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??