit viroid (CPFV), protoplast infection with, 241 Cucurnk sativus, leaf protoplasts, virus infection, 227 Cytomegalovirus, monoclonal antibodies, 101 Cytoplasmic polyhedrosis virus (CPV), 59-60, 64, 198, 210 transcriptase activity, 64

D DAS-ELISA, 161,162

Dauca carota, protoplast isolation, 224, 225 Decoration, 170, 173, 174 Dengue virus monoclonal antibodies, 101- 102 types 2 and 3, mixed infections, 104 Deoxyribonuclease, 282 Desmotubules, 315 Diabetes mellitus, 47 DNA binding protein, 42 Dolichos enation mosaic virus (DEMV), 319 as helper virus, 341 -342 Domestos, 218 Driselase, 219, 224-225

E Eastern spruce budworm, entomopoxvirus, 195, 210 Eggplant mottled crinkle virus (EMCV), immunosorbent electron microscopy, 179 Electron microscopy. See also Immunoelectron microscopy detection of rotaviruses, 180 Enzyme-linked immunosorbent assay (ELISA), 97-99, 108, 118, 144, 163, 170, 188, 193 competition, 147 indirect, 147, 148 detection of Tobamo viruses, 155-156

392

SUBJECT INDEX

multilayered sandwich, 147 in plant virology, 132 of rotavirus, in human stools, 180 sensitivity, 191- 192 vs. immunosorbent electron microscopy, 175-176,177 VS. PA-CGT, 180 of virus antigen in protoplasts, 246 Elk, chronic wasting disease in, 6 , 9 Encephalopathies, subacute transmissible spongiform. SeePrions, disease Enzeco AP 650,219 Epitope mapping, 110 reaction of antibody with, 99 Epstein-Barr virus, 113 detection of structural change, 118 new cell surface marker, 114 monoclonal antibodies, 101 Entomopoxvirus, 195- 213 acidic deoxyribonuclease, 203 Aedesaegyptis, 197 alkaline protease, 204-205 Amsactamoorei, 195, 196,198,200-204, 206,208-209 Anomalacuprea, 197 Aphdiustnsmank, 197 Camptochironomus tentans, 197 Chironomus attenuntus, 197 Chironomus decorus, 210 Chironomus luridus, 195,197 Chironomus near decorus, 206 Choristoneura, 200 Choristoneura biennis, 195,196,199,201, 202,206 Choristoneura ConfEiCtana, 196 Christoneura diuersana, 196 Choristoneura fumiferana, 195,206 components, 200-206 Demodenaboranemis, 196 Dermolepida alborhirtum, 197 DNA-dependent RNA polymerase, 204 Eunoaaudiaris, 196,201- 203,206 Figulus sublaeuis, 197 Geotrupes silvaticus, 197 Goeldichironomus holoprasinus, 197,206 hala, 200 host range, 196-198 inclusion body, enzymes, 203-205

infection, 206-209 in larvae, 207-208 adsorption, 207 maturation, 207-208 morphogenesis, 207-208 nonoccluded particles, 208 occlusion, 208 penetration, 207 Melanoplus sanguinipes, 197, 201, 202-203,206 Melolontha melolonthu, 195, 196, 198 morphological properties, 196- 197 multiplication, 206-209 neutral deoxyribonuclease, 203- 204 nucleotide phosphohydrolase, 203 Oncopera alboguttata, 210 Operophtera brumata, 196 Oreopsyche angustella, 196 Othnonius batesi, 196, 206,210 in pest control, 209-210 Phyllopertha horticola, 197 polyadenylic acid polymerase, 204 spindles, 198, 200, 201 structural features, 198-200 structural proteins, 201-203 in tissue culture cells, 208-209 virion, 198-200 components, 201- 202 enzymes, 203-205 Escherichia coli, 266,301, 305 Ethyl ether, 283

F Ferret chronic wasting disease in, 9 kuru in, 7 scrapie in, 9 Flavivirus antibody enhancement, 121 diagnosis, monoclonal antibodies in, 101 Fluorescein isothiocyanate, 245 Fluarescent-activated cell sorter, 144 Foot and mouth disease virus (FMDV), 116 assembly, 116-117 Friend leukemia virus gp, 70, 114 in persistently infected cells, 113

SUBJECT INDEX Fungi antibiotic production, effect of viral infection, 256-257 induced resistance to, 255 Furazolidone, 282

G Gerstmann-Straussler syndrome (GSS), 5-6,12 amyloid plaques, 21-22 natural host, 6 Glycine max cell culture protoplasts, virus infection, 229 protoplasts auxin requirement, 224 isolation, 225 Glycosylation, 115 Goat Cruetzfeldt-Jakob disease in, 7 , l l kuru in, 7 scrapie in, 2, 6-8, 21, 22 pathogenesis, 18 transmission, 24 transmissible mink encephalopathy in, 7-9 Gossipium hirsutum, facultative resistance, 341 Granulin, 202 Granulosis viruses, 195, 198 Grapevine stem pitting-associated virus (GSP-AV), immunosorbent electron microscopy, 178 Guinea pig, Creutzfeldt-Jakob disease in, 7, 8,11,19

H Hamster, 43 cannibalism model, 14 Creutzfeldt-Jakob disease in, 7, 11 scrapie in, 3, 7-9, 11, 15-18 pathogenesis, 19 transmissible mink encephalopathy in, 7-9

393

Hapadnaviruses, study, monoclonal antibodies in, 102 Haplopappus gracilis, protoplast yield, 224 Harvey sarcoma virus, v-ras gene product, monoclonal antibodies, 113 HeLa cells, 301, 304 Hemagglutination inhibition (HI), 99 Hemolysin inhibition (HLI), 99 Hepatitis B virus, monoclonal antibodies, 101 Herpes simplex virus immunosorbent electron microscopy, 187 new glycoprotein, 114 novel polypeptide, 114- 115 protection from, with monoclonal antibodies, 121 protein modification, 115 structural analysis, 111 type 1 antibodies, 101 early glycoprotein (GVP-11), maturation, 115 epidemiology, 104 type 2 antibodies, 101 epidemiology, 104 types 1 and 2, purification of glycoproteins, 118 virus-specific polypeptides, 114 Herpes virus immunosorbent electron microscopy, 186 rapid enzyme immune filtration test, 101 taxonomic polypeptides, 104 Hordeum uulgure, leaf protoplasts, virus infection, 227 H protein, 115, 323 Human beta herpesvirus 5, 101 Human gamma herpesvirus 4,101. See ako Epstein-Barr virus Human reovirus, 59-60 activation, 64 defective interfering particles, 81- 85 gene products, 69 - 71 subgenomic RNAs, 78- 79 transcription, temperature optimum, 64 Humans Creutzfeldt-Jakob disease in, 6, 7 , 8 Gerstmann-Straussler syndrome, 6 kuru in, 6, 7, 8

