Group Case Study Presentation Evaluation: 50 points
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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
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Replication of Replication of Reverse-Reverse-
Transcribing Transcribing VirusVirus
Replication of Replication of Reverse-Reverse-
Transcribing Transcribing VirusVirus
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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)
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Sub-Family: Spumavirinae
• “foamy” vacuoles in cell culture• Mammals, primates• Human foamy virus – first
retrovirus found in humans• “orphan virus” - no associated
disease
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Sub-Family: Oncovirinae
• “tumor”• infection leads to cell
transformation• RNA tumor virus• Avian, reptile, mammals, primates• Human T-cell leukemia virus (HTLV)
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Sub-Family: Lentivirinae
• “slow”• Persistent chronic infection• Chronic disease of CNS, lung,
immune deficiency• No cell transformation• Mammals, primates• Human immunodeficiency virus (HIV)
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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
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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)
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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HIV Spliced mRNAs• Translates for
env proteins• Translates for
regulatory & accessory proteins– Switch for
subgenomic, genomic mRNAs
– Down-regulate (nef)
– Activate (tat)– Infectivity
(vif)
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HIV Genomic/Sub-genomic mRNAs
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HIV Assembly/Releas
e• 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
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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
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Natural History of HIV Infection
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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
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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
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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
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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
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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
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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
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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
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QUESTIONS???
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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?
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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