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BRIEF CONTENTS

SECTION I: INTRODUCTION 11. 125 TO VIROLOGY Bert L. Semler, University of California, Irvine

1 2. 137 1. Introduction to Virology 2 Richard Kuhn, Purdue University Nicholas H. Acheson, McGill University 1 3. Togaviruses 148 2. Structure and Assembly 18 Milton Schlesinger, Washington University Stephen C. Harrison, Harvard University in St. Louis Sondra Schlesinger, Washington University 3. : The World in St. Louis of 31 Revised by: Richard Kuhn, Purdue University Nicholas H. Acheson, McGill University 1 4. Coronaviruses 159

4. Virus Entry 45 Mark Denison, Vanderbilt University Ari Helenius, Swiss Federal Institute of Michelle M. Becker, Vanderbilt University Technology, Zurich

SECTION II: VIRUSES OF SECTION IV: NEGATIVE-STRAND AND AND DOUBLE-STRANDED RNA VIRUSES OF 5. Single-Stranded RNA 59 1 5. Paramyxoviruses and Rhabdoviruses 175 Jan van Duin, University of Leiden Nicholas H. Acheson, McGill University 6. Microviruses 69 Daniel Kolakofsky, University of Geneva Christopher Richardson, Dalhousie University Bentley Fane, University of Arizona Revised by: Laurent Roux, University of Geneva

7. T7 77 1 6. Filoviruses 188 William C. Summers, Yale University Heinz Feldmann, Division of Intramural Research, MAID, NIH 8. Bacteriophage Lambda 85 Hans-Dieter Klenk, University of Marburg Michael Feiss, University of Iowa Nicholas H. Acheson, McGill University

9. Viruses of Archaea 97 17. Bunyaviruses 200

David Prangishvili, Institut Pasteur Richard M. Elliott, University of St. Andrews

SECTION III: POSITIVE-STRAND 1 8. Influenza Viruses 210 RNA VIRUSES OF EUKARYOTES DaliusJ. Briedis, McGill University

1 O. Cucumber Mosaic Virus 112 1 9. Reoviruses 225

Ping XΜ, J. Noble Research Institute Terence S. Dermody, Vanderbilt University Marilyn J. Roosinck, J. Noble Research Institute James D. Chappell, Vanderbilt University VI Brief Contents

SECTION V: SMALL DNA VIRUSES 29. Human Immunodeficiency OF EUKARYOTES Virus 354

Alan Cochrane, University of Toronto 20. Parvoviruses 238

Peter Beard, Swiss Institute for Experimental 30. Hepadnaviruses 365 Cancer Research Christopher Richardson, Dalhousie University

21. Polyomaviruses 247 SECTION VIII: Nicholas H. Acheson, McGill University AND

22. Papillomaviruses 263 31. Viroids and Hepatitis Greg Matlashewski, McGill University Delta Virus 378 Revised by: Lawrence Banks, International Jean-Pierre Perreault, Universite de Sherbrooke Centre for Genetic Engineering and Martin Pelchat, University of Ottawa Biotechnology, Trieste 32. Prions 387

DaliusJ. Briedis, McGill University SECTION VI: LARGER DNA VIRUSES OF EUKARYOTES SECTION IX: HOST DEFENSES 23. Adenoviruses 274 AGAINST VIRUS INFECTION Philip Branton, McGill University Richard C. Marcellus, McGill University 33. Intrinsic Cellular Defenses Against Virus Infection 398

24. Herpesviruses 285 Karen Mossman, McMaster University Pierre Genin, University Paris Descartes Bernard Roizman, University of Chicago John Hiscott, McGill University Gabriella Campadelli-Fiume, University of Bologna Richard Longnecker, Northwestern University 34. Innate and Adaptive Immune Responses to Virus Infection 415 25. Baculoviruses 302 Malcolm G. Baines, McGill University Eric B. Carstens, Queen s University Karen Mossman, McMaster University

26. Poxviruses 312

Richard C. Condit, University of Florida SECTION X: ANTIVIRAL AGENTS AND VIRUS VECTORS

27. Viruses of and 35. Antiviral Vaccines 428 Mimi virus 325 Brian Ward, McGill University Michael J. Allen, Plymouth Marine Laboratory William H. Wilson, Bigeloro Laboratory for Ocean Sciences 36. Antiviral Chemotherapy 444 Donald M. Coen, Harvard University

SECTION VII: VIRUSES THAT USE 37. Eukaryotic Virus Vectors 456 A REVERSE TRANSCRIPTASE Renald Gilbert, NRC Biotechnology Research Institute, Montreal 28. 342 Bernard Massie, NRC Biotechnology Research Institute, Alan Cochrane, University of Toronto Montreal CONTENTS