394

SUBJECT INDEX

Hybridoma, 132, 133 antibodies of immunoglobulins, 144 virus-specific, screening, 144- 145 cloning, 142- 144 methods, 144 conservation, 146 culture, 137, 139, 145 homogeneity, 146- 147 positive detection, 144 enhanced frequency, 139- 142 yields, 140 - 142 Hydroxylamine, 30, 31-32

I Ilar virus antibodies, production, 134, 135, 145 monoclonal antibody-determined antigenic properties, 150- 151 Immunocomplexes, demonstration, by immunosorbent electron microscopy, 186 Immunoelectron microscopy, 99,105 antibody binding site location, 112 in virology, 169 Immunofluorescence, 99,104 Immunoglobulin, gamma, as clumping antibodies, 193 Influenza A virus, 102 antigenic drift, 103 Influenza B virus, 102 antigenic drift, 103 variants, 104 Influenza virus, 96 amino acid alterations, 111 antibody binding site location, 112 antigenic drift, 103, 120 avian vs. seal strains, 107 characterization, with monoclonal antibodies, 103 diagnosis, monoclonal antibodies in, 101 hemagglutinin, 117 amino acid substitutions, 111 fusion protein activity, 117 study, 106 human infection, 103 immunosorbent electron microscopy, 187

mRNA, 66 NA molecule, structural analysis, 111 neuraminidase, study, 106 nucleocapsid proteins, in transcription, 108 protection from, with monoclonal antibodies, 121 protein, functional areas, 107 seal, 103 variant selection and mapping, 110 Immunosorbent electron microscopy, 188 advantages, 192 antibody-coated grid technique, 171-177, 190 effect of pH of virus extracts, 176 specificity, 174-175 VS. PA-CGT, 178-179 antigen-coated grid technique, 171, 187-188, 191 antisera, 189 buffers, 189 decoration, 190-191 definition, 170 detection of viruses, 169-194 direct trapping of viruses on grids, 191 disadvantages, 192 effect of pH, 190 grid coating, 189 incubation temperature, 190 incubation time, 189- 190 methods, 170- 188 of plant vs. animal viruses, 192-193 protein A-coated bacteria technique, 171, 182- 186, 191,192 protein A-coated grid technique, 171, 177- 182, 186,190- 193 effect of BSA in buffer on specificity of virus trapping, 181- 182 effect of time and temperature, 181- 183 protein A coating of grid, 190 in relation to SPRIA of ELISA, 192 techniques, nomenclature, 171

J Japanese encephalitis virus, 110 protection from, with monoclonal antibodies, 122

SUBJECT INDEX

K Kinetin, 242 for callus tissue culture, 223 Kommagome pipets, 222 Kuru, 3,5,10 agents, resistance to irradiation, 12 and cannibalism, 13, 14-15, 24 cause, 12 distribution, 12 duration of illness, 8 epidemiology, 13 incidence, 13 incubation periods, 7 natural host, 6 plaques, 10, 12, 21-22, 45 vs. senile plaques, 22

L Laminin, 42 Leafiopper, infection, by wound tumor virus, 72 - 73,89 Lens crystallin, 42 Lettuce mosaic virus (LMV) diagnosis, with RIA, 162 immunization of mice, 133- 136 monoclonal antibody-determined antigenic properties, 153 monoclonal antibodies, 134 Lily symptomless virus (LSV), immunosorbent electron microscopy, 176 LIP gene, 4 Liposomes, in protoplast inoculation with viral RNA, 239 - 241 Lissamine rhodamine B, 245 Lupus erythematosus, 48 Luteo virus antibodies, production, 134, 135, 145 monoclonal antibody-determined antigenic properties, 151- 152 Lycopersicon esculentum, leaf protoplasts, virus infection, 227 Lymphocytic choriomeningitis (LCM) virus, in persistently infected cells, 113 Lymphoprep, 222 Lysogeny, 298

395 M

Macerase, 219, 225 Macerocyme, 219-220,222, 224 Macerocyme R-10,220 Macrophages, in hybridoma culture, 139 Maize chlorotic mottle virus (MCMV), immunosorbent electron microscopy, 179 Maize dwarf mosaic virus (MDMV) diagnosis, with RIA, 162 immunization of mice, 133-136 monoclonal antibody-determined antigenic properties, 153 monoclonal antibodies, 134 Maize rough dwarf virus (MRDV), immunosorbent electron microscopy, 187 Marek’s associated tumor surface antigen, monoclonal antibodies, 101 Marek’s disease virus, 101, 113 Mayvill cellulase, 220 Measles virus, 112, 114 antibodies, production, 96-97 detection of structural change, 118 hemagglutinin, 118 monoclonal antibody groups, 107,109 H protein, maturation, 115 matrix (M) protein, 113 persistence, leading to SSPE, 120-121 in persistently infected cells, 113 unusual composite protein, 114 Medicago, protoplast culture, 243 Meicelase P, 219 MHV, GP-1,106 Mice Creutzfeldt-Jakob disease in, 7, 8, 11 immunization, with plant viruses, 133- 137 kuru in, 7 modulation of Prion diseases by gene in MHC, 21 scrapie in, 2-3, 7-9, 11, 21-23, 43 genetic loci controlling, 4 pathogenesis, 18-19 with Creutzfeldt-Jakob disease, 11, 23 Micrococcus, 266 Minks kuru in, 7

