Group Case Study Presentation Evaluation: 50 points

1. Group #1 = 49.4 #2 = 49.4 #3 = 49.2 #4 = 49.8 #5 = 49.0 #6 = 48.3 #7 = 48.4 #8 = 49.8 Group #9 = 49.6 #10 = 49.5 #11 = 47.6 #12 = 49.3 #13 = 49.3 #14 = 49.8 #15 = 48.7 Group Case Study Presentation Evaluation: 50 points

2. Replication of Reverse-Transcribing Virus

3. Family Retroviridae • “backward” nucleic acid synthesis • Convert genomic viral (+)RNA -> cellular dsDNA (provirus) • Uses RT (reverse transcriptase), RNA-dependent, DNA polymerase (also DNA-dependent, DNA polymerase)

4. Sub-Family: Spumavirinae • “foamy” vacuoles in cell culture • Mammals, primates • Human foamy virus – first retrovirus found in humans • “orphan virus” - no associated disease

5. Sub-Family: Oncovirinae • “tumor” • infection leads to cell transformation • RNA tumor virus • Avian, reptile, mammals, primates • Human T-cell leukemia virus (HTLV)

6. Sub-Family: Lentivirinae • “slow” • Persistent chronic infection • Chronic disease of CNS, lung, immune deficiency • No cell transformation • Mammals, primates • Human immunodeficiency virus (HIV)

7. Lentivirus: HIV • Envelope (env) - 120 nm, glycoprotein spikes • Matrix protein (gag) • Capsid -icosahedral, wedge-shape • Nucleoprotein (gag) – group-specific antigen • Genome – two copies (+)RNA • Enzymes (prot:pol:int) – protease, polymerase (RT, RNAse-H), integrase

8. HIV Genome: (+)RNA • Two RNA molecules associate by dimer linkage site • 10 kb; 5’ cap, 3’ polyA tail • Three major genes -(gag, pol, env) • Complex overlapping genes found in Lentivirus - regulatory, accesory (vif, tat, rev, vpu, vpr)

9. HIV Genome: 5’ End Region • R – terminal repeat, important for reverse transcription • U5 – unique 5’ end sequence (becomes 3’end of proviral DNA, signal for poly-A addition to mRNA) • PB – primer binding site of cell tRNA • Leader – recognition sequence for packaging genome RNA, donor site for all spliced subgenomic mRNAs

10. HIV Genome: Major Genes • gag (“group-specific antigen”) - code for structual proteins; capsid, matrix, nucleoprotein (RNA-binding) • pol (prot:pol:int) – code for enzymes • Protease cleaves viral polyprotein • RT/RNase for reverse transcription • Integrase cuts cell DNA to insert proviral DNA • env– code for envelope glycoproteins; surface, transmembrane

11. HIV Genome: 3’ End Region • PP – polypurine (A-G) tract, initiation site for viral (+)DNA synthesis • U3 – unique 3’ end sequence (becomes 5’ end of proviral DNA), regulatory sequences for mRNA transcription & DNA replication • R – terminal repeat, for reverse transcription

12. HIV Provirus (dsDNA) Replication • Uncoat in cytoplasm, viral genome (+)RNA with RT -> (-)DNA -> (±)DNA, transport into nucleus • Evidence for viral DNA: • Virus replication inhibited by actinomycin-D (blocks DNA->mRNA) • Infected cells have DNA complimentary to viral RNA • Discovery of viral RT

13. Reverse Transcription (ssRNA to dsDNA) • Cell tRNA primer at PB internal site • (-)DNA synthesis, simultaneous RNA degradation by RT • “strong stop” at end, reinitiate DNA synthesis by “jumping” to other end • PP (short RNA sequence of genome) primer for (+)DNA strand synthesis • “strong stop” at end, “jumping” to other end • Proviral dsDNA with novel ends, Long Terminal Repeat (U3, R, U5)

14. Reverse Transcription: “1st Jump” • 1. Primer tRNA anneals to PBS (genome RNA); RT makes (-)DNA (R U5) copy of 5’ end; RNase H removes hybridized RNA (R, U5) • 2.“(-)DNA strong stop” • 3. “First Jump” – (-)DNA R hybridizes to RNA R sequence at 3’end • 4.(-)DNA extended and completed (to PBS); most RNA removed, except PP tract

