Preface xvii
Acknowledgments xxi
About the Authors xxiii
Key of Repetitive Elements xxv
Part I The Science of Virology 1
1 Foundations 2
Luria's Credo 3
Viruses Defined 3
Why We Study Viruses 3
Viruses Are Everywhere 3
Viruses Infect All Living Things 4
Viruses Can Cause Human Disease 5
Viruses Can Be Beneficial 5
Viruses "R" Us 6
Viruses Can Cross Species Boundaries 6
Viruses Are Unique Tools To Study Biology 6
Virus Prehistory 7
Viral Infections in Antiquity 7
The First Vaccines 8
Microorganisms as Pathogenic Agents 9
Discovery of Viruses 11
The Defining Properties of Viruses 13
The Structural Simplicity of Virus Particles 13
The Intracellular Parasitism of Viruses 13
Cataloging Animal Viruses 18
The Classical System 18
Classification by Genome Type: the Baltimore System 19
A Common Strategy for Viral Propagation 21
Perspectives 21
References 24
Study Questions 24
2 The Infectious Cycle 26
Introduction 27
The Infectious Cycle 27
The Cell 27
Entering Cells 28
Viral RNA Synthesis 29
Viral Protein Synthesis 29
Viral Genome Replication 29
Assembly of Progeny Virus Particles 29
Viral Pathogenesis 29
Overcoming Host Defenses 30
Cultivation of Viruses 30
Cell Culture 30
Embryonated Eggs 35
Laboratory Animals 35
Assay of Viruses 35
Measurement of Infectious Units 35
Efficiency of Plating 38
Measurement of Virus Particles 40
Viral Reproduction: The Burst Concept 49
The One-Step Growth Cycle 49
One-Step Growth Analysis: a Valuable Tool for Studying Animal Viruses 52
Global Analysis 53
DNA Microarrays 54
Mass Spectrometry 56
Protein-Protein Interactions 56
Single-Cell
Virology 56
Perspectives 58
References 59
Study Questions 60
Part II Molecular Biology 61
3 Genomes and Genetics 62
Introduction 63
Genome Principles and the Baltimore System 63
Structure and Complexity of Viral Genomes 63
DNA Genomes 64
RNA Genomes 65
What Do Viral Genomes Look Like? 68
Coding Strategies 69
What Can Viral Sequences Tell Us? 69
The "Big and Small" of Viral Genomes: Does Size Matter? 71
The Origin of Viral Genomes 73
Genetic Analysis of Viruses 74
Classical Genetic Methods 75
Engineering Mutations into Viral Genomes 77
Engineering Viral Genomes: Viral Vectors 83
Perspectives 87
References 87
Study Questions 88
4 Structure 90
Introduction 91
Functions of the Virion 91
Nomenclature 92
Methods for Studying Virus Structure 92
Building a Protective Coat 95
Helical Structures 96
Capsids with Icosahedral Symmetry 99
Other Capsid Architectures 111
Packaging the Nucleic Acid Genome 112
Direct Contact of the Genome with a Protein Shell 112
Packaging by Specialized Viral Proteins 113
Packaging by Cellular Proteins 113
Viruses with Envelopes 115
Viral Envelope Components 115
Simple Enveloped Viruses: Direct Contact of External Proteins with the Capsid or Nucleocapsid 117
Enveloped Viruses with an Additional Protein Layer 118
Large Viruses with Multiple Structure Elements 119
Particles with Helical or Icosahedral Parts 120
Alternative Architectures 123
Other Components of Virions 125
Enzymes 125
Other Viral Proteins 125
Cellular Macromolecules 126
Mechanical Properties of Virus Particles 126
Investigation of Mechanical Properties of Virus Particles 126
Stabilization and Destabilization of Virus Particles 128
Perspectives 128
References 129
Study Questions 130
5 Attachment and Entry 132
Introduction 133
Attachment of Virus Particles to Cells 133
General Principles 133
Identification of Receptors for Virus Particles 135
Virus-Receptor Interactions 137
Entry into Cells 142
Virus-induced Signaling via Cell Receptors 142
Routes of Entry 143
Membrane Fusion 145
Intracellular Trafficking and Uncoating 154
Movement of Viral and Subviral Particles within Cells 154
Uncoating of Enveloped Virus Particles 155
Uncoating of Nonenveloped Viruses 155
Import of Viral Genomes into the Nucleus 159
The Nuclear Pore Complex 159
Nuclear Localization Signals 159
Nuclear Import of RNA Genomes 161
Nuclear Import of DNA Genomes 162
Import of Retroviral Genomes 162
Perspectives 164
References 165
Study Questions 166
6 Synthesis of RNA from RNA Templates 168
Introduction 169
The Nature of the RNA Template 169
Secondary Structures in Viral RNA 169
Naked or Nucleocapsid RNA 170
The RNA Synthesis Machinery 171