396

SUBJECT INDEX

scrapie in, 7 -9 transmissible mink encephalopathy in, 6-9 Mitomycin C, 282, 299 Moloney murine leukemia virus, protein distribution, 115- 116 Monkey Creutzfeldt-Jakob disease in, 7,8,11 kuru in, 7 , 8 scrapie in, 7-9 transmissible mink encephalopathy in, 7-9 Monoclonal antibodies, 193 in affinity columns, 118 against plant viruses, 131-168 application to virus diagnosis, 161-165 production, 133- 134 properties desired, 163 for analysis of epitope fine structure, 165 in tobacco mosaic virus, 155- 160 assays, 164 avidity, determination, 109 binding, monitoring, 104 binding group analysis for structure, 110 characterization, 146- 149 in characterization of protein structure and function, 105-112 cocktail, 163- 164 competitive binding, 108- 109 relative interference efficiencies, 109 steric effects, 108-109 correlation of binding sites with protein structure, 108-112 cross reactions, 122-123, 164-165 cytotoxicity with, 122 in definition of virus-specific protein, 105-118 in diagnostic virology, 100- 102 in differentiation of active areas on protein, 106-108 to enhance virus infection, 121 epitope, structure-dependent function, 158- 159 heterospecific, screening for, 145 in identification of functional protein, 106 immunoglobulin isotype, determination, 148- 149 immunological techniques applied to, 96-99

irnmunoprecipitation, detergent conditions, 97, 99 for investigation of protein synthesis in persistently infected and transformed cells, 112-115 in investigation of virus pathogenesis, 119-121 nomenclature, 149 as probes for virus protein expression, 112-118 production, 96-97 large scale, 145- 146 protective effects, 119,121- 122 purification, 146 reaction with novel virus proteins, 114-115 in serology, 123 specific for p15E, 106 specificity, 97 -99,122-123,164- 165 assay, 164 determination, 147- 148 in study of viral protein processing and maturation, 115-118 in study of viruses, 95- 130 therapeutic uses, 121-122 in vaccine preparation, 122 in viral epidemiology, 102- 104 in viral taxonomy, 102-104 for virus diagnosis, 161-164 advantages, 165 in virus identification, 99 - 104 limitations, 104 Morbilliviruses, 102 Mule deer, chronic wasting disease in, 6 , 9 Multiple sclerosis, 47 Murine fibroblasts, 301, 304 Murine leukemia virus, gp 70, structural analysis, 111 Myeloma cells conservation, 146 culture, 137 in production of monoclonal antibodies against plant viruses, 137 fusion with spleen cells, 138-142 Myzus persicae, 255

N Neoplastic disorders, 48 Neotopes, 147, 154, 157-158

SUBJECT INDEX Nepo viruses, monoclonal antibody-determined antigenic properties, 152- 153 Neutralization (NT), 99 Nicotiana benthamania, leaf protoplasts, virus infection, 227 glutinosa, 319 rustica, leaf protoplasts, virus infection, 227 syluestrk, protoplast isolation, 224, 225 tabacum cell culture protoplasts, virus infection, 229 leaf protoplasts, virus infection, 227, 240 protoplasts auxin requirement, 224 isolation, 217-219, 255 resistance to viruses, 251, 252, 339 suspension cultures, tobacco mosaic virus infection, 249-250

0 Oat sterile dwarf virus, immunosorbent electron microscopy, 187- 188 Onozuka cellulase, 219 Onozuka R-10,220,224,225 Onozuka SS, 224,225 Orthopoxviruses, 205,209

P Papovavirus, immunosorbent electron microscopy, 192 Paramyxovims, 32 HN protein, functional areas, 107 Parkinson’s disease, 47 Parvoviruses, 33 study, monoclonal antibodies in, 103 Pea enation mosaic virus (PEMV) protoplast infection, 226, 228 replication, inhibition, 254 Pectinase, 219, 225 Pectolyase-Y 23,219,225 Penicillium chrysogenurn, virus-like particles, 256-257 Peptidyltransferase, 42

397

Peronospora tabacinu, 255 Petunia sp., leaf protoplasts, virus infection, 228 Phaseolus vulgaris, leaf protoplasts, virus infection, 228 Phytophthora parasitica, 255 Picornaviruses, diagnosis, monoclonal antibodies in, 101 PID-1 gene, 4 PID gene, 23- 24 Plant antiviral substances, 251-255 cell differentiation, regulation, 87 cell growth, regulation, 87 culture, for protoplast isolation, 217-218 defense reactions, suspension, in plant virus transport, 344-345 leaves, pretreatment, for protoplast isolation, 218-219 resistance against viral infections, 251-255,314,337-348 extreme, 337-341 facultative, 337-341, 343, 346 tissue culture, 216 virus disease, monoclonal antibodies, 102 virus-infected chymeric protein, 323 differential temperature treatment, 349- 356 pathogenesis-related proteins, 322 virus infection, sequence, 338 wound-inducible hormone in tumorigenesis, 85- 87 Plant cell auxin requirements, 223-224 from callus tissue, infection, 248-251 suspension culture, 216 infection, 248-251 with viroids, 250-251 with virus particles, 248-250 isolation of protoplasts from, 223-225 Plant virus, 131-132. See also specific virus acquired resistance, 320 antisera, 132 cell-to-cell movement, 251-252, 313-314 means, 344-345 diagnosis, 161 DNA, infection of protoplasts, 241 epitopes, 132

398

SUBJECT INDEX

genome transport from infected to healthy cells, 315-337 phenomenon, 315 - 319 role of plasmodesmata, 314-319 highly purified, for hyperimmunization of mice, 136- 137 host range control, role of transport function in, 341 - 344 immunosorbent electron microscopy, 186,192- 193 infection effect on antibiotic production in fungi, 256-257 transport form, 348-359 interaction between, leading to transport function complementation, 336-337 monoclonal antibodies, 132- 168 mosaic group, 317-318 mutants, complementation between, 332-336 phloem-limited, 318 complementation with helper virus, 343-344 precipitin test, 132 in protoplasts dot molecular hybridization, 247 electron microscopy, 247 sucrose gradient assay, 247 replication bioassay, 243 - 244 determination, 243 - 247 inhibition by plants, 253-254 research, use of protoplasts and separated cells in, 215-262 RNA detection, in protoplasts, 247 infection of protoplasts, 235-241 replication, virus-coded products in, 328 in subliminal infection, 347 serological detection, 244-247 fluorescent antibody staining, 244-245 peroxidase staining, 245 - 246 quantitation of virus antigens in protoplasts, 246-247 staining methods, 244-246 subliminal symptomless infection, 345-348 systemic infection, 313