SECTION I: INTRODUCTION Analysis of viral macromolecules reveals the detailed TO VIROLOGY pathways of virus replication 13 STEPS IN THE VIRUS 1. Introduction to Virology 2 REPLICATION CYCLE 13 1. Virions bind to receptors on the cell surface 13 THE NATURE OF VIRUSES 3 2. The virion (or the viral genome) enters the cell 14 Viruses consist of a nucleic acid genome packaged in a 3. Early viral genes are expressed: the Baltimore protein coat 3 classification of viruses 14 Viruses are dependent on living cells for their replication The seven groups in the system 14 Virus particles break down and release their 4. Early viral proteins direct replication of viral genomes 15 genomes inside the cell 3 5. Late messenger RNAs are made from newly Virus genomes are either RNA or DNA, but not both 4 replicated genomes 15 WHY STUDY VIRUSES? 4 6. Late viral proteins package viral genomes and Viruses are important disease-causing agents 4 assemble virions 16 Viruses can infect all forms of 4 7. Progeny virions are released from the host cell 16 Viruses are the most abundant form of life on Earth 5 The study of viruses has led to numerous discoveries in 2. Virus Structure and Assembly 18 molecular and cell biology 5 BASIC CONCEPTS OF VIRUS A BRIEF : STRUCTURE 18 TH E STU DY OF VI RUSES 6 Virus structure is studied by electron microscopy The scientific study of viruses is very recent 6 and X-ray diffraction 19 Viruses were first distinguished from other Many viruses come in simple, symmetrical packages 19 microorganisms by filtration 6 The crystallization of tobacco mosaic virus challenged WITH ICOSAHEDRAL conventional notions about genes and the nature SYMMETRY 21 of living organisms 6 Some examples of virions with icosahedral symmetry The "phage group" stimulated studies of bacteriophages The concept of quasi-equivalence 21 and helped establish the field of molecular biology 7 Larger viruses come in more complex packages 2 3 Study of tumor viruses led to discoveries in molecular CAPSIDS WITH HELICAL SYMMETRY 25 biology and understanding of the nature of cancer 8 VIRAL ENVELOPES 26 DETECTION AND TITRATION OF VIRUSES Viral envelopes are made from lipid bilayer membranes 26 Most viruses were first detected and studied by Viral glycoproteins are inserted into the lipid infection of intact organisms 9 membrane to form the envelope 2 7 The plaque assay arose from work with bacteriophages 9 Eukaryotic cells cultured in vitro have been adapted PACKAGING OF GENOMES AND VIRION for plaque assays 9 ASSEMBLY 28 Hemagglutination is a convenient and rapid assay Multiple modes of assembly 2 8 for many viruses 10 Specific packaging signals direct incorporation of Virus particles can be seen and counted by electron viral genomes into virions 2 8 microscopy 10 Core proteins may accompany the viral genome The ratio of physical virus particles to infectious inside the capsid 28 particles can be much greater than 1 11 Formation of viral envelopes by budding is driven by interactions between viral proteins 2 8 THE VIRUS REPLICATION CYCLE: AN OVERVIEW 11 DISASSEMBLY OF VIRIONS: THE DELIVERY The single-cycle virus replication experiment 11 OF VIRAL GENOMES TO THE HOST CELL 29 An example of a virus replication cycle: mouse Virions are primed to enter cells and release polyomavirus 12 their genome 29

