I. The Nature of Viruses 8.1 What Is a Virus? 8.2 Structure of the Virion 8.3 Overview of the Virus Life Cycle 8.4 Culturing, Detecting, and Counting Viruses

8.1 What Is a Virus? Virus: subcellular genetic element that cannot replicate independently of a living (host) cell Virus particle (virion): extracellular form of a virus Exists outside host and facilitates transmission from one host cell to another Contains nucleic acid genome surrounded by a protein coat and, in some cases, other layers of material (Figure 8.1)

Viral genomes (Figure 8.2) Either DNA or RNA genomes Some are circular, but most are linear Viruses Genome: DNA RNA RNA DNA Types: ssDNA dsDNA ssRNA dsRNA ssRNA (Retroviruses) dsDNA (Hepadnaviruses) Single or double-stranded Some have ability to reverse flow of information Figure 8.2 Viral genomes.

8.1 What Is a Virus? Virus taxonomy Viruses are usually classified on the basis of the nucleic acid they contain Particle shape and host range are less important

8.2 Structure of the Virion Viral structure Genome: nucleic acid of the virus Capsid: the protein shell that surrounds the genome of a virus particle (Figure 8.3) Composed of a number of protein molecules arranged in a precise and highly repetitive pattern around the nucleic acid Capsid protein: individual protein molecule building block Capsomere: visible subunit of the capsid Smallest morphological unit visible with an electron microscope

8.2 Structure of the Virion Viral structure (cont'd) Nucleocapsid: complete complex of nucleic acid and protein packaged in the virion Enveloped virus: virus that contains additional layers around the nucleocapsid

Naked virus Enveloped virus (unenveloped) Figure 8.1 Nucleocapsid Nucleic acid Nucleic acid Capsid (composed of capsid proteins) Figure 8.1 Comparison of naked and enveloped virus particles. Naked virus (unenveloped) Enveloped virus Figure 8.1

8.2 Structure of the Virion Nucleocapsids are constructed in highly symmetric ways Helical symmetry: rod-shaped viruses (e.g., tobacco mosaic virus) Length of virus determined by length of nucleic acid Width of virus determined by size and packaging of protein subunits May or may not be surrounded by an envelope Icosahedral symmetry: near-spherical viruses (e.g., human papillomavirus; Figure 8.4) Most efficient arrangement of subunits in a closed shell

Figure 8.3 18 nm Structural subunits (capsomeres) Virus RNA Figure 8.3 The arrangement of RNA and protein coat in a simple virus, tobacco mosaic virus. Figure 8.3

Figure 8.4 5-Fold 3-Fold 2-Fold Cluster of 5 units “pentamer” Symmetry Figure 8.4 Icosahedral symmetry. Figure 8.4

Enveloped viruses (Figure 8.5) Have membrane surrounding nucleocapsid (core) Lipid bilayer with embedded proteins Envelope makes initial contact with host cell Figure 8.5 Enveloped viruses. Figure 8.5

8.2 Structure of the Virion Some virions contain enzymes critical to infection Lysozyme Makes hole in cell wall Lyses bacterial cell Nucleic acid polymerases (reverse transcriptase) Neuraminidases Enzymes that cleave glycosidic bonds Allows liberation of viruses from cell

8.3 Overview of the Virus Life Cycle Phases of viral replication (Figure 8.6) Attachment (adsorption) of the virus to a susceptible host cell Entry (penetration) of the virion or its nucleic acid Synthesis of virus nucleic acid and protein by cell metabolism as redirected by virus Assembly of capsids and packaging of viral genomes into new virions (maturation) Release of mature virions from host cell

8.3 Overview of the Virus Life Cycle (Bacteriophage) Head Protein coat remains outside Virions Virion DNA Viral DNA enters Cell (host) 1. Attachment (adsorption of phage virion) 2. Penetration of viral nucleic acid 3. Synthesis of viral nucleic acid and protein 4. Assembly and packaging of new viruses 5. Cell lysis and release of new virions Figure 8.6 The replication cycle of a bacterial virus. Virus replication is typically characterized by a one-step growth curve (Figure 8.7) Figure 8.6

Maturation Figure 8.7 Nucleic acid Protein coats Early enzymes Virus Eclipse Maturation Early enzymes Nucleic acid Protein coats Virus added Assembly and release Figure 8.7 One-step growth curve of virus replication. Latent period Figure 8.7

8.4 Culturing, Detecting, and Counting Viruses Viruses replicate only in certain types of cells or in whole organisms Bacterial viruses are easiest to grow; model systems Animal viruses (and some plant viruses) can be cultivated in tissue or cell cultures Plant viruses typically are most difficult because study often requires growth of whole plant

8.4 Culturing, Detecting, and Counting Viruses Plaques assay: are clear zones that develop on lawns of host cells Bacterial lawn or tissue culture Titer: number of infectious virus units detected per volume of fluid Plaque assay: analogous to the bacterial colony; one way to measure virus infectivity (Figure 8.8) Each plaque results from infection by a single virus particle