transport efficiency, determination of, 319-322 function, 313-364, 345 in vascular bundles, 253 tropism to cell types, in facultative resistance, 343 as vehicle for genetic information, 256 Plasmodesmata diameter, 315 role in virus transport function, 315, 344-345 structure, 315 Plum pox virus (PPV), immunosorbent electron microscopy, 175 Poliovirus, 31 assembly, 116-117 capsids, 116 strains, comparison, monoclonal antibodies in, 102 type 3, neutralization, 112 VP1, detection, 106 Polyhedin, 202 Polyhedrosis virus cytoplasmic. SeeCytoplasmic polyhedrosis virus nuclear, 195-198,210 Polymyxin B, 282 Polyoma virus dsDNA, resistance to radiation, 33 T antigen, 97 Potato leafroll virus (PLRV) complementation with helper virus, 343 - 344 immunization of mice, 136 immunosorbent electron microscopy, 173 monoclonal antibody-determined antigenic properties, 152 monoclonal antibodies, 135 yield of positive hybridomas, 140-142 Potato mop-top virus (PMTV), immunosorbent electron microscopy, 173- 174 Poty viruses, monoclonal antibody-determined antigenic properties, 153- 154 Poxviridae, 195 Prions, 1-56 adaptation upon passage, 25 in CNS, 19 definition, 4

SUBJECT INDEX diseases, 6-12. Seealso specific disease amyloid plaques, 21-22 characteristics, 7 common features, 6-8 duration of illnesses, 8 epidemiology, 12-13 experimental transmission, 24 incubation periods, 6- 7 host genes controlling, 22-24 pathogenesis, 18-22 incubation time host genes controlling, 22-24 isolates, 24-25 molecular models, 45-47 scrapie. Seealso Scrapie agent properties, 25-45 strains, 24 - 25 structures, 24 as subviral pathogens, 47 transmission, 24-25 vs. viroids, 47 Protein A, 191. Seealso Immunosorbent electron microscopy Protoplasts extreme resistance in, 339 infected, culture, 242 -243 infection addition of poly-L-ornithine, 226- 230, 232-234 direct method, 226 indirect method, 226 with viral DNA, 241 with viral nucleic acids, 235-241 with viral RNA, 235-241 buffers, 238 calcium chloride in, 237 cycloheximide in, 238 liposome-mediated, 239 - 241 osmotic stabilizer, 236-237 polycations in, 237-238 polyethylene glycol in, 239 temperature, 238-239 zinc salts, 237-238 with viroids, 241 -242 with virus particles, 226-235 buffers, 231 calcium chloride in, 235 inocula concentration for, 233- 234 osmotic stabilizer, 234

399

pH, 232 polyethylene glycol in, 232-233 protoplast concentration for, 233 temperature, 234-235 inoculation, 226-242 isolation from cell suspension cultures, 223- 225 enzyme solutions for, 219-221 from leaves, 217-223 procedures, 221-223 multiplicity of infection, 257 osmotic stabilizers, 220 in plant virus research, 215-216 source, 256 technology, applications, 256- 257 virus antigen in dilution end point, 246-247 enzyme-linked immunosorbent assay, 246 quantitative determination, 246- 247 radioimmunoassay, 246 Prune dwarf virus (PDV), 151 Prunus necrotic ringspot virus (PNRV) diagnosis, with monoclonal antibody, 161 immunization of mice, 136 monoclonal antibody-determined antigenic properties, 150- 151 monoclonal antibodies, 135 Pseudomonas, 294 acidouorans, 267 aeruginosa, 301, 304 phage 7V, 267 fluorescence, 301 tabaci, 255 Psoralen, 31, 33 Potato spindle tuber viroid, infection of plant cell culture, 250-251 of protoplast, 241- 242 Pteridium aquilinum, protoplast culture, 243 Potato virus A (PVA), monoclonal antibodies, 135 Potato virus X (PVX) complementation with phloem-limited virus, 343-344 as helper for tobacco mosaic virus mutants in cell-to-cell movement, 334-336 as helper virus, 342 protoplast infection, 228

400

SUBJECT INDEX

replication inhibition, 254 stimulation, in double infection, 336-337 RNA, in subliminal infection, 347-348 virus-specific informosome-like ribonucleoprotein, 350-352 Potato virus Y (PVY) diagnosis, with monoclonal antibody, 162- 163 in double infection with potato virus X, 336 immunosorbent electron microscopy, 172, 176 monoclonal antibody-determined antigenic properties, 153 monoclonal antibodies, 135 positive hybridomas, 140- 143 strains, 153 Potato yellow dwarf virus (PYDV), protoplast infection, 227 Pyocin, 301, 304, 305

R Rabies virus, 122 diagnosis, monoclonal antibodies in, 101 pathogenicity study, monoclonal antibodies in, 102 variants, selection, 119 Radioimmunoassay (RIA), 97-99,108,118 competition, 162 of virus antigen in protoplasts, 245 Raphnus sativus, leaf protoplasts, virus infection, 228 Raspberry ringspot virus (RRV), protoplast infection, 227-228,229,231 Rauscher virus, gp 70, structural analysis, 110- 111 Reoviridae, 57 - 58 replication, 64 transcriptases, 64 transcription, 63- 64 Reovirus protein, functional areas, 107 variants, selection, 119- 120 Respiratory syncitial virus (RSV), antibodies, production, 97 Rhapidosomes, 302

Rheumatoid arthritis, 47 Rhizopus, pectinase, 219 Rhododendron, protoplasts, 221 Rhozyme H P 150, 219-220 Ribonuclease, 282 Rift Valley fever virus, study, monoclonal antibodies in, 103 RMV, RNA, protoplast infection, 240 RNA 3’-terminal phosphate cyclase, 42 RNA tumor virus, 110 Rotavirus, 64,188, 193 activation, 64 ELISA, 180 immunosorbent electron microscopy, 176, 177,179-180 study, monoclonal antibodies in, 103 Ryegrass cryptic virus (RCV), immunosorbent electron microscopy, 178

S Saccharamycescerevisiae virus (ScV), subgenomic RNAs, 79 Saintpaulia ianuntha, protoplast isolation, 225 Salmonella, 266-294 Scrapie, 2,8-9 adaptation process, 25 antibodies, attempts to demonstrate, 20 and Creutzfeldt-Jakob disease, 11 digestion by proteinase K, 41-42 by trypsin, 41 -42 distribution, 12 duration of illness, 8 endpoint titration, 15 comparison to incubation time interval assay, 17 epidemiology, 12 - 13 fibrils found with, 43 incubation period, 7, 15, 21 host genes controlling, 4, 22-24 incubation time interval assay, 3,4,15- 18 infectivity, dependence upon protein, evidence for, 38-39 natural host, 6 nomenclature, 4 pathogenesis, 18-19 lack of immune response in, 19-21