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3. Virus Classification: The World A variety of cell surface proteins can serve as specific virus of Viruses 31 receptors 47 Receptors interact with viral glycoproteins, surface VIRUS CLASSIFICATION 31 protrusions, or "canyons" on the surface of the virion 48 Many different viruses infecting a wide variety Many viruses enter the cell via receptor-mediated of organisms have been discovered 31 endocytosis 48 Virus classification is based on molecular architecture, Passage from endosomes to the cytosol is often triggered by genetic relatedness, and host organism 3 1 low pH 49 Viruses are grouped into species, genera, and families 32 Membrane fusion is mediated by specific viral "fusion Distinct naming conventions and classification schemes proteins" 50 have developed in different domains of virology 33 Fusion proteins undergo major conformational changes MAJOR VIRUS GROUPS 33 that lead to membrane fusion 50 Study of the major groups of viruses leads to Non-enveloped viruses penetrate by membrane lysis or understanding of shared characteristics pore formation 51 and replication pathways 3 3 Virions and capsids are transported within the cell in Viruses with single-stranded DNA genomes are small and vesicles or on microtubules 52 have few genes 34 Import of viral genomes into the nucleus 52 Viruses with double-stranded DNA genomes include the The many ways in which viral genomes are largest known viruses 3 5 uncoated and released 54 Most viruses and many viruses of vertebrates have positive-strand RNA genomes 35 SECTION II: VIRUSES OF Viruses with negative-strand RNA genomes have helical BACTERIA AND ARCHAEA nucleocapsids; some have fragmented genomes 38 Viruses with double-stranded RNA genomes have fragmented genomes and capsids with icosahedral 5. Single-Stranded RNA symmetry 3 8 Bacteriophages 59 Viruses with a reverse transcription step in their replication The discovery of RNA phages stimulated cycle can have either RNA or DNA genomes 39 research into messenger RNA function and viruses and satellite nucleic acids require a helper RNA replication 59 virus to replicate 40 RNA phages are among the simplest known organisms 59 Viroids do not code for proteins, but replicate independently Two genera of RNA phages have subtle differences 60 of other viruses 40 RNA phages bind to the F-pilus and use it to insert their THE EVOLUTIONARY ORIGIN OF RNA into the cell 60 VIRUSES 40 Phage RNA is translated and replicated in a regulated The first steps in the development of life on Earth: fashion 61 the RNA world 40 RNA secondary structure controls translation of lysis and Viroids and RNA viruses may have originated in replicase genes 61 the RNA world 41 Ribosomes translating the coat gene disrupt secondary The transition to the DNA-based world 42 structure, allowing replicase translation 62 Retroviruses could have originated during the Ribosomes terminating coat translation can reinitiate at the transition to DNA-based cells 43 lysis gene start site 63 Small- and medium-sized DNA viruses could Replication versus translation: competition for the same have arisen as independently replicating genetic RNA template 64 elements in cells 43 Genome replication requires four host cell proteins Large DNA viruses could have evolved from plus the replicase 64 cellular forms that became obligatory A host ribosomal protein directs polymerase to the intracellular parasites 43 coat start site 65 These arguments about the origin of viruses are only Polymerase skips the first A residue but adds a terminal speculations 44 A to the minus-strand copy 65 Synthesis of plus-strands is less complex and more 4. Virus Entry 45 efficient than that of minus-strands 65 The start site for synthesis of maturation protein is How do virions get into cells? 45 normally inaccessible to ribosomes 65 Enveloped and non-enveloped viruses have distinct Synthesis of maturation protein is controlled by penetration strategies 46 delayed RNA folding 66 Some viruses can pass directly from cell to cell 46 Assembly and release of virions 67

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6. Microviruses 69 The X lytic transcription program is controlled by termination and antitermination of RNA synthesis at tpX174: a tiny virus with a big impact 69 specific sites on the genome 87 Overlapping reading frames allow efficient use The CI repressor blocks expression of the lytic of a small genome 70 program by regulating three nearby promoters: P], tpX174 binds to glucose residues in lipopolysaccharide PR,andPRV, 88 on the cell surface 70 Cleavage of CI repressor in cells with damaged DNA tpX174 delivers its genome into the cell through leads to prophage induction 89 spikes on the capsid surface 71 The Cro repressor suppresses CI synthesis and regulates Stage I DNA replication generates double-stranded early gene transcription 89 replicative form DNA 72 Making the decision: go lytic or lysogenize? 90 Gene expression is controlled by the strength of A quick review 90 promoters and transcriptional terminators 72 Breaking and entering: the insertion of X prophage Replicative form DNAs are amplified via a rolling circle DNA into the bacterial chromosome 90 mechanism 72 Excision of \ DNA from the bacterial chromosome 92 Summary of viral DNA replication mechanisms 73 Int synthesis is controlled by retroregulation 93 Procapsids are assembled by the use of scaffolding X DNA replication is directed by O and P, but carried proteins 73 out by host cell proteins 93 Scaffolding proteins have a flexible structure 74 Assembly of X heads involves chaperones and scaffolding Single-stranded genomes are packaged into procapsids as proteins 93 they are synthesized 74 DNA is inserted into preformed proheads by an Role of the J protein in DNA packaging 75 ATP-dependent mechanism 94 Cell lysis caused by E protein leads to release Host cell lysis 94 of phage 75 Did all icosahedral ssDNA virus families evolve from a common ancestor? 75 9. Viruses of Archaea 97 Archaea, the third domain of life 97 7. Bacteriophage T7 77 Viruses of Archaea have diverse and unusual T7: a model phage for DNA replication, transcription, morphologies 99 and RNA processing 77 are temperate viruses that produce virions T7 genes are organized into three groups based on without killing the host cell 99 transcription and gene function 78 Genomes of fuselloviruses are positively supercoiled 101 Entry of T7 DNA into the cytoplasm is powered by Transcription of SSV-1 DNA is temporally controlled 101 transcription 79 Filamentous enveloped viruses of the Transcription of class II and III genes requires a come in many lengths 102 novel T7-coded RNA polymerase 79 A droplet-shaped virus is the only known member of the Class II genes code for enzymes involved in T7 DNA (from the Latin gtitta, "droplet") 103 replication 80 Acidianiis bottle-shaped virus (ABV): its name T7 RNAs are cleaved by host cell ribonuclease III to says it all! 103 smaller, stable inRNAs 80 The genome of Pyrobaculum spherical virus has nearly all Class III gene expression is regulated by delayed open reading frames encoded on one DNA strand 104 entry and by promoter strength 80 Viruses in the family Rudiviridae (from the Latin rudis, DNA replication starts at a unique internal origin and is "small rod") are non-enveloped, helical rods 105 primed by T7 RNA polymerase 80 Rudiviruses escape from the cell by means of unique Large DNA concatemers are formed pyramidal structures 106 during replication 81 Acidianus two-tailed virus (ATV) has a virion with tails that Concatemer processing depends on transcription by T7 spontaneously elongate 106 RNA polymerase and occurs during DNA packaging into Infection with ATV at high temperatures leads to lysogeny 106 preformed proheads 82 Two related viruses of hyperhalophiles resemble Special features of the T7 family of phages 82 fuselloviruses by morphology but not by genetics 108 Two unusual viruses with icosahedral capsids and prominent spikes 108 8. Bacteriophage Lambda 85 A virus with a single-stranded DNA genome is closely Roots... 85 related to a virus with a double-stranded DNA genome 108 Phage adsorption and DNA entry depend on cellular Comparative genomics of archaeal viruses 109 proteins involved in sugar transport 86 Conclusion 1 10 XII Contents

