Principles of Virology, Volume 1

Jane Flint is Professor Emerita of Molecular Biology at Princeton University. Dr. Flint's research focused on investigation of the mechanisms by which viral gene products modulate host pathways and antiviral defenses to allow efficient reproduction in normal human cells of adenoviruses, viruses that are used in such therapeutic applications as gene transfer and cancer treatment. Vincent R. Racaniello is Higgins Professor of Microbiology & Immunology at Columbia University Vagelos College of Physicians & Surgeons. Dr. Racaniello has been studying viruses for over 40 years, including polio- virus, rhinovirus, enteroviruses, hepatitis C virus, and Zika virus. He blogs about virus-es at virology.ws and is host of This Week in Virology. Glenn F. Rall is a Professor and the Chief Academic Officer at the Fox Chase Cancer Center, and is an Adjunct Professor in the Microbiology and Immunology departments at the University of Pennsylvania, as well as Thomas Jefferson, Drexel, and Temple Universities. Dr. Rall studies viral infections of the brain and the immune responses to those infections, with the goal of defining how viruses contribute to disease. Theodora Hatziioannou is a Research Associate Professor at Rockefeller University and is actively involved in teaching programs at Albert Einstein College of Medicine. Dr. Hatziioannou has worked on multiple viruses with a focus on retroviruses and the molecular mechanisms that govern virus tropism and on the improvement of animal models for human disease. Anna Marie Skalka is a Professor Emerita and former Senior Vice President for Basic Research at the Fox Chase Cancer Center. Dr. Skalka is internationally recognized for her contributions to the understanding of the biochemical mechanisms by which retroviruses replicate and insert their genetic material into the host genome, as well as her research into other molecular aspects of retrovirus biology.

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