SUBJECT INDEX plaques, 21, 45 prions. Seealso Scrapie agent assays, 15- 18 protein (PrP), 3, 5, 35-43,47 apparent molecular weight, 46 correlation with infectious prion, 41-42,46 lack of immunogenicity, 21 microheterogeneity, 39 molecular weight, 20 nomenclature, 4 purification, 41 radiolabeling, 18, 39, 40, 42 resistance to protease digestion, 39-40,42 as structural component ofprion, 39-42 transmission, 24 Scrapie agent, 2 assay, 2, 15- 18, 27 current molecular models, 46-47 denaturation, effect on protein (PrP), 42 differential sensitivity to proteases, 41 -42 distribution, 26 electron microscopy, 43 -44 heterogeneity, 27 hydrophobicity, 27, 29 hypothesis on chemical structure, 45-46 inactivation, 39 by carbethoxylation, 32 by DEP, 36-38 by proteinase, 35-36, 38 by urea, 37-38 with KSCN, 38 with trypsin, 35-36, 38 increased sensitivity to ionizing radiation in presence of oxygen, 32 membrane hypothesis, 26 minimum molecular weight, 33 molecular size, 32-35 molecular weight, 34 peaks of infectivity, 26 possible nucleic acid core, 46-47 possible oligonucleotides, 34 -35 properties, 25-45 purification, 25-29 reaction to chemical modification, 30-32 resistance to inactivation by irradiation, 32-33 to procedures that attack nucleic acids, 29-31

401

to psoralen photoreaction, 30-31 to ultraviolet inactivation, 30 to zinc-catalyzed hydrolysis, 31 rod-like structures in purified fractions of, 43-45 search for nucleic acid, 29-32 sedimentation properties, 26-27,34 size, 47 spread, 18- 19 strains, 24 - 25 in susceptibility to nuclease digestion, 29-30 unusual properties, 3,45-46 vs. plasmids, 46 vs. potato spindle tuber viroid, 32 vs. viruses and viroids, 32, 46 Seal influenza virus, 103 Sendai virus, 112 HN protein, hemagglutination and neuraminidase activity, 96 in persistently infected cells, 113 protein, functional areas, 107 Serology, 99 Sheep breeds, comparative susceptibility to scrapie, 23 scrapie in, 2, 6-8, 12-13, 22-23, 43 elevated immunoglobulin G, 2 1 genetic loci controlling, 4 pathogenesis, 18 transmission, 24 transmissible mink encephalopathy in, 7, 8 Shigella, 266 fiexneri, 301,305 sonnei, 301,304, 305 Simian agent 12 virus, taxonomic polypeptides, 104 SINC gene, 4, 22-24 Sindbis virus, 121 antibody binding site location, 112 DI particles, 83 glycoprotein El, 116 immunosorbent electron microscopy, 181-185,190,192 protection from, with monoclonal antibodies, 121 protein, functional areas, 107 SIP gene, 4 SIP/LIP gene, 22

402

SUBJECT INDEX

Slow virus diseases, unconventional. See Prions, disease Sobemo viruses antibodies, production, 134, 135, 145 monoclonal antibody-determined antigenic properties, 154 Sodium deoxycholate, 282 Solid-phase radioimmunoassay, diagnosis of SMV, 161- 162 Somatic cell hybridization, 132 SoMV, 154 Sorrel, wound tumor virus-infected, root tumors, 87 Southern bean mosaic virus (SBMV), 154, 318 monoclonal antibodies, 135 protoplast infection, 229, 231, 232, 235 Soybean, protoplast culture, 243 Soybean mosaic virus (SMV) diagnosis, with monoclonal antibody, 161 immunization of mice, 133-136 monoclonal antibody-determined antigenic properties, 153 monoclonal antibodies, 135 Spheroidin, 202-203 Spiroplasma, and scrapie agent, 43 Spleen cells fusion with myeloma cells, 138-142 isolation, 137- 138 nonfused, longevity, 142- 143 Solid phase radioimmunoassay (SPRIA), 170,188,193 Squirrel monkeys chronic wasting disease in, 9 kuru in, 14 Staphylococcus, 294 Streptomyces, antibiotic production, 257 Streptomycin, sensitivity to vibriocin, 301 Subacute sclerosing panencephalitis (SSPE), 112-113, 120 Subterranean clover red leaf virus (SCRLV), monoclonal antibodies, 135 Subviral pathogens, 47-48 Sugar cane mosaic virus (SCMV), immunosorbent electron microscopy, 177 SV40, T antigen, 106 ATPase active site, 111

ATPase activity, function inhibition assay, 108 cross reactions, 123 Sweet clover, tumors, induced by wound tumor virus, 86-87 Swine virus, 96

T Tabomavirus, 102 Tick-borne encephalitis virus, 103 Ti-plasmids, 88 Tobacco induced resistance in, 255 leaf, protoplast culture, 243 Tobacco etch virus, in double infection with potato virus X, 336 Tobacco mosaic virus, 102, 115, 169, 215-216,256,317-319 assembly, 117 complementation with phloem-limited virus, 344 as dependent virus, helper viruses promoting transport of, 335-336 in double infection with potato virus X, 336 enzyme-linked immunosorbent assay, 246 facultative resistance to, 340, 341 genome, molecular organization, 322 -325 helper virus promoting transport over nonhosts, 341 -342 immunization of mice, 136 immunosorbent electron microscopy, 172,175, 177, 183, 187 AB-CGT, specificity, 174-175 infection detection, 245 of plant cell cultures, 248-250 infectivity, neutralization with monoclonal and polyclonal antibodies, 159-160 monoclonal antibody-determined antigenic properties, 153,154- 160 monoclonal antibodies, 135 mutants, 325-332 coat protein, 325-327, 349 complementation of ts transport function by trhelper virus, 333 -334

