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) Baltimore and Temin

"Palm" region (yellow) 20 nucleotides per second
"Thumb" region (orange) No exonuclease capability (proofreading)
"Connector" region (cyan) 10 bases change per replication cycle
Ribonuclease H region (purple)
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 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

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 Gapped intermediate Gaps are

at the target site repaired
Splicing
Translation, assembly& Maturation
 Summary of
etrovirus Replication Cycle
Viral Mediated Events:
• Virus enters cells by
direct fusion or
endocytosis.
• Icosahedral viral particle Reverse transcription

is released into the DNA copy of genome

cytoplasm and begins to genomic
RNAs 
transcribe double Integration 
stranded DNA from the into host DNA
diploid RNA genome. mRNAs

• An integration complex is RNA Synthesis Translation

transported to the
nucleus and functions to RT 
integrate the viral DNA Budding
into the host genome. Splicing Gag
Host mediated Events:
• The integrated viral DNA NUCLEUS Maturation
is transcribed by host RNA Glycoproteins
polymerase II to produce
full length viral RNAs. EUKARYOTIC HOST CELL
• These RNAs are
differentially spliced to
produce viral genomic and
mRNAs and are exported
to the cytoplasm.
Conclusion
Questions??