Identification of RNA-Dependent RNA Polymerases 171
Three-Dimensional Structures of RNA-Dependent RNA Polymerases 173
Mechanisms of RNA Synthesis 176
Initiation 176
Capping 179
Elongation 179
Functions of Additional Polymerase Domains 181
RNA Polymerase Oligomerization 181
Template Specificity 182
Unwinding the RNA Template 182
Role of Cellular Proteins 183
Paradigms for Viral RNA Synthesis 183
(+) Strand RNA 184
Synthesis of Nested Subgenomic mRNAs 184
(¿) Strand RNA 185
Ambisense RNA 189
Double-Stranded RNA 189
Unique Mechanisms of mRNA and Genome Synthesis of Hepatitis Delta Virus 190
Do Ribosomes and RNA Polymerases Collide? 192
Origins of Diversity in RNA Virus Genomes 193
Misincorporation of Nucleotides 193
Segment Reassortment and RNA Recombination 193
RNA Editing 194
Perspectives 195
References 196
Study Questions 197
7 Synthesis of RNA from DNA Templates 198
Introduction 199
Properties of Cellular RNA Polymerases That Transcribe Viral DNA 199
Some Viral Genomes Must Be Converted to Templates Suitable for Transcription 200
Transcription by RNA Polymerase II 201
Regulation of RNA Polymerase II Transcription 203
Common Properties of Proteins That Regulate Transcription 206
Transcription of Viral DNA Templates by the Cellular Machinery Alone 208
Viral Proteins That Govern Transcription of DNA Templates 209
Patterns of Regulation 209
The Human Immunodeficiency Virus Type 1 Tat Protein Autoregulates Transcription 211
The Transcriptional Cascades of DNA Viruses 217
Entry into One of Two Alternative Transcriptional Programs 226
Transcription of Viral Genes by RNA Polymerase III 230
The VA-RNA I Promoter 231
Inhibition of the Cellular Transcriptional Machinery 232
Unusual Functions of Cellular Transcription Components in Virus-Infected Cells 233
Viral DNA-Dependent RNA Polymerases 233
Perspectives 234
References 235
Study Questions 236
8 Processing 238
Introduction 239
Covalent Modification during Viral Pre-mRNA Processing 240
Capping the 5¿ Ends of Viral mRNA 240
Synthesis of 3¿ Poly(A) Segments of Viral mRNA 243
Internal Methylation of Adenosine Residues 245
Splicing of Viral Pre-mRNA 246
Regulated Processing of Viral Pre-mRNA 249
Editing of Viral mRNAs 255
Export of RNAs from the Nucleus 257
The Cellular Export Machinery 257
Export of Viral mRNA 258
Posttranscriptional Regulation of Viral or Cellular Gene Expression by Viral Proteins 262
Temporal Control of Viral Gene Expression 262
Viral Proteins Can Inhibit Cellular mRNA Production 264
Regulation of Turnover of Viral and Cellular mRNAs in the Cytoplasm 266
Intrinsic Turnover 266
Regulation of mRNA Stability by Viral Proteins 267
mRNA Stabilization Can Facilitate Transformation 267
Nonsense-Mediated mRNA Decay 267
Noncoding RNAs 271
Small Interfering RNAs and Micro-RNAs 271
Long Noncoding RNAs 276
Circular RNAs 278
Perspectives 278
References 279
Study Questions 281
9 Replication of DNA Genomes 282
Introduction 283
DNA Synthesis by the Cellular Replication Machinery 284
Eukaryotic Replicons 284
Cellular Replication Proteins 287
Mechanisms of Viral DNA Synthesis 287
Lessons from Simian Virus 40 288
Replication of Other Viral DNA Genomes 290
Properties of Viral Replication Origins 294
Recognition of Viral Replication Origins 296
Viral DNA Synthesis Machines 301
Resolution and Processing of Viral Replication Products 301
Exponential Accumulation of Viral Genomes 302
Viral Proteins Can Induce Synthesis of Cellular Replication Proteins 303
Synthesis of Viral Replication Machines and Accessory Enzymes 304
Viral DNA Replication Independent of Cellular Proteins 304
Delayed Synthesis of Structural Proteins Prevents Premature Packaging of DNA Templates 305
Inhibition of Cellular DNA Synthesis 305
Synthesis of Viral DNA in Specialized Intracellular Compartments 305
Limited Replication of Viral DNA Genomes 308
Integrated Parvoviral DNA Can Be Replicated as Part of the Cellular Genome 308
Different Viral Origins Regulate Replication of Epstein-Barr Virus 310
Limited and Amplifying Replication from a Single Origin: the Papillomaviruses 313
Origins of Genetic Diversity in DNA Viruses 315
Fidelity of Replication by Viral DNA Polymerases 315
Modulation of the DNA Damage Response 316
