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

Diploid (+)RNA

Long Terminal Region (LTR)      

Repeat (R) Region: Unique (U5): Primer Binding Site: Leader: Polypurine Tract: Unique (U3):

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

Virus Replication 1). Receptor binding and membrane fusion 2). Internalization and un-coating of virion 3). Reverse transcription of RNA to form double-stranded DNA 4). Entry of viral DNA into host nucleus 5). Integration of viral DNA into cellular DNA to form the provirus 6). Transcription of provirus to form viral RNA 7). Splicing of viral RNAs and export from host nucleus 8). Translation of viral RNAs to form viral proteins 9). Assembly of virion and packaging of viral RNA genome 10). Budding and release of new virions 11). Proteolytic processing of proteins and virion maturation

Binding, fusion, Internalization, un-coating & Reverse transcription

Reverse Transcriptase

"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 Transcription

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 3) 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 cont.

2) RNA portion of the RNA-DNA hybrid is digested by the RNase H activity of RT, resulting in a singlestranded 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 3) 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.

3) (+) 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.

2) 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).

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:

Steps in retroviral DNA integration 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 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

Splicing

Translation, assembly& Maturation

Summary of etrovirus Replication Cycle 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.

Reverse transcription DNA copy of genome Integration  into host DNA RNA Synthesis

genomic RNAs  mRNAs Translation RT 

Splicing

NUCLEUS

Gag

Glycoproteins

EUKARYOTIC HOST CELL

Budding

Maturation

Conclusion

Questions??