Basic Virology

Third
Edition
Edward K. Wagner
Department of Molecular Biology and Biochemistry
University of California, Irvine

MartinezJ. Hewlett
Department of Molecular and Cellular Biology
University of Arizona

David C. Bloom
Department of Molecular Genetics and Microbiology
University of Florida

David Camerini
Department of Molecular Biology and Biochemistry
University of California, Irvine

Blackwell
Publishing

Brief Contents

Preface xxi
Acknowledgments

xxix

PARTI

VIROLOGY AND VIRAL DISEASE

Chapter
Chapter
Chapter
Chapter

Introduction The Impact of Viruses on Our View of Life 3

An Outline of Virus Replication and Viral Pathogenesis 15
Virus Disease in Populations and Individual Animals 27
Patterns of Some Viral Diseases of Humans 41
*

1
2
3
4

PART II BASIC PROPERTIES OF VIRUSES AND VIRUSCELL INTERACTION 63

Chapter 5
Chapter 6
Chapter 7
Chapter 8

PART III

Virus Structure and Classification 65

The Beginning and End of the Virus Replication Cycle 79
Host Immune Response to Viral Infection - The Nature of the Vertebrate
Immune Response 97
Strategies to Protect Against and Combat Viral Infection 119

WORKING W I T H VIRUS

145

Chapter 9
Visualization and Enumeration of Virus Particles 147
Chapter 10 Replicating and Measuring Biological Activity of Viruses 155
Chapter 11 Physical and Chemical Manipulation of the Structural Components of
Viruses 173
Chapter 12 Characterization of Viral Products Expressed in the Infected Cell 193
Chapter 13 Viruses Use Cellular Processes to Express their Genetic
Information 213

PART IV
VIRUSES
Chapter 14
Chapter 15
Chapter 16

R E P L I C A T I O N PATTERNS O F SPECIFIC
243
Replication of Positive-sense RNA Viruses 245
Replication Strategies of RNA Viruses Requiring RNA-directed mRNA
Transcription as the First Step in Viral Gene Expression 273
Replication Strategies of Small and Medium-sized DNA Viruses 303

VI

BRIEF CONTENTS

Chapter 17
Chapter 18
Chapter 19
Chapter 20
Chapter 21

Replication of Some Nuclear-replicating Eukaryotic DNA Viruses with

Large Genomes 331
Replication of Cytoplasmic DNA Viruses and "Large" Bacteriophages 359
Retroviruses: Converting RNA to DNA 381
Human Immunodeficiency Virus Type 1 (HIV-1) and Related
Lentiviruses 399
Hepadnaviruses: Variations on the Retrovirus Theme 413

PART V VIRUSES: NEW APPROACHES AND NEW

PROBLEMS 433
Chapter
Chapter
Chapter
Chapter

22
23
24
25

The Molecular Genetics of Viruses 435

Molecular Pathogenesis 463
Viral Bioinformatics and Beyond 473
Viruses and the Future - Problems and Promises

Appendix
Resource Center
Technical Glossary 507
Index 533

501

485

Contents

Preface xxi
Preface to the second edition xxii
Preface to the third edition xxii
Text organization xxiii
Specific features of this text designed to aid instructors and
students in pursuing topics in greater depth xxvi
Depth of coverage xxvi
Sources for further study xxvi
The Internet xxvii
Chapter outlines xxvii
Case studies xxvii
Review material xxvii
Glossary xxvii
Acknowledgments xxix

PART I

VIROLOGY AND VIRAL DISEASE

CHAPTER 1

Introduction - The Impact of Viruses on Our View of Life 3

The science of virology 3
The effect of virus infections on the host organism and populations
viral pathogenesis, virulence, and epidemiology 4
The interaction between viruses and their hosts 6
The history of virology 7
Examples of the impact of viral disease on human history 8
Examples of the evolutionary impact of the virus-host
interaction 9
The origin of viruses 9
Viruses have a constructive as well as destructive impact on
society 12
Viruses are not the smallest self-replicating pathogens 13
Questions for Chapter 1 14

CHAPTER 2

An Outline of Virus Replication and Viral Pathogenesis

Virus replication 15
Stages of virus replication in the cell 17
Pathogenesis of viral infection 19
Stages of virus-induced pathology 19