1. The cell–phage mixture is poured onto a solidified nutrient agar plate. Mixture containing molten top agar, bacterial cells, and diluted phage suspension Nutrient agar plate 2. The mixture is left to solidify. Plaques Sandwich of top agar and nutrient agar 3. Incubation allows for bacterial growth and phage replication. Phage plaques Figure 8.8 Quantification of bacterial virus by plaque assay. Lawn of host cells Figure 8.8

Figure 8.9 Confluent monolayer of tissue culture cells Viral plaques Figure 8.9 Animal cell cultures and viral plaques. Confluent monolayer of tissue culture cells Viral plaques Figure 8.9

II. Bacteriophage Life Cycles 8.5 Attachment and Entry of Bacteriophage T4 8.7 Replication of Bacteriophage T4 8.8 Temperate Bacteriophages and Lysogeny

8.5 Attachment and Entry of Bacteriophage T4 Attachment of virion to host cell is highly specific Requires complementary receptor molecules on the surface of a susceptible host and its infecting virus (Figure 8.10) Receptors on host cell carry out normal functions for cell (e.g., uptake proteins, cell-to-cell interaction) Receptors include proteins, carbohydrates, glycoproteins, lipids, lipoproteins, or complexes

Figure 8.10 Chi M13 T4 Flagellum Iron transport protein T1 Pilus ϕX174 MS2 LPS Figure 8.10 Bacteriophage receptors. Outer membrane Peptidoglycan Cytoplasmic membrane Figure 8.10

8.5 Attachment and Entry of Bacteriophage T4 The attachment of a virus to its host cell results in changes to both virus and cell surface that facilitate penetration Permissive cell: host cell that allows the complete replication cycle of a virus to occur

8.5 Attachment and Entry of Bacteriophage T4 Bacteriophage T4: virus of E. coli; one of the most complex penetration mechanisms (Figure 8.11) Virions attach to cells via tail fibers that interact with polysaccharides on E. coli cell envelope Tail fibers retract, and tail core makes contact with E. coli cell wall Lysozyme-like enzyme forms small pore in peptidoglycan Tail sheath contracts, and viral DNA passes into cytoplasm

Figure 8.11 T4 virion Tail fibers Tail pins Outer membrane Tail tube Site of tail lysozyme activity Peptidoglycan Figure 8.11 Attachment of bacteriophage T4 to an Escherichia coli cell. Cytoplasmic membrane Cytoplasm T4 genome Figure 8.11

8. 7 Replication of Bacteriophage T4 T4 genome can be divided into three parts: early, middle, and late proteins (Figure 8.13) Early and middle proteins: enzymes needed for DNA replication and transcription Late proteins: head and tail proteins and enzymes required to liberate mature phage particles

Figure 8.13 Figure 8.13 Time course of events in phage T4 infection. T4 nucleases, DNA polymerase, and new sigma factors Phage T4 DNA Phage head proteins Tail, collar, base plate, and tail fiber proteins Mature T4 virion T4 lysozyme production Infection Phage DNA replication Transcription Early mRNA Middle mRNA Late mRNA Self-assembly Translation Lysis Figure 8.13 Time course of events in phage T4 infection. Early proteins Middle proteins Late proteins 5 10 15 20 25 Minutes Figure 8.13

8. 7 Replication of Bacteriophage T4 Production of viral proteins Early proteins (needed before replication Enzyme for the synthesis and glucosylation of the T4 base hydroxymethylcytosine Enzymes that function in T4 replisome Proteins that modify host RNA polymerase Middle proteins Additional proteins that modify host RNA polymerase

8. 7 Replication of Bacteriophage T4 Production of viral proteins (cont'd) Late proteins (needed after replication) Synthesized later Include proteins of virus coat Typically structural components Synthesized in larger amounts

8. 7 Replication of Bacteriophage T4 Stepwise Packaging the T4 genome (Figure 8.14) Precursor of bacteriophage head is assembled Packaging motor is assembled Double-stranded DNA is pumped into head under pressure using ATP After head is filled with DNA, T4 tail, tail fibers, and other components are added

Figure 8.14 Figure 8.14 Packaging of DNA into a T4 phage head. ATP Prohead Motor Packaging motor complex Mature virion ATP Scaffold proteins Packaging motor attaches to prohead. Scaffold proteins discarded Other assembly steps Packaging motor discarded Capsid proteins dsDNA Portal proteins Figure 8.14 Packaging of DNA into a T4 phage head. Figure 8.14

8.8 Temperate Bacteriophages and Lysogeny Viral life cycles Virulent mode: viruses lyse host cells after infection-T4 is an example (aka lytic pathway or cycle) Temperate mode: viruses replicate their genomes in tandem with host genome and without killing host-phage lambda is an example (aka lysogenic pathway or cycle)