SUBJECT INDEX influencing synthesis of genomic or subgenomic RNAs, 327-330 non-coat-protein, 327-332 temperature-sensitive (ts), 326-327, 330 in transport gene, 330-332 plant resistance to, 251- 252 positive hybridomas, 139-142, 144 protein molecule, folding, 157 protoplast infection, 227-229, 231, 232, 234,235 stimulation by polyethyleneimine, 230 protoplasts infected with, culture, 242 replication, 252 -253 inhibition, 254 resistance to, 339 RNA, 322 -323 cell suspension inoculation with, 239 low-molecular-weight component, 322 - 324 mutants influencing synthesis of, 327- 330 nucleotide sequences, 323-325 protoplast infection with, 236-240 in subliminal infection, 347-348 RNA polymerase, 328 RNA replicase, 328 strains, 175 common epitope, 158 structure, 160 subliminal symptomless infection, 345 translocation protein, 251-252 transport form, 349 virus-specific informosome-like ribonucleoprotein, 350 - 353 polypeptides, 350- 351 Tobacco necrotic dwarf virus (TNDV), 253,343 protoplast infection, 228 Tobacco rattle virus (TRV) immunosorbent electron microscopy, 187 protoplast infection, 227- 228, 229, 231 RNA, 330 Tobacco ringspot virus (TobRV), monoclonal antibody-determined antigenic properties, 152 Tobacco streak virus (TSV) immunization of mice, 136

403

monoclonal antibody-determined antigenic properties, 150- 151 monoclonal antibodies, 135 Tobamo viruses antibodies, production, 134, 135, 145 monoclonal antibody-determined antigenic properties, 154-160 Toluene, 283 Tomato facultative resistance, 340, 346 protoplasts, 217, 219 resistance to viruses, 339 virus complementation in, 342 Tomato black ring virus (TBRV), protoplast infection, 228 Tomato bushy stunt virus (TBSV), immunosorbent electron microscopy, 174-175,183,186,187 Tomato ringspot virus (TomRV) monoclonal antibody-determined antigenic properties, 152 monoclonal antibodies, 135 Tomato spooted wilt virus (TSWV), transport function complementation, 337 Transformation-sensitive membrane glycoprotein, 42 Transmissible mink encephalopathy, 9, 24 adaptation process, 25 duration of illness, 8 incubation periods, 7 natural host, 6 Trichoderma viride, pectinase, 219 Tris, 283 Triticum aestivum, leaf protoplasts, virus infection, 228 TRSV, RNA, protoplast infection with, 239 Tulip breaking virus (TBV), monoclonal antibodies, 135 Turkey ( H l N l ) virus, 96 Turnip rosette virus (TRosV), 154 protoplast infection, 227, 232, 234, 235 replication, inhibition, 254 RNA, protoplast infection, 240 Turnip yellow mosaic virus (TMYV), 318 immunosorbent electron microscopy, 174-175, 183, 186 protoplast infection, 227

404

SUBJECT INDEX

replication, inhibition, 524 RNA, in subliminal infection, 347

U Ultraviolet light, effect on BMV replication, 339

V Varicella zoster virus, monoclonal antibodies, 101 Venezuelan equine encephalitis (VEE), 117 gp 56, detection, 106 Vesticular stomatitis virus (VSV) DI particles, 83 G protein, structure, 109-110 nucleocapsid protens, in transcription, 108 Vibrio, 264- 265 alginolyticus, 264 antigenic structures, 264 cholerae, 264-266,288,304-305 bacteriocins, 299 biotypes, 265 classical, 265 classical and El Tor, classification, 272,277,291-293 El Tor, 265 Celebes-type, 294-295, 297 Classic-Ubon type, 294, 297 diagnosis, 297 kappa-type, 294- 295 phage-induced mutation, 298 strains change, 298 kappa phage, 295 liberation, 296 - 297 morphology, 296 resistance, 297 NAG, 266 parahaemolyticus, 264, 266 morphology groups, 284-285 phages, 283 phage typing, 294 serotyping, 294 species, 264 Vibriocins, 265, 298-306 bacteria typing, 304 chemical properties, 302-303 detection, 299-301

diagnostic use, 306 discovery, 299 host range, 301 mode of action, 303-304 morphology, 301-302 physical properties, 302 -303 practical use, 304-306 production, 299-300 streptomycin-resistant, 299 - 300 streptomycin-sensitive, 299 therapeutic use, 306 Vibriophage, 263-298 biological properties, 266- 267 classification, 286, 290 components, 287-290 nucleic acids, 287 - 289 proteins, 288-290 dimensions, 287-288 host range, 266-267 inactivation, 281-282 by chemicals, 282 by heat, 278-280 pH, 283 inoviridae, 291 - 292 intracellular, 272- 276 lysogenic, 294 - 298 biological properties, 295 - 297 for diagnosis and tracing of cholera El Tor carriers, 297-298 host range, 295 liberation of Kappa phage, 296-297 morphology, 296 practical use, 297 resistance, 297 serological properties, 295 - 296 for typing of El Tor vibrios, 297 morphology, 283 - 287 multiplication, 272, 276 myoviridae, 286, 291-292 phage adsorbtion, 267-272 photoreactivation, 279-285 plaque morphology, 267 podoviridae, 286, 291 -292 research, historical background, 265 -266 sedimentation properties, 287,289 sensitivity to physical and chemical agents, 278-283 serological properties, 276- 277 styloviridae, 286, 291 - 292 ultraviolet inactivation, 279 - 285

SUBJECT INDEX uses, 292-294 epidemiological,294 prophylactic, 293 therapeutic, 293 Vigna sesquepedalis, leaf protoplasts, virus infection, 228 sinensis. SeeCowpea sinensis unguicullata facultative resistance, 340 resistance to viruses, 339 virus infection, 228 Vincarosea cell suspension, inoculation with TMV-RNA, 239 protoplasts auxin requirement, 224 isolation, 225 Virino, 4 -5 Viroids, 256 plant cell culture infection with, 250-251 protoplast infection with, 241- 242 Virology, monoclonal antibodies in, 95- 130 Virus. Seealso Plant virus; specific virus architecture, 117-118 examination, 117 assembly, 116 identification, monoclonal antibodies in, 99-104 infection, alteration in course of, 120-121 mutations, 118 pathogenicity, 119-120 protection from, with monoclonal antibodies, 121- 122 protein conformational changes and complex formation, 116-118 maturation, 115- 116 processing, 115 purification, 118 quantitation, 118 transformation-specific proteins, 113 variants isolation, 119-120 monoclonal antibody selection, 110 Virus-antibody interaction, IEM observation, 169 Virus-induced proteins, 322 Virus-specific informosome-like ribonucleoprotein

