1. PowerPoint ® Lecture Presentations prepared by John Zamora Middle Tennessee State University C H A P T E R © 2015 Pearson Education, Inc. Viruses and Virology 8

2. © 2015 Pearson Education, Inc. I. The Nature of Viruses 8.1What Is a Virus? 8.2Structure of the Virion 8.3Overview of the Virus Life Cycle 8.4Culturing, Detecting, and Counting Viruses

3. © 2015 Pearson Education, Inc. 8.1 What Is a Virus? Virus: genetic element that cannot replicate independently of a living (host) cell Virology: the study of viruses 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

4. © 2015 Pearson Education, Inc. Figure 8.1 Nucleocapsid Envelope Nucleic acid Capsid Nucleic acid Capsid (composed of capsomeres) Naked virus Enveloped virus 8.1 What Is a Virus?

5. © 2015 Pearson Education, Inc. 8.1 What Is a Virus? Viral genomes Either DNA or RNA genomes Some are circular, but most are linear Figure 8.2 Genome: Types: ssDNA dsDNAssRNA dsRNAssRNA (Retroviruses) dsDNA (Hepadnaviruses) DNARNA DNA Viruses

6. © 2015 Pearson Education, Inc. 8.1 What Is a Virus? Viral hosts and taxonomy Viruses can be classified on the basis of the hosts they infect Bacterial viruses (bacteriophages) Archaeal viruses Animal viruses Plant viruses Other viruses

7. © 2015 Pearson Education, Inc. 8.2 Structure of the Virion Capsid: the protein shell that surrounds the genome of a virus particle Capsomere: subunit of the capsid Smallest morphological unit visible with EM Nucleocapsid: complete complex of nucleic acid and protein packaged in the virion Figure 8.3 TMV

8. © 2015 Pearson Education, Inc. 8.2 Structure of the Virion 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 Figure 8.3 TMV

9. © 2015 Pearson Education, Inc. Figure 8.4 Icosahedral symmetry: spherical viruses Most efficient arrangement of subunits in a closed shell 8.2 Structure of the Virion

10. © 2015 Pearson Education, Inc. 8.2 Structure of the Virion Enveloped viruses Have membrane surrounding nucleocapsid Lipid bilayer with embedded proteins Envelope makes initial contact with host cell Figure 8.5 InfluenzaVaccinia

11. © 2015 Pearson Education, Inc. 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 Neuraminidases Enzymes that cleave glycosidic bonds Allows liberation of viruses from cell Tamiflu

12. © 2015 Pearson Education, Inc. Head Figure 8.6 Virion DNA Cell (host) 1. Attachment (adsorption of phage virion) Penetration of viral nucleic acid 2. Synthesis of viral nucleic acid and protein 3. Assembly and packaging of new viruses 4. Cell lysis and release of new virions 5. Virions Protein coat remains outside Viral DNA enters 8.3 Overview of the Virus Life Cycle Phases of viral replication

13. © 2015 Pearson Education, Inc. Figure 8.7 Virus replication is typically characterized by a one-step growth curve 8.3 Overview of the Virus Life Cycle Burst size: number of virions released

14. © 2015 Pearson Education, Inc. 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 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

15. © 2015 Pearson Education, Inc. 8.4 Culturing, Detecting, and Counting Viruses Titer: number of infectious units per volume of fluid Figure 8.8 Mixture containing molten top agar, bacterial cells, and diluted phage suspension Phage plaques Nutrient agar plate Sandwich of top agar and nutrient agar Lawn of host cells The cell–phage mixture is poured onto a solidified nutrient agar plate. 1. The mixture is left to solidify. 2. 3. Incubation allows for bacterial growth and phage replication. Plaques Bacteriophage T4 infection

16. © 2015 Pearson Education, Inc. Viral plaques Confluent monolayer of animal tissue cells Figure 8.9 8.4 Culturing, Detecting, and Counting Viruses

17. © 2015 Pearson Education, Inc. 8.4 Culturing, Detecting, and Counting Viruses Plating efficiency is used in quantitative virology The number of plaque-forming units is almost always lower than direct counts by electron microscopy Inactive virions Conditions not appropriate for infectivity

18. © 2015 Pearson Education, Inc. 8.5 Attachment and Entry of Bacteriophage T4 Attachment of virion to host cell is highly specific Requires complementary receptors on the surface of a susceptible host and its infecting virus 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

19. © 2015 Pearson Education, Inc. Chi Flagellum Pilus MS2 M13 T1 Iron transport protein T4 ϕ X174 LPS Outer membrane PeptidoglycanCytoplasmic membrane Figure 8.10 8.5 Attachment and Entry of Bacteriophage T4

20. © 2015 Pearson Education, Inc. 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

21. © 2015 Pearson Education, Inc. Figure 8.11 8.5 Attachment and Entry of Bacteriophage T4 a) Virions attach to cells via tail fibers that interact with polysaccharides on E. coli cell envelope b) Tail fibers retract, and tail core makes contact with E. coli cell wall c) Lysozyme-like enzyme forms small pore in peptidoglycan. Tail sheath contracts, and viral DNA passes into cytoplasm

22. © 2015 Pearson Education, Inc. 8.6 The T4 Genome Many eukaryotes possess mechanisms to diminish viral infections: for example, immune defense mechanisms, RNA interference Prokaryotes also possess mechanisms CRISPR Similar to RNA interference Nucleases guided to specific DNA sequence Prokaryotic immune system

