I. The Nature of Viruses 8.1 What Is a Virus? Plus 9.1, 9.2 (parts) 8.2 Structure of the Virion 8.3 Overview of the Virus Life Cycle 8.8 Temperate Bacteriophages and Lysogeny 8.10, 9.6, 9.7 Overview of Animal 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) a variety of forms 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.

9.1 Size and Structure of Viral Genomes-Range Viral genome size (Figure 9.1) Smallest circovirus: 1.75-kilobase single strand Largest megavirus: 1.25-megabase pairs (Pandoravirus)

Figure 9.1 Comparative genomics.

9.1 Size and Structure of Viral Genomes Taxonomy Based mainly on idea proposed by David Baltimore (Baltimore Scheme) Depends on relationship of genome to mRNA Genome structure is the key feature

Class I & VII Class II Class III Class IV Class V Class VI dsDNA (±) virus ssDNA (+) virus dsRNA (±) virus ssRNA (+) virus ssRNA (–) virus ssRNA (+) retrovirus Transcription of the minus strand Synthesis of the minus strand Transcription of the minus strand Used directly as mRNA Transcription of the minus strand Reverse transcription dsDNA intermediate (replicative form) dsDNA intermediate Transcription mRNA (+) Transcription of the minus strand DNA Viruses RNA Viruses Class I classical semiconservative Class II classical semiconservative, discard (–) strand Class VII transcription followed by reverse transcription Class III make ssRNA (+) and transcribe from this to give ssRNA (–) complementary strand Class IV make ssRNA (–) and transcribe from this to give ssRNA (+) genome Class V make ssRNA (+) and transcribe from this to give ssRNA (–) genome Class VI make ssRNA (+) genome by transcription of (–) strand of dsDNA Genome replication Figure 9.2 The Baltimore classification of viral genomes. Figure 9.2

9.2 Viral Evolution No one knows where viruses came from Viruses may date from before the evolution of cells or Viruses may have evolved after cells Or both Do some viruses represent a 4th domain?

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: subunit of the capsid that can be observed microscopically Smallest morphological unit visible with an electron microscope

8.2 Structure of the Virion Viral structure (cont'd) Nucleocapsid: nucleic acid and protein packaged in the virion-different in different viruses Enveloped virus: virus that contains additional layers around the nucleocapsid-lipids derived from host

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 (capsid proteins) 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 (aka 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 or steps of viral replication (Figure 8.6) Attachment (adsorption) of the virus to a susceptible host cell via a receptor 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 = bacterial virus) 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 Virus replication is typically characterized by a one-step growth curve (Figure 8.7) Figure 8.6 The replication cycle of a bacterial virus. 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

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)

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

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

Lambda can cause specialized transduction

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) Starts with ss break = “nick” Not standard replication fork as used for host DNA Complementary strands synthesized at different times

Rolling Circle Replication

II. Viruses with DNA Genomes 8.10 An Overview of Animal Viruses 9.6 Uniquely Replicating DNA Animal Viruses 9.7 DNA Tumor 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

Figure 8.21 Diversity of animal viruses.

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

9.6 Uniquely Replicating DNA Animal Viruses Double-stranded DNA animal viruses that have unusual replication strategies Pox viruses Adenoviruses

Pox viruses are ds DNA viruses that replicate in the cytoplasm What do they need to do this? Figure 9.11 Smallpox virus.

9.6 Uniquely Replicating DNA Animal Viruses Adenoviruses Major group of icosahedral, linear, double-stranded DNA viruses, no envelope Cause mild respiratory infections in humans DNA replicates in the nucleus Replication requires protein primers and avoids synthesis of a lagging strand (Figure 9.12) Two unusual characteristics

Covalently linked protein TP pTP is primer Strand displacement replication Viral proteins

9.7 DNA Tumor Viruses Some DNA viruses can induce cancer (“tumor viruses or “oncoviruses” Human Papilloma Viruses (HPV) Herpesviruses

9.7 DNA Tumor Viruses HPV Nonenveloped icosahedral virion-no enzymes in the virion; replicates in host nucleus Basal skin cells 8 kbp DNA is circular (Figure 9.13a) Small genome, has overlapping genes

9.7 DNA Tumor Viruses Some papilloma viruses cause cancer In most infected host cells, virus infection results in the formation of new virions and the release from host cell In a few infected host cells, part or all of the virus DNA becomes integrated into host DNA (analogous to a prophage), genetically altering cells in the process (Figure 9.13b) Integrated virus DNA can interfere with normal cell division

Interfere with regulation Infection Papilloma virus DNA + Host DNA Accidental Viral DNA integrates into host DNA. All or part Viral DNA Transcription of tumor-inducing genes Interfere with regulation mRNA Transport of mRNA to cytoplasm and translation Figure 9.13b Polyomaviruses and tumor induction. tumor-induction proteins Transformation of cell to tumor state Cell transformation Figure 9.13b

9.7 DNA Tumor Viruses Genes involved in cancer are oncogenes C-oncogenes are plain old cellular genes that somehow lead to transformation and cancer: tumor suppressor or tumor activator genes-sometimes called proto-oncogenes before they start trouble V-oncogenes are viral versions of c-oncogenes

9.7 DNA Tumor Viruses Herpesviruses dsDNA, enveloped, nuclear replication Invertebrates, fish, reptiles, birds, mammals At least 8 types infect humans: HHV’s HHV-3 = VZV, HHV-4 = EBV Very widespread Cause latent infections