15

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CONTENTS

Initial stages of infection - entry of the virus into the host 20

The incubation period and spread of virus through the host 21
Multiplication of virus to high levels occurrence of disease
symptoms 23
The later stages of infection the immune response 24
The later stages of infection virus spread to the next
individual 24
The later stages of infection fate of the host 24
Questions for Chapter 2 25

CHAPTER 3

Virus Disease in Populations and Individual Animals 27

The nature of virus reservoirs 27
Some viruses with human reservoirs 28
Some viruses with vertebrate reservoirs 30
Viruses in populations 30
Viral epidemiology in small and large populations 30
Factors affecting the control of viral disease in populations 33
Animal models to study viral pathogenesis 34
A mouse model for studying poxvirus infection and spread 35
Rabies: where is the virus during its long incubation period? 37
Herpes simplex virus latency 37
Murine models 39
Rabbit models 40
Guinea pig models 40
Questions for Chapter 3 40

CHAPTER 4

Patterns of Some Viral Diseases of Humans 41

The dynamics of human-virus interactions 42
The stable association of viruses with their natural host places
specific constraints on the nature of viral disease and mode of
persistence 42
Classification of human disease-causing viruses according to
virushost dynamics 44
Viral diseases leading to persistence of the virus in the host are
generally associated with viruses having long associations with
human populations 44
Viral diseases associated with acute, severe infection are
suggestive of zoonoses 48
Patterns of specific viral diseases of humans 49
Acute infections followed by virus clearing 49
Colds and respiratory infections 49
Influenza 49
Variola 49
Infection of an "accidental" target tissue leading to permanent
damage despite efficient clearing 50
Persistent viral infections 50
Papilloma and polyomavirus infections 50
Herpesvirus infections and latency 52
Other complications arising from persistent infections 52
Viral and subviral diseases with long incubation periods 53
Rabies 53

CONTENTS

HIV-AIDS 53
Prion diseases 54
Some viral infections targeting specific organ systems 54
Viral infections of nerve tissue 54
Examples of viral encephalitis with grave prognosis 55
Rabies 55
Herpes encephalitis 55
Viral encephalitis with favorable prognosis for recovery 56
Viral infections of the liver (viral hepatitis) 56
Hepatitis A 57
Hepatitis B 57
Hepatitis C 57
Hepatitis D 57
Hepatitis E 58
Questions for Chapter 4 58
Problems for Part I 59
Additional Reading for Part I 61

PART II

BASIC PROPERTIES OF VIRUSES AND

VIRUS-CELL INTERACTION 63

CHAPTER 5

Virus Structure and Classification

65

The features of a virus 65

Viral genomes 69
Viral capsids 69
Viral envelopes 72
Classification schemes 72
The Baltimore scheme of virus classification 75
Disease-based classification schemes for viruses 75
The virosphere 77
Questions for Chapter 5 78

CHAPTER 6

The Beginning and End of the Virus Replication Cycle 79

Outline of the virus replication cycle 79
Viral entry 80
Animal virus entry into cells the role of the cellular receptor 80
Mechanisms of entry of nonenveloped viruses 83
Entry of enveloped viruses 84
Entry of virus into plant cells 85
Injection of bacteriophage DNA into Escherichia coli 87
Nonspecific methods of introducing viral genomes into cells 89
Late events in viral infection: capsid assembly and virion release 89
Assembly of helical capsids 89
Assembly of icosahedral capsids 92
Generation of the virion envelope and egress of the enveloped
virion 93
Questions for Chapter 6 96

CHAPTER 7

Host Immune Response to Viral Infection - The Nature of the

Vertebrate Immune Response 97
The innate immune response - early defense against pathogens 98
Toll-like receptors 99

IX

CONTENTS

Defensins 99
The adaptive immune response and the lymphatic system 100
Two pathways of helper T response - the fork in the road 101
The immunological structure of a protein 102
Role of the antigen-presenting cell in initiation of the immune
response 104
Clonal selection of immune reactive lymphocytes 107
Immune memory 108
Complement-mediated cell lysis 108
Control and dysfunction of immunity 108
Specific viral responses to host immunity 109
Passive evasion of immunity antigenic drift 110
Passive evasion of immunity internal sanctuaries for
infectious virus 110
Passive evasion of immunity immune tolerance 110
Active evasion of immunity - immunosuppression 111
Active evasion of immunity - blockage of MHC antigen
presentation 111
Consequences of immune suppression to virus infections 112
Measurement of the immune reaction 112
Measurement of cell-mediated (T-cell) immunity 112
Measurement of antiviral antibody 112
Enzyme-linked immunosorbent assays (ELISAs) 113
Neutralization tests 114
Inhibition of hemagglutination 114
Complement fixation 115
Questions for Chapter 7 ^117
C H A PTE R 8