23. © 2015 Pearson Education, Inc. 8.6 The T4 Genome Restriction modification DNA destruction system; effective only against double-stranded DNA viruses Restriction enzymes cleave DNA at specific sequences Modification of host's own DNA at restriction enzyme recognition sites prevents cleavage of own DNA CH 3 | 5’--- G A T C ---3’ 3’--- C T A G ---5’ | CH 3 DAM 5’--- G A T C ---3’ 3’--- C T A G ---5’ DpnIII

24. © 2015 Pearson Education, Inc. 8.6 The T4 Genome Viral mechanisms to evade bacterial restriction systems Production of proteins that inhibit host cell restriction system Chemical modification of viral DNA (glycosylation or methylation)

25. © 2015 Pearson Education, Inc. 8.6 The T4 Genome T4 DNA contains the modified base 5-hydroxymethylcytosine Cytosine 5-hydroxymethyl- cytosine Site of glucosylation An unusual base in T4 DNA Figure 8.12b

26. © 2015 Pearson Education, Inc. Infection T4 nucleases, DNA polymerase, and new sigma factors Phage T4 DNAPhage head proteins Tail, collar, base plate, and tail fiber proteins Mature T4 virion T4 lysozyme production Early mRNA Middle mRNALate mRNA Phage DNA replication Early proteins Transcription Translation Late proteinsMiddle proteins Minutes 0510152025 Self-assembly Lysis Figure 8.13 8. 7 Replication of Bacteriophage T4 T4 genome can be divided into three parts: early, middle, and late proteins

27. © 2015 Pearson Education, Inc. Figure 8.14 Prohead Motor Scaffold proteins Capsid proteins Portal proteins dsDNA Packaging motor attaches to prohead. Packaging motor complex Scaffold proteins discarded Other assembly steps Packaging motor discarded Mature virion ATP 8. 7 Replication of Bacteriophage T4 Packaging the T4 genome

28. © 2015 Pearson Education, Inc. 8.8 Temperate Bacteriophages and Lysogeny Viral life cycles Virulent mode: viruses lyse host cells after infection Temperate mode: viruses replicate their genomes in tandem with host genome and without killing host 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. © 2015 Pearson Education, Inc. Figure 8.15 8.8 Temperate Bacteriophages and Lysogeny

30. © 2015 Pearson Education, Inc. 8.8 Temperate Bacteriophages and Lysogeny Bacteriophage lambda Infects E. coli; Linear, dsDNA genome Figure 8.16

31. © 2015 Pearson Education, Inc. Figure 8.17a Complementary, single-stranded regions 12 nucleotides long at the 5′ terminus of each strand Upon penetration, DNA ends base-pair, forming the cos site, and the DNA ligates and forms double-stranded circle When lambda is lysogenic, its DNA integrates into E. coli chromosome at the lambda attachment site (attλ) 8.8 Temperate Bacteriophages and Lysogeny

32. © 2015 Pearson Education, Inc. 8.8 Temperate Bacteriophages and Lysogeny When Bacteriophage lambda enters lytic pathway, lambda synthesizes long, linear concatemers of DNA by rolling circle replication Figure 8.17b Rolling Circle Video https://www.youtube.com/watch?v=fB1OkshHAVw

33. © 2015 Pearson Education, Inc. 8.8 Temperate Bacteriophages and Lysogeny Regulation of lytic vs. lysogenic events in lambda is controlled by a complex genetic switch Key elements are two repressor proteins cI protein (the lambda repressor): causes repression of lambda lytic events Cro repressor: controls activation of lytic events

34. © 2015 Pearson Education, Inc. Figure 8.19 8.9 An Overview of Bacterial Viruses Bacteriophages are very diverse and can be classified on the basis of their genome structure

35. © 2015 Pearson Education, Inc. 8.9 An Overview of Bacterial Viruses 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)

36. © 2015 Pearson Education, Inc. Figure 8.20 8.9 An Overview of Bacterial Viruses Best-studied bacteriophages infect enteric bacteria (e.g. 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

37. © 2015 Pearson Education, Inc. 8.10 An Overview of Animal Viruses Entire virion enters the animal cell, unlike in prokaryotes Eukaryotic cells contain a nucleus, 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

38. © 2015 Pearson Education, Inc. Figure 8.21 8.10 An Overview of Animal Viruses

39. © 2015 Pearson Education, Inc. Figure 8.22 8.10 An Overview of Animal Viruses

40. © 2015 Pearson Education, Inc. 8.10 An Overview of Animal Viruses Retroviruses: RNA viruses that replicate through a DNA intermediate Figure 8.23a

41. © 2015 Pearson Education, Inc. 8.10 An Overview of Animal Viruses Retroviruses have a unique genome Two identical ssRNA molecules that resemble mRNA Contain specific genes gag: encode structural proteins pol: encode reverse transcriptase and integrase env: encode envelope proteins Figure 8.23b

42. © 2015 Pearson Education, Inc. Figure 8.24 Replication of a retrovirus Process of replication of a retrovirus: Entrance into the cell Removal of virion envelope at the membrane Reverse transcription of one of the two RNA genomes Integration of retroviral DNA into host genome Transcription of retroviral DNA Assembly and packaging of genomic RNA Budding of enveloped virions; release from cell

43. © 2015 Pearson Education, Inc. 8.11 The Virosphere and Viral Ecology About 10 6 prokaryotes per ml of seawater About 10 7 viruses per ml of seawater Figure 8.25

44. © 2015 Pearson Education, Inc. 8.11 The Virosphere and Viral Ecology Bacteriophages thought to have major impact on evolution of Bacteria (Chapter 9) 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

45. © 2015 Pearson Education, Inc. 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