Ebola virus uses RNA editing to make two glycoproteins Nucleocapsids enter the nucleus, where mRNA synthesis and from the same gene 194 RNA replication occur 215 Do the secreted glycoproteins play a role in virus Capped 5' ends of cellular premessenger RNAs are used as pathogenesis? 195 primers for synthesis of viral mRNAs 215 Minor nucleocapsid protein VP30 activates viral mRNA Viral mRNAs terminate in poly(A) tails generated by synthesis in Ebola virus 195 "stuttering" transcription 216 Matrix protein VP40 directs budding and formation of Two influenza A mRNAs undergo alternative splicing filamentous particles 195 in the nucleus 216 Most filovirus outbreaks have occurred in equatorial Genome replication begins when newly synthesized NP Africa 196 protein enters the nucleus 217 Filovirus infections are transmitted to humans from an Nucleocapsids are exported from the nucleus in a complex unknown origin 197 with matrix protein and NS2 218 Spread of filovirus infections among humans is limited to The NS1 protein interferes with polyadenylation of cellular close contacts 197 mRNAs 218 Pathogenesis of filovirus infections 197 The NS1 protein also suppresses a variety of host cell Clinical features of infection 198 antiviral response pathways 219 PB1-F2 may contribute to suppression of the host immune 1 7. Bunyaviruses 200 response 219 Viral envelope proteins assemble in the plasma inembrane Most bunyaviruses are transmitted by arthropod vectors, and direct budding of virions 219 including mosquitoes and ticks 200 Neuraminidase cleaves sialic acid, the cellular receptor that Some bunyaviruses cause severe hemorrhagic fever, binds to HA 220 respiratory disease, or encephalitis 201 Influenza virus strains vary in both transmissibility and Bunyaviruses encapsidate a segmented RNA genome in a pathogenicity 220 simple enveloped particle 202 Genetic variability generates new virus strains that can cause Bunyavirus protein coding strategies: negative-strand and pandemics 220 ambisense RNAs 203 The 1918 pandemic influenza A virus was probably not a L RNA codes for viral RNA polymerase 203 reassortant virus 221 M RNA codes for virion envelope glycoproteins 203 Genome sequences from some previous influenza A virus S RNA codes for nucleocapsid protein and a strains confirm the antigenic shift hypothesis 221 nonstructural protein 204 Highly pathogenic avian influenza A H5N1 strains in poultry After attachment via virion glycoproteins, bunyaviruses enter farms are a potential threat but are poorly transmitted the cell by endocytosis 204 among humans 221 Bunyavirus mRNA synthesis is primed by the capped 5' ends A new pandemic strain of influenza A virus arose by genetic of cellular mRNAs 204 shift and spread worldwide in 2009 222 Coupled translation and transcription may prevent premature termination of mRNAs 206 19. Reoviruses 225 Genome replication begins once sufficient N protein is made 206 Reoviruses were the first double-stranded RNA viruses Virus assembly takes place at Golgi membranes 206 discovered 225 Evolutionary potential of bunyaviruses via genome Some members of the are important reassortment 207 pathogens 226 Reoviridae have segmented genomes made of double- stranded RNA 226 18. Influenza Viruses 210 Reovirus virions contain concentric layers of capsid Influenza viruses cause serious acute disease in humans, and proteins 227 occasional pandemics 210 The attachment protein binds to one or two cellular Influenza virus infections of the respiratory tract can lead to receptors 228 secondary bacterial infections 211 During entry, the outer capsid is stripped from virions and Orthomyxoviruses are negative-strand RNA viruses with the core is released into the cytoplasm 229 segmented genomes 211 Enzymes in the viral core synthesize and cap messenger Eight influenza virus genome segments code for a total of 11 RNAs 230 different viral proteins 2 12 Translation of reovirus mRNAs is regulated 231 Hemagglutinin protein binds to cell receptors and mediates Interferon and PKR: effects on viral and cellular protein fusion of the envelope with the endosomal membrane 214 synthesis 231 M2 is an ion channel that facilitates release of nucleocapsids Synthesis of progeny double-stranded genomes occurs from the virion 214 within subviral particles 232 Contents XIII

