1. Group Case Study Presentation Evaluation: 50 points 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

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 U 5 ) copy of 5’ end; RNase H removes hybridized RNA (R, U 5 ) 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 U 3 RU 5 ) 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