15. Reverse Transcription: “2nd Jump” • 5.PP primer for (+)DNA (5’ end U3RU5) synthesis; RNase H degrades PP tract • 6.“(+)DNA strong stop” • 7. “2nd Jump” – (+)DNA binds to PBS near 3’ end of (-)DNA • 7a. RNase H degrades PBS/tRNA of (-)DNA • 8. Both strands extended & Provirus completed: • dsDNA • LTR at ends

16. HIV Provirus Integration Into Cell DNA • Requires viral LTR on ends of DNA • Viral integrase (endonuclease) nicks cell DNA at random sites • Viral DNA ligated into cell DNA • Integration required for retrovirus infection • Free viral RNA / DNA degraded by host cell

17. HIV Provius Gene Expression • Uses host cell RNA pol II • Genome length mRNA: • Translates for gag or gag-pol proteins (by translational frame shift) • Genome for progeny virus • Multiple splicing for subgenomic mRNAs

18. HIV Spliced mRNAs • Translates for env proteins • Translates for regulatory & accessory proteins • Switch for subgenomic, genomic mRNAs • Down-regulate (nef) • Activate (tat) • Infectivity (vif)

19. HIV Genomic/Sub-genomic mRNAs

20. HIV Assembly/Release • Viral genome mRNA in cytoplasm associates with viral nucleoprotein and viral pol proteins • Capsid formation, insert genome RNA, migrate to matrix protein at cell plasma membrane • Capsid picks up envelope by budding through plasma membrane, exits cell

21. HIV Pathogenesis • Infects macrophage (phagocytic defense) & helper T cell (regulates both humoral & cell-mediated immunity) • Persistent chronic infection in lymphoid tissue (clinical symptom of PGL = persistent generalized lymphadenopathy) • Virus held in low level by host defense • Over time, virus replicates to high level, destroys T cells, host immunity impaired • Clinical AIDS disease, opportunistic infections, and death • Follow course of infection by: CD4+T cells, HIV (RNA), clinical disease in patient

22. Natural History of HIV Infection

23. Retrovirus Oncogene • Oncogene: gene encoding the proteins originally identified as the transforming agents of oncogenic viruses, some of which were shown to be normal components of cells (growth control proteins) • v-onc is viral version of an oncogene • c-onc is cellular version of same gene • Most likely v-onc subverted from cell

24. Oncornavirus: Three Mechanisms for Cell Transformation • 1. Oncogene Transforming Protein • 2. Alter Host Cell Regulation • 3. Stimulate Host Cell Growth • Useful models in study of cell regulation and cell transformation • Most human cell cancers due to chemical carcinogens

25. Oncornavirus: 1. Oncogene Transforming Protein • Rapid transforming • Rous sarcoma virus in chickens • “src” (v-onc) • Gene product - tyrosine kinase, up-regulates cell metabolism • Leads to rapid cell transformation

26. Oncornavirus: 2. Alter Host Cell Growth Regulation • Slow transforming • Virus does not have oncogene • Murine leukemia virus integrates into cell DNA • Turns on c-onc, up-regulates host cell • Continued cell activation, over period of time, leads to cell transformation

27. Oncornavirus: 3. Stimulate Host Cell Growth • Slow transforming • Virus does not have oncogene • Human T-cell leukemia virus (HTLV) • Infects T lymphocyte, release of cytokines, stimulates growth of neighboring T cells • Continued T cell activation, over time leads to cell transformation

28. Cellular Retrovirus-Like Genetic Elements • 1940’s - Barbara McClintock propose “moveable genes” by genetic studies of maize • Remove & insert circular genetic elements • Allow for genetic diversity • Bacterial transponsons: drug resistance • Retrotransposons: yeast, drosophila • Retroposons: humans

29. Reading & Questions • Chapter 19: Retroviruses: Converting RNA to DNA • Omit Chapter 20: Human Immunodeficiency Virus Type 1 (HIV-1) and Related Lentiviruses • Questions: 1, 2, 8, 9

30. QUESTIONS???

31. Class Discussion – Chapter 12 • 1. How does reverse transcriptase (RT) synthesize RNA into DNA utilizing three different enzyme activities? • 2. Why must the retrovirus DNA replication complex make two “jumps”? How is it able to “jump”? Seriously, does DNA really “jump”? • 3. Is reverse transcription unique to viruses?

32. MICR 401 Final Exam • Tuesday, Dec. 4, 2012 • 1:30 – 3:00pm • Papovavirus thru Hepadnavirus • Case Study and Questions #9-15 • Lecture & Discussion Questions, Reading & Chapter Questions • Exam: • Objective Questions (MC, T/F, ID) • Short Essay Questions