Reoviruses induce apoptosis via activation of innate immune Four early mRNAs are made by differential splicing of a response transcription factors NF-KB and IRF-3 233 common transcript 253 Studies of reovirus pathogenesis in mice 234 T antigens share common N-terminal sequences but have different C-terminal sequences 254 T antigens bring resting cells into the DNA synthesis (S) SECTION V: SMALL DNA VIRUSES phase of the cell cycle 254 OF EUKARYOTES Small T antigen inhibits protein phosphatase 2A and induces cell cycling 254 20. Parvoviruses 238 Middle T antigen stimulates protein tyrosine kinases that signal cell proliferation and division 255 Parvoviruses have very small virions and a linear, Large T antigen activates or suppresses transcription of single-stranded DNA genome 238 cellular genes by binding to a number of important cellular Parvoviruses replicate in cells that are going through regulatory proteins 255 the cell cycle 239 Large T antigen hexamers bind to the origin of DNA Discovery of mammalian parvoviruses 2 3 9 replication and locally unwind the two DNA strands 257 Parvoviruses have one of the simplest-known virion Large T antigen assembles the cellular DNA synthesis structures 239 machinery to initiate viral DNA replication 257 Parvoviruses have very few genes 239 High levels of late transcripts are made after DNA Single-stranded parvovirus DNAs have unusual replication begins 259 terminal structures 240 Three late mRNAs are made by alternative splicing 260 Uncoating of parvovirus virions takes place in the How do polyomaviruses transform cells in vitro and nucleus and is cell-specific 240 cause tumors in vivo} 260 DNA replication begins by extension of the 3' end Only non-permissive cells can be transformed 261 of the terminal hairpin 241 Transformed cells integrate viral DNA into the cell The DNA "end replication" problem 241 chromosome 261 Steps in DNA replication 243 Nonstructural proteins are multifunctional 243 Adenovirus functions that help replication of 22. Papillomaviruses 263 adeno-associated virus 244 Papillomaviruses cause warts and other skin and In the absence of helper virus, adeno-associated mucosal lesions 263 virus DNA can integrate into the cell genome 244 Oncogenic human papillomaviruses are a major cause of Parvovirus pathogenesis: the example of B19 virus 244 genital tract cancers 264 Papillomaviruses are not easily grown in cell culture 264 21. Polyomaviruses 247 Papillomavirus genomes are circular, double-stranded DNA 264 The infectious cycle follows differentiation of epithelial cells 265 Mouse polyomavirus was discovered as a tumor-producing Viral mRNAs are made from two promoters and two infectious agent 247 polyadenylation signals 266 Simian virus 40 was found as a contaminant of Salk Viral El and E2 proteins bind to the replication origin and poliovirus vaccine 247 direct initiation of DNA replication 267 Human polyomaviruses are widespread but cause disease Viral E7 protein interacts with cell-cycle regulatory proteins, only rarely 248 particularly Rb 267 Polyomaviruses are models for studying DNA virus Viral E6 protein controls the level of cellular p53 protein 268 replication and tumorigenesis 248 Synergism between E6 and E7 and the predisposition Polyomavirus capsids are constructed from pentamers of the to cancer 269 major capsid protein 248 Cells transformed by papillomaviruses express E6 and E7 The circular DNA genome is packaged with cellular gene products from integrated viral DNA 270 histones 249 Future prospects for diagnosis and treatment of diseases Circular DNA becomes supercoiled upon removal caused by papillomaviruses 270 of histones 249 Supercoiled DNA can be separated from relaxed or linear DNA molecules 250 SECTION VI: LARGER DNA Polyomavirus genes are organized in two divergent VIRUSES OF EUKARYOTES transcription units 250 Virions enter cells in caveolae and are transported to the 23. Adenoviruses 274 nucleus 2 51 The viral minichromosome is transcribed by cellular RNA Adenoviruses cause respiratory and enteric infections in polymerase II 252 humans 274 XIV Contents

Adenoviruses can be oncogenic, but do not cause DNA replication initially proceeds in a bidirectional cancer in humans 274 fashion from a replication origin 291 Virions have icosahedral symmetry and are studded with Rolling circle replication subsequently produces knobbed fibers 275 multimeric concatemers of viral DNA 292