Recombination of Viral Genomes 318
Perspectives 321
References 321
Study Questions 323
10 Reverse Transcription and Integration 324
Retroviral Reverse Transcription 325
Discovery 325
Impact 325
The Process of Reverse Transcription 326
General Properties and Structure of Retroviral Reverse Transcriptases 334
Other Examples of Reverse Transcription 337
Retroviral DNA Integration 340
The Pathway of Integration: Integrase-Catalyzed Steps 341
Integrase Structure and Mechanism 347
Hepadnaviral Reverse Transcription 350
A DNA Virus with Reverse Transcriptase 350
The Process of Hepadnaviral Reverse Transcription 352
Perspectives 358
References 359
Study Questions 360
11 Protein Synthesis 362
Introduction 363
Mechanisms of Eukaryotic Protein Synthesis 363
General Structure of Eukaryotic mRNA 363
The Translation Machinery 364
Initiation 365
Elongation and Termination 375
The Diversity of Viral Translation Strategies 378
Polyprotein Synthesis 378
Leaky Scanning 378
Reinitiation 381
StopGo Translation 382
Suppression of Termination 382
Ribosomal Frameshifting 383
Bicistronic mRNAs 384
Regulation of Translation during Viral Infection 385
Inhibition of Translation Initiation after Viral Infection 385
Regulation of eIF4F 389
Regulation of Poly(A)-Binding Protein Activity 392
Regulation of eIF3 392
Interfering with RNA 392
Stress-Associated RNA Granules 393
Perspectives 395
References 396
Study Questions 397
12 Intracellular Trafficking 398
Introduction 399
Assembly within the Nucleus 400
Import of Viral Proteins for Assembly 401
Assembly at the Plasma Membrane 403
Transport of Viral Membrane Proteins to the Plasma Membrane 404
Sorting of Viral Proteins in Polarized Cells 419
Disruption of the Secretory Pathway in Virus-Infected Cells 421
Signal Sequence-Independent Transport of Viral Proteins to the Plasma Membrane 422
Interactions with Internal Cellular Membranes 426
Localization of Viral Proteins to Compartments of the Secretory Pathway 426
Localization of Viral Proteins to the Nuclear Membrane 426
Transport of Viral Genomes to Assembly Sites 427
Transport of Genomic and Pregenomic RNA from the Nucleus to the Cytoplasm 427
Transport of Genomes from the Cytoplasm to the Plasma Membrane 429
Perspectives 430
References 431
Study Questions 432
13 Assembly, Release, and Maturation 434
Introduction 435
Methods of Studying Virus Assembly and Egress 435
Structural Studies of Virus Particles 436
Visualization of Assembly and Exit by Microscopy 436
Biochemical and Genetic Analyses of Assembly Intermediates 436
Methods Based on Recombinant DNA Technology 439
Assembly of Protein Shells 439
Formation of Structural Units 439
Capsid and Nucleocapsid Assembly 441
Self-Assembly and Assisted Assembly Reactions 445
Selective Packaging of the Viral Genome and Other Components of Virus Particles 447
Concerted or Sequential Assembly 447
Recognition and Packaging of the Nucleic Acid Genome 448
Incorporation of Enzymes and Other Nonstructural Proteins 458
Acquisition of an Envelope 459
Sequential Assembly of Internal Components and Budding from a Cellular Membrane 459
Coordination of the Assembly of Internal Structures with Acquisition of the Envelope 460
Release of Virus Particles 460
Assembly and Budding at the Plasma Membrane 461
Assembly at Internal Membranes: the Problem of Exocytosis 464
Release of Nonenveloped Virus Particles 470
Maturation of Progeny Virus Particles 470
Proteolytic Processing of Structural Proteins 470
Other Maturation Reactions 474
Cell-to-Cell Spread 475
Perspectives 479
References 479
Study Questions 481
14 The Infected Cell 482
Introduction 483
Signal Transduction 483
Signaling Pathways 483
Signaling in Virus-Infected Cells 485
Gene Expression 489
Inhibition of Cellular Gene Expression 489
Differential Regulation of Cellular Gene Expression 492
Metabolism 496
Methods To Study Metabolism 496
Glucose Metabolism 497
The Citric Acid Cycle 501
Electron Transport and Oxidative Phosphorylation 502
Lipid Metabolism 504
Remodeling of Cellular Organelles 507
The Nucleus 509
The Cytoplasm 511
Perspectives 516
References 518
Study Questions 519
Appendix Structure, Genome Organization, and Infectious Cycles of Viruses Featured in This Book 521
Glossary 557
Index 563
