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

Replication of Replication of Reverse-Reverse-

Transcribing Transcribing VirusVirus

Replication of Replication of Reverse-Reverse-

Transcribing Transcribing VirusVirus

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)

Sub-Family: Spumavirinae

• “foamy” vacuoles in cell culture• Mammals, primates• Human foamy virus – first

retrovirus found in humans• “orphan virus” - no associated

disease

Sub-Family: Oncovirinae

• “tumor”• infection leads to cell

transformation• RNA tumor virus• Avian, reptile, mammals, primates• Human T-cell leukemia virus (HTLV)

Sub-Family: Lentivirinae

• “slow”• Persistent chronic infection• Chronic disease of CNS, lung,

immune deficiency• No cell transformation• Mammals, primates• Human immunodeficiency virus (HIV)

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

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)

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

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

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

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

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)

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

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

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

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

HIV Spliced mRNAs• Translates for

env proteins• Translates for

regulatory & accessory proteins– Switch for

subgenomic, genomic mRNAs

– Down-regulate (nef)

– Activate (tat)– Infectivity

(vif)

HIV Genomic/Sub-genomic mRNAs

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

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

Natural History of HIV Infection

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

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

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

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

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

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

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

QUESTIONS???

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?

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