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1. Chapter 9 Virology

3. Viral Genomes Genetic material = DNA or RNA (only retroviruses contain both at a certain stage of their reproductive cycle) Genetic material may be ss or ds, ex. ssDNA, dsDNA, ssRNA, dsRNA. Viruses are often classified according to what type of genetic material they contain. All viruses obey the Central Dogma: DNA ? RNA ? protein (although, retroviruses carry out an extra step called reverse transcription)

4. Viral Hosts Viruses are also classified based on what type of host they infect. Animals, plants, and bacteria can all be infected by viruses. Bacterial viruses are called bacteriophages (or phages for short = “to eat”) There is a formal system of viral taxonomy featuring orders, families, genera, and species. Virus family names have the suffix -viridae.

5. Viral Structure Viruses are composed of: (1) genome, (2) protein coat (= capsid, coat, shell) (3) optional envelope (lipid membrane). Capsid subunits = capsomers, which undergo self-assembly to provide a protective coat surrounding the genome. The finished genome surrounded by capsid = nucleocapsid. Viruses vary widely in size and shape. They range from 0.02 ?m (20 nm) - 0.3 ?m (300 nm) in size.

6. Virus Symmetry Viruses are geometrical and highly symmetrical in shape. 2 types of symmetry: 1. Helical - rod-shaped viruses 2. Icosahedral - spherical viruses.

7. Enveloped Viruses The virus envelope consists of a lipid bilayer with proteins embedded in it. The lipids of the membrane are derived from the membranes of the host cell but the proteins are encoded by the virus.

8. Complex Viruses Some virions are even more complex, ex. T4 bacteriophage of E. coli, with possesses an icosahedral head and helical tails. Refer to Fig. 9.5 of the text.

9. Viral Enzymes Virions do not carry on metabolic processes, although some virions contain enzymes that play roles in the infection process. Ex. some virions contain lysozyme which makes holes in the bacterial cell wall to allow the viral nucleic acid to enter the cell. Viruses may contain their own nucleic acid polymerases or enzymes that the host cell does not have, ex. reverse transcriptase of retroviruses. Some viruses contain enzymes to aid in the release of new viruses from the host cell.

10. Studying Viruses Requires the Host Because viruses replicate only inside living cells, research on viruses requires use of appropriate hosts. Bacteriophages are the easiest to study in the lab. One must grow the host in order to study the virus. Animal cells or tissues can be grown in tissue or cell culture in order to study animal viruses without having to provide the whole animal.

11. Cell Culture 1. Remove tissue from animal 2. Dissociate cells by treating with enzyme 3. Spread suspension onto bottom of flat bottle or Petri dish (cells will adhere to surface) 4. Monolayer of cells is overlaid with suitable medium and culture is incubated This establishes a primary cell culture. Some primary cell cultures can grow indefinitely and be established as permanent cell lines.

12. Quantification of Viruses Viruses are so small that they cannot be quantified directly without a electron microscope. Generally, viruses are quantified by measuring their effects on the host cells they infect. Can calculate the # of virus infectious units this way.

13. Plaque assay When a virion initiates an infection on a layer or lawn of host cells growing on a flat surface, a zone of lysis or zone of growth inhibition may occur, resulting in a clear area in the lawn of growing host cells. This clearing = a plaque. (Where have we seen this concept before?) This procedure permits the isolation of pure virus strains because if a plaque has arisen from a single virion, all the virions inthis plaque are probably genetically identical. In some cases, the virus may not destroy the host cells, but cause changes in morphology or growth rate called transformation. Transformed cells may lose contact inhibtion - what is this?

14. Efficiency of Plating Counts made by plaque assay are always lower than counts made the the EM because virions don’t usually infect hosts with 100% efficiency. When the plaque method is using to quantify virus, the conc. (titer) of the virus suspension should be expressed as the # of plaque-forming units (what does this term sound like?).

15. Animal Infectivity Methods Some viruses do not cause recognizable effects in cell cultures but result in the death of the whole animal. With animal infectivity methods, the virus suspension is serially diluted, injected into susceptible animals, incubated for a period of time, and the fraction of live and dead animals is calculated at each dilution in order to get an end point dilution. This method is less accurate than cell culture methods, but necessary to study some viruses.

16. Viral Replication Viruses: hijackers of the molecular world! They hold host cells “hostage,” forcing them to make more viruses. 1. Attachment (adsorption) of a virion to a susceptible host cell. 2. Penetration of the virion or its nucleic acid into the cell. 3. Synthesis of nucleic acid and protein: virus redirects cell metabolism to synthesize new virus parts. 4. Assembly of structural subunits and packaging of nucleic acid into new virus particles. 5. Release of mature virions from the cell.

17. Viral Replication

18. Viral Replication (cont.) Viruses have a one-step growth curve. After attachment and penetration, the virus uncoats, undergoing an eclipse period, during which its infectivity is greatly decreased. Maturation occurs in the assembly/packaging stage during which the infectivity of the virus titer increases. 2 mechanisms of release: (1) cell lysis (the virus titer reaches a burst size.) or (2) budding/excretion.

19. Attachment Viruses have 1+ proteins on their surfaces which interact with specific receptors on the surface of target host cells in order to gain entry into the cells. The receptors on the surface of the host are normal surface components of the host (ex. proteins, carbs., lipids, etc.), which carry out normal functions for the cell (i.e., they aren’t there just for viruses to gain entry). Receptors determine which type(s) of cells will be susceptible to which virus(es). If a receptor is altered it can make the cell resistant to infection. However, viruses may use many receptor, so one that is changed won’t prevent binding. Also, viruses can mutate to cope with a changed receptor.