9.7 DNA Tumor Viruses Some Herpesviruses cause cancer Diverse mechanisms lead to cancer HHV-8 (Kaposi’s Sarcoma) Epstein-Barr Virus HHV-4 EBV causes Burkitt’s Lymphoma by disrupting regulation of the c-myc oncogene

III. Viruses with RNA Genomes 9.8 Positive-Strand RNA Viruses 9.9 Negative-Strand RNA Animal Viruses 9.11 Viruses That Use Reverse Transcriptase

9.8 Positive-Strand RNA Viruses Poliovirus (Figure 9.16a and b) Small virus Viral RNA is translated directly, producing a single long, giant protein (polyprotein) that undergoes self-cleavage to generate ~20 smaller proteins necessary for nucleic acid replication and virus assembly (Figure 9.16c)

Figure 9.16a Poliovirus. Figure 9.16a

Figure 9.16b Poliovirus. Figure 9.16b

Figure 9.16c AAAA 5′ + 3′ Replicase-mediated 3′ – 5′ 5′ + AAAA 3′ VPg Synthesis of the new plus strand Replicase-mediated 3′ – 5′ Synthesis of the minus strand Poliovirus genome 5′ + AAAA 3′ VPg Poly(A) Translation Polyprotein Proteases cleave the polyprotein. Figure 9.16c Poliovirus. Structural coat proteins Proteases RNA replicase Figure 9.16c

Replication of positive-strand RNA viruses requires a negative-strand RNA intermediate from which new positive strands are synthesized RNA replicase makes the viral RNAs

9.9 Negative-Strand RNA Animal Viruses Negative-strand RNA viruses Negative-strand RNAs are complementary to the mRNA They are copied into mRNA by an enzyme present in the virion Only those that infect Eukarya are known

9.9 Negative-Strand RNA Animal Viruses Influenza A Enveloped, pleomorphic virus (Figure 9.19) Segmented genome-8 pieces ss - RNA Surface proteins interact with host cell surface, elicit immune response

9.9 Negative-Strand RNA Animal Viruses Influenza Nuclear replication Genome segments transcribed and transcripts processed Cap-stealing ensures preferential treatment for influenza mRNAs Antigenic shift-new pandemic strains Antigenic drift-seasonal strains

Reassortment mechanism, New combinations of genes Produces new pandemic strains 2009 strain was a shift strain

9.11 Viruses That Use Reverse Transcriptase Retroviruses (RNA viruses) and hepadnaviruses (DNA viruses) use reverse transcriptase for replication Key step in virus replication cycle Reverse Transcriptase Enzyme activity that converts ss RNA into ds DNA

9.11 Viruses That Use Reverse Transcriptase Hepadnaviruses Virions small, irregular-shaped particles (Figure 9.23a) Unusual genomes partially double-stranded (Figure 9.23b) Include hepatitis B virus HBV Complex symptoms, may progress to chronic phase with liver cancer Viral replication occurs through an RNA intermediate Transmission through exposure to body fluids

9.11 Viruses That Use Reverse Transcriptase Retroviruses Genome RNA reverse transcribed to dsDNA Aka “cDNA” inserts into chromosome Provirus Gene expression and protein processing are complex (Figure 9.22) Gag, pol, env gene regions common to all retroviruses Retroviruses that cause cancer often have an extra gene region “src”

9.11 Viruses That Use Reverse Transcriptase Gag, pol, env regions common to all retroviruses Retroviruses that cause cancer often have an extra gene region “src” Rous sarcoma virus (chickens) has acquired a src gene Host cell gene acquired by defective excision “v-src” is the name given to the gene

IV. Subviral Agents 9.12 Viroids 9.13 Prions

9.12 Viroids Viroids: infectious RNA molecules that lack a protein coat Smallest known pathogens (246–399 bp) Cause a number of important plant diseases (Figure 9.24) Small, circular, ssRNA molecules (Figure 9.25) Do not encode proteins; completely dependent on host-encoded enzymes

Figure 9.24 Viroids and plant diseases.

9.13 Prions Prions: infectious proteins whose extracellular form contains no nucleic acid Known to cause disease in animals (transmissible spongiform encephalopathies) Host cell contains gene (PrnP) that encodes native form of prion protein that is found in healthy animals (Figure 9.27) Prion misfolding results in neurological symptoms of disease (e.g., resistance to proteases, insolubility, and aggregation)

Figure 9.27 Figure 9.27 Prions. Neuronal cell Prnp Nucleus DNA Transcription Translation Normal function Figure 9.27 Prions. PrPc (normal prion) PrPSc-induced misfolding of PrPC Abnormal function PrPSc (misfolded prion) Figure 9.27

Chronic Wasting Disease in deer and elk Mad Cow Disease Kuru, KJD in humans

10.12 Preserving Genome Integrity: CRISPR Interference CRISPR: Clustered Regulatory Interspaced Short Palindromic Repeats Type of prokaryotic "immune system" Region of bacterial chromosome containing DNA sequences similar to foreign DNA (spacers) alternating with identical repeated sequences (Figure 10.28) CRISPR-associated proteins (Cas proteins) Obtain and store segments of foreign DNA as spacers Recognize and destroy foreign DNA

Figure 10.28 Figure 10.28 Operation of the CRISPR system. Unique spacer sequences Bacterial chromosome DNA cas genes Repeat Repeat Cas proteins Transcription and translation Transcription CRISPR RNA Cutting of RNA by Cas proteins Foreign sequence is recognized by CRISPR RNA. Figure 10.28 Operation of the CRISPR system. Viral infection or conjugation Viral or plasmid DNA Cas proteins cut and destroy foreign nucleic acid. Figure 10.28