Fibers make contact with cellular receptor proteins to DNA replication leads to activation of ^ and y2 genes 292 initiate infection 276 Viral nucleocapsids are assembled on a scaffold in Expression of adenovirus genes is controlled at the the nucleus 293 level of transcription 276 Envelopment and egress: three possible routes 294 El A proteins are the kingpins of the adenovirus Many viral genes are involved in blocking host growth cycle 277 responses to infection 295 E1A proteins bind to the retinoblastoma protein and establishes latent infection in neurons 296 activate E2F, a cellular transcription factor 277 Latency-associated transcripts include stable 296 El A proteins also activate other cellular EPSTEIN-BARR VIRUS 296 transcription factors 278 Epstein-Barr virus was discovered in lymphomas in African El A proteins indirectly induce apoptosis by activation of children 296 cellular p53 protein 279 Epstein-Barr virus infects mucosal epithelial cells and E1B proteins suppress ElA-induced apoptosis and target B-lymphocytes 297 key proteins for degradation, allowing virus Epstein-Barr virus expresses a limited set of proteins in replication to proceed 279 latently infected B lymphocytes 298 The preterminal protein primes DNA synthesis carried Epstein—Barr virus nuclear antigens direct limited out by viral DNA polymerase 280 replication of the viral genome and activate viral Single-stranded DNA is circularized via the inverted and cellular genes 299 terminal repeat 2 80 Latent membrane proteins mimic receptors on B The major late promoter is activated after DNA lymphocytes 299 replication begins 2 81 Small, untranslated viral RNAs expressed during latent Five different poly(A) sites and alternative splicing infections target host defense mechanisms 300 generate multiple late mRNAs 281 The tripartite leader ensures efficient transport of late mRNAs to the cytoplasm 2 81 25. Baculoviruses 302 The tripartite leader directs efficient translation Insect viruses were first discovered as pathogens of of late adenovirus proteins 282 silkworms 302 Adenovirus-induced cell killing 283 Baculoviruses are used for pest control and to express Cell transformation and oncogenesis by human eukaryotic proteins 303 adenoviruses 283 Baculovirus virions contain an elongated nucleocapsid 303 Baculoviruses produce two kinds of particles: "budded" and 24. Herpesviruses 285 "occlusion-derived" virions 304 Baculoviruses have large, circular DNA genomes and Herpesviruses are important human pathogens 285 encode many proteins 305 Most herpesviruses can establish latent infections 286 Insects are infected by ingesting occlusion bodies; infection HERPES SIMPLEX VIRUS 286 spreads within the insect via budded virions 306 Herpes simplex virus genomes contain both unique Viral proteins are expressed in a timed cascade regulated at and repeated sequence elements 286 the transcription level 306 Nomenclature of herpes simplex virus genes Immediate early gene products control expression of early and proteins 288 genes 307 The icosahedral capsid is enclosed in an envelope Early gene products regulate DNA replication, late along with tegument proteins 288 transcription, and apoptosis 307 Entry by fusion is mediated by envelope glycoproteins and Late genes are transcribed by a novel virus-coded RNA may occur at the plasma membrane or in endosomes 288 polymerase 308 Viral genes are sequentially expressed during the Baculoviruses are widely used to express foreign proteins 3 08 replication cycle 289 Tegument proteins interact with cellular machinery to 26. Poxviruses 312 activate viral gene expression and to degrade cellular messenger RNAs 289 Smallpox was a debilitating and fatal worldwide disease 312 Immediate early (a) genes regulate expression of other Variolation led to vaccination, which has eradicated smallpox herpesvirus genes 291 worldwide 313 β gene products enable viral DNA replication 291 Poxviruses remain a subject of intense research interest 313 Contents XV

Linear vaccinia virus genomes have covalently sealed hairpin PHAEOVI RUSES 335 ends and lack introns 314 Seaweed viruses 3 3 5 Two forms of vaccinia virions have different roles in have a temperate life cycle and integrate their spreading infection 3 15 genomes into the host 3 3 5 Poxviruses replicate in the cytoplasm 316 PRYMNESIOVIRUSES AND Poxvirus genes are expressed in a regulated transcriptional RAPHIDOVIRUSES 335 cascade controlled by viral transcription factors 317 The lesser-known 3 3 5 Virus-coded enzymes packaged in the core carry out early RNA synthesis and processing 3 18 336 Enzymes that direct DNA replication are encoded by early The world's largest known virus 336 mRNAs 318 Mimivirus is unquestionably a virus 3 3 6 Poxviruses produce large concatemeric DNA molecules that Why such a large genome? 3 3 7 are resolved into monomers 3 18 Mimivirus has a unique mechanism for releasing its core 3 3 7 Postreplicative mRNAs have 5' end poly(A) extensions and Virus replication occurs exclusively in the cytoplasm 3 3 7 3' end heterogeneity 319 Genome replication 338 Mature virions are formed within virus "factories" 320 Genes coding for translation factors and DNA repair Extracellular virions are extruded through the plasma enzymes 338 membrane by actin tails 321 Ancestors of mimivirus may have transferred genes from Poxviruses make several proteins that target host defenses bacteria to eukaryotes 339 against invading pathogens 321 Conclusion 340