20. Penetration Attachment of a virus to a host cell results in changes to the virus and/or the host cell that allow penetration of the virus into the cell. A cell that allows multiplication of a virus to take place = permissive for that virus. Strategies for penetration: enveloped virus can fuse with cell membrane and uncoat there, viruses can enter cells through endocytosis – what’s that? Penetration is more complex in bacterial cells with cell walls than in other cells, ex. animal cells, without cell walls.

21. T4 Bacteriophage Has a head and a tail + tail fibers. Head contains DNA. Tail fibers attach to cell surface via core polysaccharides in the gram neg. cell wall. Tail fibers retract, tail makes contact with cell surface. A lysozyme-like enzyme from the virus bore a hole in the cell wall. The tail sheath contracts, extruding DNA from the head into the cell.

22. Host Cell vs. Virus Modification of host cell receptor. Destruction of viral DNA within the host cell via restriction endonucleases (= restriction enzymes) that cleave the DNA at specific points. Host cell DNA is protected from its own restriction enzymes by modifying its own DNA, ex. methylation of purines or pyrimidines. Viruses can fight back by modifying their own DNA by methylation or glucosylation or by inhibiting host restriction systems. However, some restriction enzymes only restrict modified DNA.

23. Viral Replication Schemes Class I = dsDNA: production of mRNA occurs as it would in host genome. Class II = ssDNA: have to make complementary strand of DNA ? dsDNA ? transcription into mRNA. Class III-VII: special virus situations in which viral polymerases or other special viral enzymes are often required to replicate the virus

24. Viral Proteins 1. Early proteins: synthesized soon after infection, necessary for replication of virus nucleic acid. 2. Late proteins: synthesized later, include proteins for the virus coat.

25. Overview of Bacterial Viruses Infect Bacteria and Archaea. Most common in the environment have dsDNA. Most don’t have lipid envelopes. Most are structurally complex.

26. Virulent vs. Temperate Viruses Virulent viruses lyse or kill their hosts after infection. Temperate viruses achieve a state where their genome replicates along with the host genome without killing the host.

27. Virulent Bacteriophage T4 Bacteriophages with linear, dsDNA that infect E. coli and related Bacteria are called T1, T2, … T7 Bacteriophages. The most studies is the T4 Bacteriophage. T4 encodes over 250 proteins, some of which are nucleases that destroy the host DNA to obtain building blocks for viral DNA. The T4 phage also codes for T4 lysozyme, which attacks the PG of the host cell.

28. Temperate Bacteriophage Lambda Temperate bacteriophages or animal viruses can enter into a state called lysogeny, in which most viral genes are not expressed. The virus genome is duplicated along with the genome of the host. During cell division, the virus is passed from one generation of bacteria to the next. These bacteria are called lysogens and spontaneously produce virions under certain conditions. Lytic pathway: expression of the viral genome leading to the production of virions and cell death. Provirus or prophage: latent state inside host cell. Phage-encoded repressor protein represses the expression of other viral genes and gives the host cell immunity against infection by the same type of virus. The lytic vs. lysogenic pathways are controlled by a genetic switch. Agents that damage DNA can cause a temperate virus in the lysogenic state to become lytic.

29. Temperate Bacteriophage Lambda (cont.)

30. Overview of Animal Viruses For many bacteriophages, only the genome and one or two proteins enters the cytoplasm of the host cell. For animal viruses, the entire virion, or at least the nucleocapsid, typically enters the cytoplasm by endocytosis and must be uncoated. Replication strategies differ in prok. vs. euk. due to the presence of the nucleus, ex. a DNA virus that uses host polymerases must replicate in the nucleus. + strand RNA viruses must have appropriate modifications (like euk. RNA has) in order to serve directly as mRNA.

31. Overview of Animal Viruses (cont.) Persistent infections vs. latent infections - what are they? Viruses can cause normal cells to undergo transformation into cancer cells. Cancer = uncontrolled cell growth. Neoplasm, tumor, benign, malignant, metastasis - what do these terms mean? How do proto-oncogenes and tumor suppressor genes affect the occurrence of cancer?

32. Retroviruses RNA genome ? DNA copy through process called reverse transcription catalyzed by an enzyme called reverse transcriptase. Ex. HIV ? AIDS Enzymes found in retroviruses: reverse transcriptase, DNA endonuclease (integrase), and protease.

33. Retroviruses (cont.) 1. Entrance into the host cell via fusion with cell membrane at sites of specific receptors. 2. Virion uncoating at membrane (genome and enzymes remain in core). 3. Reverse transcription of one of the two RNA genomes into ssDNA ? converted to linear dsDNA by reverse transcriptase, which then enters the nucleus. 4. Integration of DNA copy of virus into host genome. 5. Transcription of viral DNA ? formation of viral mRNAs and progeny viral RNA. 6. Encapsidation of the viral RNA into nucleocapsids in the cytoplasm. 7. Budding of enveloped virions at the cytoplasmic membrane and release from the cell.

34. Retroviruses (cont.)

35. Viroids and Prions Viroids: small, circular, ssRNA molecules that mimic dsDNA, smallest known pathogens, extracellular form = naked RNA (no capsid), contains no protein-encoding genes, is totally dep. on the host cell for replication. Prions: extracellular form = protein only, contains no nucleic acid, infectious protein, cause serious diseases, ex. scrapie in sheep, bovine spongiform encephalopathy in cattle = mad cow disease, Creutzfeldt-Jakob disease in humans = mad cow disease in humans (prion “jumped” the species barrier, though it is inefficiently transmitted). Prions subvert host enzymes and cause a normal host gene to produce more copies of the pathogenic protein.