405

detection, 349-350 functions, 352 - 358 properties, 349-350 structure, 350-353 Virus-specific proteins, 322 Virus-specific ribonucleoprotein particles, 314

W Wound tumor virus, 64 capsomeres, 59 discovery, 58 enzymatic activities, 63 exvectorial isolates, 74 characterization, 74- 78 genome patterns, 85 resemblance to defective interfering particles, 81-85 subgenornic RNAs associated with, characterization, 78- 84 gene expression, in cultured vector cells, regulation, 71-72 genome, 59-61 segment responsible for tumor induction, 88 growth rate in vector, 72-73 infection of plant host, 85-88 isolates, 73 - 74 loss of transmissibility, 73 -74, 75-78, 85 molecular biology, 57-93 morphology, 58-59 physical properties, 58-59 polypeptides, 61 -63 apparent molecular weights, 62, 71 encoded by segment 5, identification, 78 expression in cell-free systems, 69-71 in cultured vector cells, 67-69 locations, 61 molecular weights, 61-63 outer-protein coat, 78 regulated infection, in vector cells, 89 research on, 89 root tumors, 85 segments, 59 - 60 studies, 58 subvectorial isolates, 74, 74- 75 transcriptase activity, 75 properties, 63 - 65

406

SUBJECT INDEX

transcription, 63 -67, 75 transcripts, 64-67 isolation, 66 - 67 nomenclature, 66-67 translation, 67 -72 transmission, 72-85 by leafhopper vector, 72 - 73 outer-protein coat polypeptides in, 78 studies, 89 tumor induction, 85-87, 89 role of growth hormones, 89-90 tumors growth independent of exogenous growth hormone, 88 tissue culture studies, 87-88 in vector cells, regulated infection, 72

vectorial isolates, 74 genome patterns, 85 virion, 58-63

Y Yellow fever virus, strains, differentiation, monoclonal antibodies in, 102

z Zea mays,leaf protoplasts, virus infection, 229 Zinga virus, study, monoclonal antibodies in, 103

CONTENTS OF RECENT VOLUMES

Volume 15 The Replication Cycle of RNA Bacteriophages Raymond C. Valentine, Richard Ward, and Mette Strand Double-Stranded Viral RNA R. K. Ralph The Translation of Viral Messenger RNA in Vitro P. L. Bergquist and D. J . W . Burns Conformation of Viral Nucleic Acids in Situ T.I. Tikehonenko Studies on the Herpes-Type Virus Recovered from the Burkitt’s Tumor and Other Human Lymphomas Sarah E . Stewart The Morphology of V i m - Antibody Interaction June D. A l m i d a and A . P. Waterson Beetle Transmission of Plant Viruses H . J. Walters Alfalfa Mosaic Virus Roger Hull

Virus-Induced Polykaryocytosis and the Mechanism of Cell Fusion George Poste Australia (Hepatitis-Associated) Antigen: Physicochemical and Immunological Characteristics George L. Le Bouvier and Robert W. McCollum Immunosuppression and Experimental Virus Infection of the Nervous System Neal Nathanson and Gerald A. Cole

AUTHOR INDEX-SUBJECT INDEX

Volume 16 Structural Defects of T-Even Bacteriophages Donald J . Cummings, Nancy L. Cause, and Gerald L. Forrest Structure and Assembly of Simple RNA Bacteriophages Thomas Hohn and Barbara Hohn The Self-Assembly of Spherical Plant Viruses J . B. Bancroft Mycoplasma Diseases of Plants and Insects Kart Maramorosch, Robert R , Granados, and Hiroyuki Hirumi Vesicular Stomatitis and Related Viruses A. F. Howatson Rabies Virus Sehchi Matsumoto 407

AUTHOR INDEX-SUBJECT INDEX

Volume 17 Virus-Erythrocyte Interactions C .Howe and L .T.Lee Tobacco Mosaic Virus with Emphasis on the Events within the Host Cell Following Infection K . K .Reddi Characteristics of Tumors Induced in Mammals, Especially Rodents, by Viruses of the Avian-Leukosis Sarcoma Group D.SimkoviE RNA-Directed DNA Synthesis and RNA Tumor Viruses Howard M . Temin and David Baltimore Growth of Arboviruses in Arthropod Tissue Culture K. R . P .Singh Small DNA Densonucleosis Virus (DNV) Edouard Kurstak Algal Viruses R .Malcolm Brown,Jr. Measles Vaccine and Its Use in Developing Countries K . Naficy and R. Nategh Viroids T. 0.Diewr AUTHOR INDEX-SUBJECT INDEX

408

CONTENTS OF RECENT VOLUMES

Volume 18 Structure of the Influenza Virion Irene T. Schulze The Polypeptides of Influenza V i s e s W. G. Laver The Mode of Action of Rifamycins and Related Compounds of Poxvirus E . A. C. Follett and T. H. Pennington Relationship of Sigma Virus to Vesicular Stomatitis Virus P. Printz Initial Effects of Viral Infection in Bacterial and Animal Host Cells Alexander Kohn and Pinhas Fuchs Nucleocapsids of Large RNA Viruses As Functionally Active Units in Transcription A . G. Bukrinskaya Plant Rhabdoviruses R. I . B. Francki The Yellowing Virus Diseases of Beet James E. Duffis AUTHOR INDEX-SUBJECT INDEX

Volume 19 The Use of Protoplasts and Separated Cells in Plant Virus Research Milton Zaitlin and Roger N . Beachy Plant Viruses with a Multipartite Genome E . M . J. Jaspars The Bromoviruses Leslie C . Lane Remarks on the Ultrastructure of Type A, B, and C Virus Particles Etienne de Harven Activation of Mammalian Leukemia Viruses Martin S. Hirsch and Paul H. Black Structural Components of RNA Tumor Viruses Dani P. Bolognesi Viruses as a Factor of Evolution: Exchange of Genetic Information in the Biosphere V .M. Zhdanov and T. I . Tikchonenko 4UTHOR INDEX-SUBJECT INDEX

Volume 20 The Structure of Tubular Viruses Roger Hull Structural Interactions Between Viruses as a Consequence of Mixed Infections J. A. Dodds and R. I . Hamilton