27. Viruses of Algae and Mimivirus 325 SECTION VII: VIRUSES THAT USE A REVERSE TRANSCRIPTASE Aquatic environments harbor large viruses 3 2 5 Phycodnaviruses are diverse and probably ancient 326 28. Retroviruses 342 Phycodnavirology: a field in its infancy 326 Conserved structure, diverse composition 327 Retroviruses have a unique replication cycle based on reverse transcription and integration of their genomes 342 327 Viral proteins derived from the gag, pol, and env genes are Known chloroviruses replicate in isolated from incorporated in virions 343 symbiotic hosts 327 Retroviruses enter cells by the fusion pathway 344 The linear genomes of chloroviruses contain hundreds of genes, Viral RNA is converted into a double-stranded DNA copy and each virus species encodes some unique proteins 327 by reverse transcription 345 capsids are constructed from many capsomers A copy of proviral DNA is integrated into the cellular and have a unique spike 328 genome at a random site 347 Virus entry begins by binding to and degradation of the host Sequence elements in the long terminal repeats direct cell wall 329 transcription and polyadenylation by host cell Transcription of viral genes is temporally controlled and enzymes 348 probably occurs in the cell nucleus 329 Differential splicing generates multiple mRNAs 348 Progeny virions are assembled in the cytoplasm 329 The Gag/Pol polyprotein is made by suppression of Small and efficient proteins 3 30 termination and use of alternative reading frames 348 A virus family with a penchant for sugar metabolism: Virions mature into infectious particles after budding hyaluronan and chitin 330 from the plasma membrane 349 COCCOLITHOVIRUSES 331 Acute transforming retroviruses express mutated forms of Viruses that control the weather 331 cellular growth signaling proteins 350 Many genes looking for a function 332 Retroviruses lacking oncogenes can transform cells by Expression of genes is temporally insertion of proviral DNA near a proto-oncogene 3 51 regulated 332 Cheshire Cat dynamics: sex to avoid virus infection 3 3 3 29. Human Immunodeficiency Virus 354 Survival of the fattest: the giant coccolithovirus genome Human immunodeficiency virus type 1 (HIV-1) and encodes sphingolipid biosynthesis 3 3 3 acquired immunodeficiency syndrome (AIDS) 355 PRASINOVIRUSES 334 HIV-1 was probably transmitted to humans from Small host, big virus 334 chimpanzees infected with SIVcpz 3 5 5 Viral genomes contain multiple genes for capsid proteins 3 34 HIV-1 infection leads to a progressive loss of cellular It works both ways 334 immunity and increased susceptibility to Not much room for maneuver 335 opportunistic infections 355 XVI Contents

Antiviral drugs can control HIV-1 infection and prevent Viroids replicate via linear multimeric RNA disease progression, but an effective vaccine has yet to be intermediates 380 developed 356 Three enzymatic activities are needed for HIV-1 is a complex 3 57 replication 380 HIV-1 targets cells of the immune system by recognizing How do viroids cause disease? 382 CD4 antigen and chemokine receptors 357 Interaction of viroid RNA with cellular RNAs or proteins Virus mutants arise rapidly because of errors generated may disrupt cell metabolism 382 during reverse transcription 3 5 8 RNA interference could determine viroid pathogenicity and Unlike other retroviruses, HIV-1 directs transport of proviral cross-protection 382 DNA into the cell nucleus 359 Circular plant satellite RNAs resemble viroids but are Latent infection complicates the elimination of HIV-1 359 encapsidated 383 The Tat protein increases HIV-1 transcription by Hepatitis delta virus is a human viroid-like satellite virus 383 stimulating elongation by RNA polymerase II 360 Hepatitis delta virus may use two different cellular RNA The Rev protein mediates cytoplasmic transport of viral polymerases to replicate 383 mRNAs that code for HIV-1 structural proteins 360 RNA editing generates two forms of hepatitis delta Together, the Tat and Rev proteins strongly upregulate viral antigen 384 protein expression 3 61 Conclusion: viroids may be a link to the ancient The Vif protein increases virion infectivity by counteracting RNA world 384 a cellular deoxcytidine deaminase 361 The Vpr protein enhances HIV-1 replication at 32. Prions 387 multiple levels 362 The Vpu protein enhances release of progeny virions from Prions are proteins that cause fatal brain diseases 387 infected cells 362 diseases were first detected in domestic The Nef protein is an important mediator of pathogenesis 362 ruminants 388 Bovine spongiform encephalopathy ("mad cow disease") 30. Hepadnaviruses 365 developed in Britain and apparently spread to humans 388 Human prion diseases can be either inherited or At least seven distinct viruses cause human hepatitis 365 transmitted 388 The discovery of hepatitis B virus 366 The infectious agent of prion diseases contains protein but Dane particles are infectious virions; abundant no detectable nucleic acid 389 non-infectious particles lack nucleocapsids 366 PrPSc is encoded by a host cell gene 390 The viral genome is a circular, partly single-stranded DNA Differences between PrPc and PrPSt 390 with overlapping reading frames 367 The prion hypothesis: formation of infectious and Nucleocapsids enter the cytoplasm via fusion and are pathogenic prions from normal PrPc 391 transported to the nucleus 367 Is the prion hypothesis correct? 392 Transcription of viral DNA gives rise to several Pathology and diagnosis of prion diseases 392 mRNAs and a pregenome RNA 368 Proteins of yeast and other fungi can form self-propagating The roles of hepatitis B virus proteins 369 states resembling prions 393 The pregenome RNA is packaged by interaction with Genetics of prion diseases: mutations in the prion gene can polymerase and core proteins 371 increase occurrence of disease 393 Genome replication occurs via reverse transcription of Prion diseases are not usually transmitted among different pregenome RNA 372 species 393 Virions are formed by budding in the endoplasmic Strain variation and crossing of the species barrier 394 reticulum 3 7 3 The nature of the prion infectious agent 394 Hepatitis B virus can cause chronic or acute hepatitis, cirrhosis, and liver cancer 374 Hepatitis B virus is transmitted by blood transfusions, SECTION IX: HOST DEFENSES contaminated needles, and unprotected sex 374 AGAINST VIRUS INFECTION A recombinant vaccine is available 375 Antiviral drug treatment has real success 375 33. Intrinsic Cellular Defenses Against Virus Infection 398 SECTION VIM: VIROIDS AND PRIONS INTRODUCTION 399 31. Viroids and Hepatitis Delta Virus 378 DETECTION OF VIRUS INFECTION BY HOST CELLS 399 Viroids are small, circular RNAs that do not encode Host cells sense virus infection with toll-like receptors and a proteins 379 variety of other molecular detection systems 399 The two families of viroids have distinct properties 379 Several toll-like receptors recognize viral nucleic acids 400 i XVIII Contents