Strategies to Protect Against and Combat Viral Infection

Vaccination induction of immunity to prevent virus
infection 120
Antiviral vaccines 120
Smallpox and the history of vaccination 120
How a vaccine is produced 122
Live-virus vaccines 122
Killed-virus vaccines 123
Recombinant virus vaccines 124
Capsid and subunit vaccines 124
DNA vaccines 125
Edible vaccines 125
Problems with vaccine production and use 125
Eukaryotic cell-based defenses against virus replication 126
Interferon 126
Induction of interferon 127
The antiviral state 128
Measurement of interferon activity 128
Other cellular defenses against viral infection 130
Small RNA-based defenses 130
Enzymatic modification of viral genomes 131
Antiviral drugs 131

119

CONTENTS

Targeting antiviral drugs to specific features of the virus

replication cycle 131
Acyclovir and the herpesviruses 132
Blocking influenza virus entry and virus maturation 132
Chemotherapeutic approaches for HIV 134
Multiple drug therapies to reduce or eliminate mutation to
drug resistance 134
Other approaches 135
Bacterial antiviral systems - restriction endonucleases 135
Questions for Chapter 8 136
Problems for Part II 139
Additional Reading for Part II 143

PART III

WORKING WITH VIRUS

145

CHAPTER 9

Visualization and Enumeration of Virus Particles 147

Using the electron microscope to study and count viruses 147
Counting (enumeration) of virions with the electron
microscope 149
Atomic force microscopy a rapid and sensitive method for
visualization of viruses and infected cells, potentially in real
time 151
Indirect methods for "counting" virus particles 152
Questions for Chapter 9 154

CHAPTER 10

Replicating and Measuring Biological Activity of Viruses 155

Cell culture techniques 156
Maintenance of bacterial cells 156
Plant cell cultures 156
Culture of animal and human cells 157
Maintenance of cells in culture 157
Types of cells 157
Loss of contact inhibition of growth and immortalization of
primary cells 159
The outcome of virus infection in cells 160
Fate of the virus 160
Fate of the cell following virus infection 162
Cell-mediated maintenance of the intra- and intercellular
environment 162
Virus-mediated cytopathology changes in the physical
appearance of cells 163
Virus-mediated cytopathology changes in the biochemical
properties of cells 163
Measurement of the biological activity of viruses 164
Quantitative measure of infectious centers 164
Plaque assays 164
Generation of transformed cell foci 165
Use of virus titers to quantitatively control infection conditions 166
Examples of plaque assays 167
Statistical analysis of infection 168

XI

XII

CONTENTS

Dilution endpoint methods 169

The relation between dilution endpoint and infectious units of
virus 169
Questions for Chapter 10 170

CHAPTER 11

Physical and Chemical Manipulation of the Structural

Components of Viruses 173
Viral structural proteins 173
Isolation of structural proteins of the virus 174
Size fractionation of viral structural proteins 176
Determining the stoichiometry of capsid proteins 177
The poliovirus capsid - a virion with equimolar capsid
proteins 178
Analysis of viral capsids that do not contain equimolar numbers
of proteins 179
Characterizing viral genomes 179
Sequence analysis of viral genomes 180
Measuring the size of viral genomes 184
Direct measure of DNA genome lengths in the electron
microscope 185
Rate zonal sedimentation and gel electrophoresis for measuring
viral genome size 185
The polymerase chain reaction detection and characterization of
extremely small quantities of viral genomes or transcripts 187
Real time PCR for precise quantitative measures of viral
DNA 189
PCR detection of RNA 190
PCR as an epidemiological tool 190
Questions for Chapter 11 191

