Retrovirus Replication by bhuvanesh kalal
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Transcript of Retrovirus Replication by bhuvanesh kalal
Retrovirus Replication
Bhuvanesh SK
2nd Sem Microbiology
DSBS Bangalore
Retrovirus replication
Index
General structure Retrovirus replication
-Reverse transcription
-Integration
Conclusion Questions??
Retroviral structure and genome
Retroviral Proteins
Gag protein - MA (matrix) CA (capsid) NC (nucleocapsid)
Pol protein encodes enzymes- PR (protease) RT (DNA polymerase and RNase H activities) IN (Integrase)
Env protein encodes- SU surface glycoprotein TM transmembrane protein
“Accessory” genes (in Complex Retroviruses) - regulate and coordinate virus expression; function in immune escape
Long Terminal Region (LTR) Repeat (R) Region: Unique (U5): Primer Binding Site: Leader: Polypurine Tract: Unique (U3):
Diploid (+)RNA
Retrovirus Virions Thin Section EM of Some Retroviruses
Type A (donut)
Type B (eccentric)MMTV
Type C (central)ALV,RSV
Type D (bar)
Lentivirus (cone)HIV
1). Receptor binding and membrane fusion2). Internalization and un-coating of virion3). Reverse transcription of RNA to form double-stranded DNA4). Entry of viral DNA into host nucleus5). Integration of viral DNA into cellular DNA to form the provirus6). Transcription of provirus to form viral RNA7). Splicing of viral RNAs and export from host nucleus8). Translation of viral RNAs to form viral proteins9). Assembly of virion and packaging of viral RNA genome10). Budding and release of new virions11). Proteolytic processing of proteins and virion maturation
Virus Replication
Binding, fusion, Internalization, un-coating &Reverse transcription
"Fingers" region (red) "Palm" region (yellow) "Thumb" region (orange) "Connector" region (cyan) Ribonuclease H region (purple)
Baltimore and Temin 20 nucleotides per second No exonuclease capability (proofreading)10 bases change per replication cycle
Reverse Transcriptase
Reverse Transcription
1) (-) strand synthesis starts near the 5’ end of the (+) strand RNA genome with a specific host tRNA as a primer and runs out of template after ~100 nt
2) Synthesis proceeds to the 5’ end of the RNA genome through the u5 region ending after the r region, forming the (-) strand strong stop DNA (-ssDNA)
Reverse transcription
2) RNA portion of the RNA-DNA hybrid is digested by the RNase H activity of RT, resulting in a single-stranded DNA product
3) This facilitates hybridization with the r region at the 3’ end of the same or second RNA genome, resulting in the first template exchange for RT
Reverse transcription cont.
4) (-) strand DNA terminates at the primer binding site
5) When (-) strand elongation passes the polypurine tract (ppt) region, the RNA template escapes digestion by RNase H and serves as a primer for (+) strand synthesis by DNA dependent DNA polymerization (DDDP)
Reverse transcription cont.
6) (+) strand synthesis then continues back to the U5 region with the (-) strand DNA as the template and terminates after copying the first 18 nt of the primer tRNA and stops, forming the (+) strand strong stop product (+ssDNA)
Reverse transcription cont.
7) The tRNA is then removed by RNase H activity of RT
8) The exposed PBS anneals to the PBS sequence at the 3’ end of the (-) strand DNA, allowing the second template exchange.
Product of the second template exchange is a circular DNA molecule with overlapping 5’ ends.
Reverse transcription cont.
9) DNA synthesis is terminated at the breaks in the template strands at the PBS and PPT ends, producing a linear molecule with long terminal repeats (LTRs).
Reverse transcription cont.
http://pathmicro.med.sc.edu/flash/hiv-ltr-fn.html
Transcription inhibitors
Actinomycin D and alpha-amanitin Nucleoside analogues : 5-bromodeoxyuridine,
cytosine arabinoside
Three forms of provirus DNA are found in all infected cells:
Endonucleolytic nicking and removal of 2 nt and formation of a new 3’ recessed end
Joining of 3’ ends to phosphates at the target site
Gapped intermediate Gaps are repaired
The two ends of viral DNA are recognized, nicked and then joined covalently to host DNA in random locations at staggered nicks also introduced by Integrase
Steps in retroviral DNA integration
Splicing
Translation, assembly& Maturation
Reverse transcription
DNA copy of genome
Integration into host DNA
Splicing Gag
Glycoproteins
mRNAs
Translation
RNAs
RT
NUCLEUS
EUKARYOTIC HOST CELL
genomic
Maturation
Budding
RNA Synthesis
Replication of a Typical Retrovirus
Viral Mediated Events:• Virus enters cells by direct fusion or endocytosis.• Icosahedral viral particle is released into the cytoplasm and begins to transcribe double stranded DNA from the diploid RNA genome.• An integration complex is transported to the nucleus and functions to integrate the viral DNA into the host genome.
Host mediated Events:• The integrated viral DNA is transcribed by host RNA
polymerase II to produce full length viral RNAs. • These RNAs are differentially spliced to produce viral genomic and mRNAs and are exported to the cytoplasm.• Viral proteins are translated and assemble to form virions.
Summary ofRetrovirus Replication Cycle
Conclusion
Questions??