8.8 Temperate Bacteriophages and Lysogeny Temperate viruses: can undergo a stable genetic relationship within the host (Figure 8.15) Lysogeny: state where most virus genes are not expressed and virus genome (prophage) is replicated in synchrony with host chromosome Lysogen: a bacterium containing a prophage Under certain conditions, lysogenic viruses may revert to the lytic pathway and begin to produce virions

Temperate virus Host DNA Viral DNA Attachment of the virus to the host cell Cell (host) Injection of viral DNA Lytic pathway Lysogenic pathway Lytic events are initiated. Induction Phage components are synthesized and virions are assembled. Viral DNA is integrated into host DNA. Lysogenized cell Figure 8.15 Consequences of infection by a temperate bacteriophage. Prophage Lysis of the host cell and release of new phage virions Viral DNA is replicated with host DNA at cell division. Figure 8.15

8.8 Temperate Bacteriophages and Lysogeny Lambda DNA forms a circle Attaches to host DNA and integrates itself into DNA Lambda “att” site Lambda repressor protein keeps lambda genes shut off Unfavorable cell growth conditions tend to favor lysogenic pathway Lysogeny stable until cell is injured or damaged

8.8 Temperate Bacteriophages and Lysogeny Unusual mode of replication for lambda DNA When it enters lytic pathway, lambda synthesizes long, linear concatemers of DNA by rolling circle replication (Figure 8.17b) Not standard replication fork as used for host DNA

Figure 8.17b 3′ RNA primers Roll One lambda genome Figure 8.17b Integration of lambda DNA and rolling circle replication. Rolling circle replication of lambda genome Figure 8.17b

III. Viral Diversity and Ecology 8.9 An Overview of Bacterial Viruses 8.10 An Overview of Animal Viruses 8.11 The Virosphere and Viral Ecology

8.9 An Overview of Bacterial Viruses Bacteriophages are very diverse (Figure 8.19) Best-studied bacteriophages infect enteric bacteria Examples of hosts: E. coli, Salmonella enterica Most phages contain dsDNA genomes Most are naked, but some possess lipid envelopes They are structurally complex, containing heads, tails, and other components (Figure 8.20)

Figure 8.19 Schematic representations of the main types of bacterial viruses.

8.10 An Overview of Animal Viruses Often, entire virion enters the animal cell, unlike in prokaryotes Nucleus is the site of replication for many animal viruses Animal viruses contain all known modes of viral genome replication (Figure 8.21) There are many more kinds of enveloped animal viruses than enveloped bacterial viruses As animal viruses leave host cell, they can remove part of host cell's lipid bilayer for their envelope

Figure 8.21 Diversity of animal viruses.

8.10 An Overview of Animal Viruses Consequences of virus infection in animal cells (Figure 8.22) Persistent infections: release of virions from host cell does not result in cell lysis Infected cell remains alive and continues to produce virus Latent infections: delay between infection by the virus and lytic events Transformation: conversion of normal cell into tumor cell

Figure 8.22 Formation of proviral state and transformation into tumor cell Transformation Tumor cell division Cell Virus Death of the cell and release of the virus Lysis Virus multiplication Slow release of virus without causing cell death Persistent infection Figure 8.22 Possible effects that animal viruses may have on cells they infect. Virus present but not replicating May revert to lytic infection Latent infection Figure 8.22

8.10 An Overview of Animal Viruses Retroviruses: RNA viruses that replicate through a DNA intermediate Enveloped viruses (Figure 8.23a) Contain a reverse transcriptase (copies information from its RNA genome into DNA), integrase, and protease DNA copy is called a cDNA Integrates into host DNA

Integrated retrovirus cDNA is called a provirus 1. Entry and uncoating of the retrovirus ssRNA (viral genome) 2. Reverse transcriptase activity (two steps) Integrated retrovirus cDNA is called a provirus dsDNA 3. Viral DNA enters nucleus and integrates into the host genome. Host DNA Viral DNA 4. Transcription by host RNA polymerase forms viral mRNA and genome copies. ssRNA 5. Translation of mRNA forms viral proteins; new nucleocapsids assembled and released by budding. Figure 8.24 Replication of a retrovirus. Host cytoplasmic membrane

8.11 The Virosphere and Viral Ecology About 106 prokaryotes per ml of seawater (Figure 8.25) About 107 viruses per ml of seawater Bacteriophages thought to have major impact on evolution of Bacteria May confer new metabolic or other beneficial properties Many of the prokaryotes in seawater are Archaea Many of the viruses in seawater may infect marine Archaea

8.11 The Virosphere and Viral Ecology Most of Earth's genetic diversity resides in viruses Most viruses are believed to be bacteriophages Viral metagenome: the sum total of all viral genes in a particular environment Most viruses are undiscovered Most viral genes have unknown function