NEW DEVELOPMENTS IN ANTIVIRAL Virus vectors are used to produce high levels of specific VACCINES 437 proteins in cultured cells 457 New approaches to vaccine development show great promise 437 Gene therapy is an expanding application of New adjuvants are being developed 437 virus vectors 458 New delivery systems for viral antigens 437 Virus vectors are produced by transfection of cells with Vaccination with defined proteins 43 7 containing deleted genomes 458 Use of live viruses with defined attenuation Virus vectors are engineered to produce optimal characteristics 438 levels of gene products 459 Use of live vectors and chimeric viruses 439 ADENOVIRUS VECTORS 460 Vaccines that can break tolerance 439 Adenovirus vectors are widely used in The changing vaccine paradigm 439 studies of gene transfer and antitumor ADVERSE EVENTS AND ETHICAL ISSUES 439 therapy 460 Vaccine-associated adverse events 439 Replication-defective adenovirus vectors are propagated Ethical issues in the use of antiviral vaccines 441 in complementing cell lines 460 Replication-competent adenovirus vectors are useful 36. Antiviral Chemotherapy 444 tools in antitumor therapy 461 Advantages and limitations of adenovirus vectors 461 The discovery and widespread use of antiviral compounds began relatively recently 444 RETROVIRUS VECTORS 462 Antiviral drugs are useful for discoveries in basic Retrovirus vectors incorporate transgenes into the cell research on viruses 445 chromosome 462 How are antiviral drugs obtained? 445 Packaging cell lines express retrovirus enzymatic and Antiviral drugs are targeted to specific steps of virus structural proteins 462 infection 445 Strategies for controlling transgene Drugs preventing attachment and entry of virions 446 transcription 463 Amantadine blocks ion channels and inhibits uncoating of Lentivirus vectors are used for gene delivery to influenza virions 447 non-dividing cells 463 Nucleoside analogues target viral DNA polymerases 447 Production of lentivirus vectors requires additional Acyclovir is selectively phosphorylated by herpesvirus «V-acting sequences 463 thymidine kinases 448 Applications of retrovirus vectors: treatment of Acyclovir is preferentially incorporated by blood disorders 464 herpesvirus DNA polymerases 449 Applications of retrovirus vectors: treatment of encodes a protein kinase that neurological disorders 465 phosphorylates ganciclovir 450 Advantages and limitations of retrovirus vectors 465 HIV-1 reverse transcriptase preferentially incorporates ADENO-ASSOCIATED VIRUS azidothymidine into DNA, leading to chain termination 450 VECTORS 465 Non-nucleoside inhibitors selectively target viral Adeno-associated virus vectors can insert transgenes replication enzymes 451 into a specific chromosomal locus 465 Protease inhibitors can interfere with virus assembly and Production of AAV vectors usually requires a maturation 452 helper virus 466 Ritonavir: a successful protease inhibitor of HIV-1 Clinical trials using adeno-associated virus vectors 467 that was developed by rational inethods 452 Advantages and limitations of AAV vectors 467 Neuraminidase inhibitors inhibit release and spread of influenza virus 453 Antiviral chemotherapy shows promise for the future 453 GLOSSARY 471 CREDITS 484 37. Eukaryotic Virus Vectors 456 NAME INDEX 489 Many viruses can be engineered to deliver and express specific genes 456 SUBJECT INDEX 491

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