CHAPTER 12

Characterization of Viral Products Expressed in the Infected

Cell 193
Characterization of viral proteins in the infected cell 193
Pulse labeling of viral proteins at different times following
infection 194
Use of immune reagents for study of viral proteins 195
Working with antibodies 196
Detection of viral proteins using immunofluorescence 198
Related methods for detecting antibodies bound to
antigens 201
Detecting and characterizing viral nucleic acids in infected
cells 205
Detecting the synthesis of viral genomes 205
Characterization of viral mRNA expressed during infection 205
In situ hybridization 207
Further characterization of specific viral mRNA
molecules 209
Use of microarray technology for getting a complete picture of the
events occurring in the infected cell 210
Questions for Chapter 12 212

CONTENTS

CHAPTER 13

Viruses Use Cellular Processes to Express Their Genetic

Information 213
Prokaryotic DNA replication is an accurate enzymatic model for
the process generally 215
The replication of eukaryotic DNA 216
The replication of viral DNA 217
The effect of virus infection on host DNA replication 217
Expression of mRNA 217
Prokaryotic transcription 219
Prokaryotic RNA polymerase 219
The prokaryotic promoter and initiation of
transcription 220
Control of prokaryotic initiation of transcription 220
Termination of prokaryotic transcription 221
Eukaryotic transcription 221
The promoter and initiation of transcription 221
Control of initiation of eukaryotic transcription 223
Processing of precursor mRNA 224
Visualization and location of splices in eukaryotic
transcripts 226
Posttranscriptional regulation of eukaryotic mRNA
function 231
Virus-induced changes in transcription and posttranscriptional
processing 232
The mechanism of protein synthesis 232
Eukaryotic translation 233
Prokaryotic translation 234
Virus-induced changes in translation 236
Questions for Chapter 13 236
Problems for Part III 239
Additional Reading for Part III 241

PART IV

REPLICATION PATTERNS OF SPECIFIC

VIRUSES 243

CHAPTER 14

Replication of Positive-sense RNA Viruses 245

RNA viruses general considerations 246
A general picture of RNA-directed RNA replication 246
Replication of positive-sense RNA viruses whose genomes are
translated as the first step in gene expression 248
Positive-sense RNA viruses encoding a single large open reading
frame 249
Picornavirus replication 249
The poliovirus genetic map and expression of poliovirus
proteins 249
The poliovirus replication cycle 252
Picornavirus cytopathology and disease 254
Flavivirus replication 256
Positive-sense RNA viruses encoding more than one translational
reading frame 257

XIII

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CONTENTS

Two viral mRNAs are produced in different amounts during

togavirus infections 258
The viral genome 258
The virus replication cycle 258
Togavirus cytopathology and disease 262
A somewhat more complex scenario of multiple translational
reading frames and subgenomic mRNA expression: coronavirus
replication 263
Coronavirus replication 264
Cytopathology and disease caused by coronaviruses 266
Replication of plant viruses with RNA genomes 267
Viruses with one genome segment 268
Viruses with two genome segments 268
Viruses with three genome segments 269
Replication of bacteriophage with RNA genomes 269
Regulated translation of bacteriophage mRNA 269
Questions for Chapter 14 272
C H A PT E R 1 5

Replication Strategies of RNA Viruses Requiring RNA-directed

mRNA Transcription as the First Step in Viral Gene
Expression 273
Replication of negative-sense RNA viruses with a monopartite
genome 275
Replication of vesicular stomatitis virus - a model for
Mononegavirales 275
Vesicular stomatitis virus virion and genome 275
Generation, capping, and polyadenylation of mRNA 276
Generation of new negative-sense virion RNA 278
Mechanism of host shutoff by vesicular stomatitis virus 279
Cytopathology and diseases caused by rhabdoviruses 280
Paramyxoviruses 280
Pathogenesis of paramyxoviruses 280
Filoviruses and their pathogenesis 282
Bornaviruses 282
Influenza viruses - negative-sense RNA viruses with a
multipartite genome 283
Involvement of the nucleus in flu virus replication 284
Generation of new flu nucleocapsids and maturation of the
virus 285
Influenza A epidemics 285
Other negative-sense RNA viruses with multipartite
genomes 288
Bunyaviruses 288
Virus structure and replication 288
Pathogenesis 291
Arenaviruses 291
Virus gene expression 292
Pathogenesis 292
Viruses with double-stranded RNA genomes 292

