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General Virology

General Virology. VIRUS STRUCTURE. Virion vs virus. Virion is the infectious particle composed of nucleic acid, protein capsid, +/- envelope may be extracellular or intracellular Virus is any stage of infection. How do we know that NA is genetic material?. Hershey-Chase.

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General Virology

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  1. General Virology VIRUS STRUCTURE

  2. Virion vs virus • Virion is the infectious particle • composed of nucleic acid, protein capsid, +/- envelope • may be extracellular or intracellular • Virus is any stage of infection

  3. How do we know that NA is genetic material? Hershey-Chase Fraenkel-Conrat Experiment TRANSFECTION EXPTS

  4. TRANSFECTION FAILS FOR SOME VIRUSES WHY?

  5. Capsid • Functions • Protection of NA • Attachment for naked viruses • Enzyme • Helical vs Icosahedral Symmetry - Why do most viruses look alike? • Tobacco mosaic virus is a ssRNA virus composed of 6000 nucleotides. The capsid is made of 2100 copies of a single protein subunit that contain 158 amino acids. Calculate the percentage of the genome that is used for structure.

  6. How do helical viruses differ? • Helical- one axis of symmetry down center • Multiple structural units

  7. Icosahedral symmetry • 20 identical equilateral triangles • Structural units on faces to give morphological capsomers • Pentons (5 fold axis of symmetry) • Hexons • 3 fold through face • 2 fold through edge How do spherical viruses differ?

  8. Envelope • Attachment • Entry • Assembly- matrix proteins • Release • Proteins are viral • Lipids are host • Rare in plants or bacteria - why? • If the membrane envelope is destroyed, the virus becomes noninfectious. Why?

  9. Herpesvirus complexity • Tegument proteins - 12/84 viral proteins in HSV • Potential role? • Virion mRNA • DNAase virion nucleic acids • RT-PCR • probe genome array • Potential role?

  10. Genome - DNA or RNA How do we experimentally show that DNA or RNA is the virus genetic material? • strandedness - (single) (double) • linear or circular, partial double stranded circle • number (single, segmented, multicomponent)

  11. RNA Genomes • sense (positive-sense, negative-sense, ambisense) • presence or absence of 5'-terminal cap or 5'-covalently-linked protein • presence or absence of 3'-terminal poly (A) tract • Retroviruses - replication strategy

  12. Some viruses have high degree of secondary structure • Poliovirus - 5’ internal ribosome entry site (IRES) Guest et al. 2004. J. Virol. 78: 11097.

  13. SARS/coronaviruses have conserved 3’region • SARS s2m in red • a - green = 530 loop of 16S RNA • Similar binding properties: • b - blue = S12 • magenta = IF1 • Possible role for s2m • Hijacks protein synthesis from cell(binding cell factors) • Needed to bind to similar viral protein for transcription • Potential drug target in red tunnel Robertson et al. 2005. PLOsBiology:3.

  14. DNA Viruses may be large genomes • PolyDNAvirus (PDV) - contain many DNA segments • Mimivirus - larger than small bacteria

  15. Host-induced modification • Viral property that varies depending on the host • Phage DNA hydroxymethyl cytosine (HMC) replaces C • Viral enzymes: C to HMC • Viral DNA polymerase: adds HMC not C • What is advantage of HMC? • Glucose is attached to HMC • Host enzyme needed to prepare glucose • Protects against host nuclease

  16. What would happen if virus without glucose enters host with RE? • What would happen if virus with glucose enters host w/o enzyme to create UDP- glucose? Host enzyme makes

  17. Proteins • structural proteins • non-structural virion proteins • transcriptase, • protease • integrase

  18. How to identify virion proteins • Purify KSHV virions • Run on SDS PAGE • Excise bands, digest - get sequence and compare to database

  19. Chemical synthesis of poliovirus: What are the implications? • Small genome positive strand RNA - sequence known • Synthesized small DNA segments (~ 69 nucleotides) with overlapping complementary segments • Added a T7 phage promotor to DNA • Used DNA to make genome RNA in HeLa cell lysate with T7 polymerase • Results: How do you show success?

  20. Morphology virion size enveloped or naked nucleocapsid capsid symmetry and structure Genome characteristics Replication strategy Antigenic Properties International Congress on Taxonomy of Viruseshttp://www.ncbi.nlm.nih.gov/ICTV/

  21. Baltimore classification

  22. WHY TRANSFECTION FAILS

  23. ONE STEP GROWTH CURVE • 1939- Ellis and Delbruck: • Infection with a high multiplicity of infection (MOI): ratio of virus to host cell • Simultaneous infection • Single replication cycle • Sample at time intervals by plaque count for plaque-forming units (PFU), • Identification of latent phase • Determination of burst size/viral yield

  24. Measuring Intracellular Events • Sample at time intervals after lysing cells (1952 - Doermann) • Chloroform • Lysis from without • Identification of eclipse and maturation phases Maturation phase

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