1 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 8.4 Culturing, Detecting, and Counting Viruses 8.8 Temperate Bacteriophages and Lysogeny

2 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)

3 Viral genomes (Figure 8.2) a variety of formsEither 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.

4 9.1 Size and Structure of Viral Genomes-RangeViral genome size (Figure 9.1) Smallest circovirus: 1.75-kilobase single strand Largest megavirus: 1.25-megabase pairs (Pandoravirus)

5 Figure 9.1 Comparative genomics.

6 8.1 What Is a Virus? Virus taxonomyViruses are usually classified on the basis of the nucleic acid they contain-see Baltimore Scheme Particle shape and host range are less important

7 9.1 Size and Structure of Viral GenomesTaxonomy Based mainly on idea proposed by David Baltimore (Baltimore Scheme) Depends on relationship of genome to mRNA Genome structure is the key feature

8 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 9.2 Viral Evolution No one knows where viruses came fromViruses may date from before the evolution of cells or Viruses may have evolved after cells Or both

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11 8.2 Structure of the VirionViral 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

12 8.2 Structure of the VirionViral 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

13 Naked virus Enveloped virus (Unenveloped) Figure 8.1 NucleocapsidNucleic 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

14 8.2 Structure of the VirionNucleocapsids 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

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

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

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18 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

19 8.2 Structure of the VirionSome 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

20 8.3 Overview of the Virus Life CyclePhases 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

21 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

22 Maturation Figure 8.7 Nucleic acid Protein coats Early enzymes VirusEclipse 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

23 8.4 Culturing, Detecting, and Counting VirusesViruses 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

24 8.4 Culturing, Detecting, and Counting VirusesPlaques: are clear zones that develop on lawns of host cells Bacterial lawn or animal cell 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

25 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

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

27 8.8 Temperate Bacteriophages and LysogenyViral 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)

28 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

29 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

30 Lambda DNA forms a circleAttaches 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

31 Lambda can cause specialized transduction

32 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

33 Rolling Circle Replication

34 II. Viruses with DNA Genomes8.10 An Overview of Animal Viruses 9.6 Uniquely Replicating DNA Animal Viruses 9.7 DNA Tumor Viruses

35 8.10 An Overview of Animal VirusesOften, 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

36 Figure 8.21 Diversity of animal viruses.

37 8.10 An Overview of Animal VirusesConsequences 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

38 9.6 Uniquely Replicating DNA Animal VirusesDouble-stranded DNA animal viruses that have unusual replication strategies Pox viruses Adenoviruses

39 9.6 Uniquely Replicating DNA Animal VirusesPox viruses Among the most complex and largest animal viruses known (Figure 9.11) Infect many types of animals Monkeypox, camelpox, cowpox Variola virus (Smallpox virus) most famous-extremely lethal up to 20th century Vaccinia virus-vaccine strain derived from cowpox DNA replicates in the cytoplasm

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

41 9.6 Uniquely Replicating DNA Animal VirusesAdenoviruses 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

42 Covalently linked protein TPpTP is primer Strand displacement replication Viral proteins

43 9.7 DNA Tumor Viruses Some DNA viruses can induce cancer (“tumor viruses or “oncoviruses” Human Papilloma Viruses (HPV) Herpesviruses

44 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

45 9.7 DNA Tumor Viruses Some papilloma viruses cause cancerIn 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

46 Interfere with regulationInfection 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

47 9.7 DNA Tumor Viruses Genes involved in cancer are oncogenesC-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

48 9.7 DNA Tumor Viruses HerpesvirusesdsDNA, 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

49 9.7 DNA Tumor Viruses Some Herpesviruses cause cancerDiverse mechanisms lead to cancer HHV-8 (Kaposi’s Sarcoma) Epstein-Barr Virus EBV causes Burkitt’s Lymphoma by disrupting regulation of the c-myc oncogene

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51 III. Viruses with RNA Genomes9.8 Positive-Strand RNA Viruses 9.9 Negative-Strand RNA Animal Viruses 9.11 Viruses That Use Reverse Transcriptase

52 9.8 Positive-Strand RNA VirusesPoliovirus (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)

53 Figure 9.16a Poliovirus. Figure 9.16a

54 Figure 9.16b Poliovirus. Figure 9.16b

55 Figure 9.16c AAAA 5′ + 3′ Replicase-mediated 3′ – 5′ 5′ + AAAA 3′ VPgSynthesis 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

56 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

57 9.9 Negative-Strand RNA Animal VirusesNegative-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

58 9.9 Negative-Strand RNA Animal VirusesInfluenza A Enveloped, pleomorphic virus (Figure 9.19) Segmented genome-8 pieces ss - RNA Surface proteins interact with host cell surface, elicit immune response

59 9.9 Negative-Strand RNA Animal VirusesInfluenza Nuclear replication Genome segments transcribed and transcripts processed Cap-stealing ensures preferential treatment for influenza mRNAs

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

61 9.11 Viruses That Use Reverse TranscriptaseRetroviruses (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

62 9.11 Viruses That Use Reverse TranscriptaseHepadnaviruses 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

63 9.11 Viruses That Use Reverse TranscriptaseRetroviruses 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”

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65 9.11 Viruses That Use Reverse TranscriptaseGag, 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

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71 IV. Subviral Agents 9.12 Viroids 9.13 Prions

72 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

73 Figure 9.24 Viroids and plant diseases.

74 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)

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

76 Chronic Wasting Disease in deer and elkMad Cow Disease Kuru, KJD in humans

77 10.12 Preserving Genome Integrity: CRISPR InterferenceCRISPR: 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

78 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 Operation of the CRISPR system. Viral infection or conjugation Viral or plasmid DNA Cas proteins cut and destroy foreign nucleic acid. Figure 10.28