CONTENTS

Reovirus structure 292

Reovirus replication cycle 294
Pathogenesis 295
Subviral pathogens 295
Hepatitis delta virus 296
Viroids 297
Prions 298
Questions for Chapter' 15 301

CHAPTER 16

Replication Strategies of Small and Medium-Sized DNA

Viruses 303
DNA viruses express genetic information and replicate their
genomes in similar, yet distinct, ways 304
Papovavirus replication 305
Replication of SV40 virus - the model polyomavirus 305
The SV40 genome and genetic map 309
Productive infection by SV40 310
Abortive infection of cells nonpermissive for SV40
replication 312
Replication of papillomaviruses 314
The HPV-16 genome 316
Virus replication and cytopathology 316
Replication of adenoviruses 319
Physical properties of adenovirus 319
Capsid structure 319
The adenovirus genome 319
The adenovirus replication cycle 319
Early events 319
Adenovirus DNA replication 321
Late gene expression 321
VA transcription and cytopathology 321
Transformation of nonpermissive cells by adenovirus 323
Replication of some single-stranded DNA viruses 323
Replication of parvoviruses 323
Dependovirus DNA integrates in a specific site in the host cell
genome 324
Parvoviruses have potentially exploitable therapeutic
applications 325
DNA viruses infecting vascular plants 325
Geminiviruses 325
Single-stranded DNA bacteriophage OX174 packages its genes
very compactly 326
Questions for Chapter 16 328

CHAPTER 17

Replication of Some Nuclear-Replicating Eukaryotic DNA Viruses

with Large Genomes 331
Herpesvirus replication and latency 332
The herpesviruses as a group 332
Genetic complexity of herpesviruses 333

XV

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CONTENTS

Common features of herpesvirus replication in the host 333

The replication of the prototypical alpha-herpesvirus HSV 334
The HSV virion 334
The viral genome 334
HSV productive infection 338
HSV latency and LAT 347
HSV transcription during latency and reactivation 348
How do LAT and other specific HSV genes function - may be
to accommodate reactivation? 350
EBV latent infection of lymphocytes, a different set of problems
and answers 351
Pathology of herpesvirus infections 354
Herpesviruses as infectious co-carcinogens 354
Baculovirus, an insect virus with important practical uses in
molecular biology 355
Virion structure 355
Viral gene expression and genome replication 356
Pathogenesis 356
Importance of baculoviruses in biotechnology 356
Questions for Chapter 17 357
C H A P T E R 18

Replication of Cytoplasmic DNA Viruses and "Large"

Bacteriophages 359
Poxviruses DNA viruses that replicate in the cytoplasm of
eukaryotic cells 360
The pox virion is complex and contains virus-coded transcription
enzymes 360
The poxvirus replication cycle 361
Early gene expression 363
Genome replication 363
Intermediate and late stages of replication 363
Pathogenesis and history of poxvirus infections 364
Is smallpox virus a potential biological terror weapon? 364
Replication of "large" DNA-containing bacteriophages 365
Components of large DNA-containing phage virions 365
Replication of phage T7 367
The genome 367
Phage-controlled transcription 367
The practical value of T7 367
T4 bacteriophage: the basic model for all DNA viruses 367
The T4 genome 368
Regulated gene expression during T4 replication 369
Capsid maturation and release 370
Replication of phage X: a "simple" model for latency and
reactivation 370
The phage X genome 372
Phage X gene expression immediately after infection 372
Biochemistry of the decision between lytic and lysogenic
infection in E. coli 375

CONTENTS

A group of algal viruses shares features of its genome structure with

poxviruses and bacteriophages 376
Questions for Chapter 18 377

CHAPTER 19

Retroviruses: Converting RNA to DNA 381

Retrovirus families and their strategies of replication 382
The molecular biology of retrovirus 383
Retrovirus structural proteins 383
The retrovirus genome 384
Genetic maps of representative retroviruses 386
Replication of retroviruses: an outline of the replication
process 386
Initiation of infection 386
Capsid assembly and maturation 389
Action of reverse transcriptase and RNase-H in synthesis of
cDNA 389
Virus gene expression, assembly, and maturation 391
Transcription and translation of viral mRNA 391
Capsid assembly and morphogenesis 391
Mechanisms of retrovirus transformation 392
Transformation through the action of a viral oncogene a
subverted cellular growth control gene 392
Oncornavirus alteration of normal cellular transcriptional control
of growth regulation 393
Oncornavirus transformation by growth stimulation of
neighboring cells 395
Cellular genetic elements related to retroviruses 395
Retrotransposons 396
The relationship between transposable elements and viruses 397
Questions for Chapter 19 397