Protozoal Viruses Louis S. Diamond and Carl F. T. Mattern

Hepatitis in Primates Friedrich Deinhardt Oncogenic Viruses in Vertebrates Transmitted by Hematophagous Arthropods J. Rehacek, R . G. Fischer, and D. H. heeke Infection and Replication of Insect Pathogenic Viruses in Tissue Culture Robert R. Granados Insect Viruses: Serological Relationships H. M . Mazzone and G. H . T i F r Viruses Attacking the Honey Bee L. Bailey DNA Virus of Higher Plants Robert J .Shepherd AUTHOR INDEX-SUBJECT INDEX

Volume 21 Epidemiology of Fox Rabies Bernard Toma and Louis Andral Ecology of Western Equine Encephalomyelitis in the Eastern United States C. G. Hayes and R. C. Wallis The Genome of Simian Virus 40 Thomas J . Kelly, Jr. and Daniel Nathaw Plant Virus Inclusion Bodies Giovanni P. Martelli and Marcello Russo Maize Rough Dwarf and Related Viruses Robert G. Milne and Osvaldo Lowisolo Viruses of Mycoplasmas and Spiroplasmas Jack Maniloff, Jyotirmoy Das, and J. R. Christensen AUTHOR INDEX-SUBJECT INDEX

Volume 22 Mycoviruses: Viruses That Infect Fungi Michael Hollings Viruses and Virus Diseases Associated with Whiteflies Julio Bird and Karl Mammorosch The Granulosis Virus of Pieris brassicae (L.) and Its Relationship with Its Host W. A. L.David Comparative Strategies of Animal Virus Replication Howard L. Bachrach

CONTENTS OF RECENT VOLUMES Halobenzimidazole Ribosides and RNA Synthesis of Cells and Viruses Igor Tamm and Prawinkumar B. Sehgal Markers and Vaccines Miroslaw Karitoch The Interplay between Lymphocytic Choriomeningitis Virus, Immune Function, and Hemopoieses in Mice Klaus Bro-JBrgensen

409

Polyamines and Virus Multiplication Seymour S . Cohen and Frank P .MeCmmick AUTHOR INDEX-SUBJECT INDEX

Volume 25 Translation of Plant Virus Messenger RNAs J .G. Atabekov and S . Yu. Morozou The Closteroviruses: A Distinct Group of AUTHOR INDEX-SUBJECT INDEX Elongated Plant Viruses Volume 23 M . Bar-Joseph, S. M . Garnsey, and D. Guidelines for Bacteriophage CharacterizaGonsalves tion Host Passage Effects with Plant Viruses C .E . Yarwood Ham-W. Ackerrnunn, Andri A d u r i e r , Laurent Berthiaume, L i l y A . Jones, Vector Cell Monolayers and Plant Viruses John A. Mayo, and Anne K. Vidaver L. M . Black The Structural Properties and IdentiiicaThe Tobraviruses B. D. Harrison and D. J . Robinson tion of Insect Viruses The Origin of Multicomponent Small RiboK. A. Harrap and C. C .Payne nucleoprotein Viruses Adenovirus Proteins and Their Messenger L. Reijnders RNAs Small Isometric Viruses of Invertebrates Lennart Philipson J . F. Longworth Adeno-Associated Viruses Reptilia-Related Viruses K. I . Berns and W. W. Hauswirth Philip D. Lungerand H . Fred Clark Envelope Antigens of Oncoviruses Neutralization of Animal Viruses An a t oli D . Altste in and Victor M . Benjamin Mandel Zhdanov AUTHOR INDEX-SUBJECT INDEX Genetics of Resistance of Animals to Viruses: I. Introduction and Studies in Mice Fredenk B. Bang Volume 26 Sendai Virus Processing of Adenovirus Nuclear RNA to Nakao Ishida and Mono Homma mRNA AUTHOR INDEX-SUBJECT INDEX Joseph R . Nevins and Selina Chen-Kiang Persistent Viral Infections as Models for Volume 24 Research in Virus Chemotherapy Tumors and Viruses in Nonhuman Primates G. Streissle S .R . S. Rangan and R. E . Gallagher Viruses as Probes for Development and DifViruses and Parasitism in Insects ferentiation Donald B . Stoltz and S . Bradleigh VinWarren Maltzman and Arnold J . Levine son The Biology and Ecology of Strains of an InThe Formation of Virus Crystalline and sect Small RNA Virus Complex Paracrystalline Arrays for Electron MicrosP. D.Scotti, J. F. Longworth, N . Plus, copy and Image Analysis G. Croizier, and C .Reinganum R. W. Hmne Tobacco Mosaic Virus: Model for Structure and Function of a Simple Virus Early Interactions of Viruses with Cellular Membranes L. Hirth and K. E . Richards Alexander Kohn Plant-Virus Interactions Related to ResistStructural Components and Replicatin of ance and Localization of Viral Infections Arenaviruses Ilan Sela I b Ro& Pedersen AUTHOR INDEX-SUBJECT INDEX

410

CONTENTS OF RECENT VOLUMES

Volume 27 Diagnostic Virology Using Electron Microscopic Techniques A n n e M . Field Studies of Japanese Encephalitis in China C. H . Huang Planlago as a Host of Economically Important Viruses John Hammond Penetration of Viral Genetic Material into Host Cell A. G. Bukrinskaya Comparative Biology and Evolution of Bacteriophages D a r r y l C. R e a n n e y and H a n s - W . Ackemann Mechanisms of Viral Tumorigenesis Raymond V . Gilden and Harvey Rabin AUTHOR INDEX-SUBJECT INDEX

Volume 28 Genetic Engineering with Plant Viruses, and Their Potential as Vectors R. Hull and J. W. Dauies

The Molecular Biology of Coronaviruses Lawrence S. Sturman and Kathryn V. Holmes Sternorrhynchous Vectors of Plant Viruses: Virus-Vector Interactions and Transmission Mechanisms Kerry F. Harris Granulosis Viruses, with Emphasis on the GV of the Indian Meal Moth, Plodia interpunctella R. A. Consigli, K. A . Tweeten, D. K. Anderson, and L. A. Bulla, Jr. Virus Structure: High-Resolution Perspectives Stephen C. Harrison Viroids T. 0. Diener Chickenpox Virus Michiaki Takahashi Mosquitoes and the Incidence of Encephalitis Paul R. Grimstad AUTHOR INDEX-SUBJECT INDEX