CHAPTER 20

Human Immunodeficiency Virus Type 1 (HIV-1) and Related

Lentiviruses 399
HIV-1 and related lentiviruses 399
The origin of HIV-1 and AIDS 399
HIV-1 and lentiviral replication 400
Destruction of the immune system by HIV-1 406
Questions for Chapter 20 411

CHAPTER 21

Hepadnaviruses: Variations on the Retrovirus Theme 413

The virion and the viral genome 413
The viral replication cycle 415
The pathogenesis of hepatitis B virus 415
A plant "hepadnavirus": cauliflower mosaic virus 416
Genome structure 416
Viral gene expression and genome replication 416
The evolutionary origin of hepadnaviruses 417
Questions for Chapter 21 419
Problems for Part IV 421
Additional Reading for Part IV 429

XVII

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CONTENTS

PART V

VIRUSES: NEW APPROACHES AND NEW

PROBLEMS 433

CHAPTER 22

The Molecular Genetics of Viruses 435

Mutations in genes and resulting changes to proteins 437
Analysis of mutations 438
Complementation 438
Recombination 439
Isolation of mutants 440
Selection 440
HSV thymidine kinase - a portable selectable marker 440
Screening 441
A tool kit for molecular virologists 441
Viral genomes 441
Locating sites of restriction endonuclease cleavage on the viral
genome - restriction mapping 442
Cloning vectors 443
Cloning of fragments of viral genomes using bacterial
plasmids 444
Cloning using phage X 449
Cloning single-stranded DNA with bacteriophage M13 451
DNA animal virus vectors 451
RNA virus expression systems 453
Defective virus particles 454
Directed mutagenesis of viral genes 454
Site-directed mutagenesis 456
Generation of recombinant viruses 456
Bacterial artificial chromosomes 458
Questions for Chapter 22 461

CHAPTER 23

Molecular Pathogenesis 463

An introduction to the study of viral pathogenesis 463
Animal models 464
Choosing a model: natural host vs. surrogate models 464
Development of new models: transgenic animals 464
Hybrid models: the SCID-hu mouse 464
Considerations regarding the humane use of animals 465
Methods for the study of pathogenesis 466
Assays of virulence 466
Analysis of viral spread within the host 467
Resolving the infection to the level of single cells 470
Characterization of the host response 470
Immunological assays 470
Use of transgenic mice to dissect critical components of the host
immune response that modulate the viral infection 471
Question for Chapter 23 471

CHAPTER 24 Viral Bioinformatics and Beyond 473

Bioinformatics 473
Bioinformatics and virology
Biological databases 474

473

CONTENTS

Primary databases 474

Secondary databases 475
Composite databases 475
Other databases 476
Biological applications 476
Similarity searching tools 476
Protein functional analysis 478
Sequence analysis ,478
Structural modeling 478
Structural analysis 478
Systems biology and viruses 479
Viral internet resources 481
Questions for Chapter 24 484

CHAPTER 25

Viruses and the Future - Problems and Promises 485

Clouds on the horizon emerging disease 485
Sources and causes of emergent virus disease 488
The threat of bioterrorism 489
What are the prospects of using medical technology to eliminate
specific viral and other infectious diseases? 490
Silver linings viruses as therapeutic agents 490
Viruses for gene delivery 491
Using viruses to destroy other viruses 493
Viruses and nanotechnology 493
The place of viruses in the biosphere 494
Why study virology? 494
Questions for Chapter 25 495
Problems for Part V 497
Additional Reading for Part V 499

APPENDIX

Resource Center 501

Books of historical and basic value 501
Books on virology 501
Molecular biology and biochemistry texts 503
Detailed sources 503
Sources for experimental protocols 503
The Internet 504
Virology sites 504
Important websites for organizations and facilities of
interest 505

Technical Glossary
Index 533

507

XIX