10 - Retrovirus & Hepadnavirus Replication Flashcards
Retroviridae
- Enveloped (+)ssRNA genomes
- USe RT to synthesise cDNA intermediate
- Includes HIV and HTLV
- Exist in human genomes in endogenous or exogenous form
Endogenous form of retroviral genomes
- Common in the genomes of humans (4-8%)
- Ancient relics of germ line infection that occurred millions of years ago
Exogenous
Horizontally transmitted, infectious viruses (eg HIV) that integrate dsDNA, complimentary to their genome, into a host
chromosome for gene expression
Retrovirus endogenisation and human endogenous retrovirus (HERV) formation
- During replication, retroviral RNA is reverse-transcribed into a double stranded DNA (dsDNA) provirus and integrated into the cellular genome
- All current human retroviruses target somatic cells, showing a horizontal transmission from an infected individual to new hosts
- The exogenous retroviruses that gave rise to HERVs were
also able to infect germ line cells
Key components of HIV
- gp120-gp41
- tRNA lys
- Integrase
- Capsid (cone shaped)
- Matrix
- Reverse Transcriptase
- Nucleocapsid
- Diploid genome
Diploid retroviral genome
- Diploid RNA genome (both copies identical)
- Coated with nucleoproteins
- Bound to tRNA, that is bound
to viral RNA at the primer binding site PBS and serves as primer for reverse transcription
High genetic variability of HIV-1
- Due to fast replication cycle and high mutation rate
- May cause the generation of many viral variants in a single infected patient during a single day
Major HIV group
Group M (main)
HIV replication cycle part 1
- HIV-1 infects CD4+ T cells via binding of surface gp120 to CD4 receptor
- This triggers a conformational change that initiates gp120 binding to a co-receptor CCR5/CXCR4
- This triggers rearrangement of the gp120-gp41 complex such that the fusion peptide of gp41 inserts into the plasma membrane and the two fuse.
- The HIV nucleocapsid releases into the cytoplasm, releasing the viral RNA genome and enzymes (RT, integrase, protease) into the cytoplasm
- RT converts ssRNA into dsDNA which is transported into the nucleus where it integrates into a host chromosome using viral integrase
HIV replication part 2
- Viral genes are transcribed by host pol II to produce eukaryotic mRNA and mRNA is transported to cytoplasm where translation occurs
- Some proteins enter the nucleus to affect gene expression
- Major structural proteins that form the internal parts of the virion are synthesised as polyproteins Gag, Gag-pol and Env.
- Protease cleaves Gag/Gag-pol polyproteins to produce infectious virus particles with a morphologically distinct core
- HIV-1 nef/vpu mediate translocation of cell-surface CD4 to the cytoplasm/lysosol allowing new virions to leave the cell
Receptor binding in HIV entry
gp120 binds to CD4
Two major domains of retrovirus RT enzyme
p66 and p51
Two active sites of RT
- The polymerase active site
- The RNase H active site
RNase H
- Binds to RNA in duplex (either RNA:RNA or RNA:DNA) and makes internal cleavages to break apart RNA template
- Essential in synthesis of complimentary dsDNA
First 5 steps of reverse transcription in HIV-1
- (First strand (-) cDNA synthesis is initiated in a 5’ direction.
- RNase H activity degrades the small fragment of complimentary template RNA
- This allows for the first jump to occur. Complimentary R
(R’) within the newly synthesised (-)cDNA is complimentary to R in the 3’ end of the RNA template, allowing for RT extension - (-)cDNA extension occurs towards the 5’ end of the template
- With RNase H activity degrading the template RNA, leaving behind only the RNA complimentary to PPT
Steps 6-8 of reverse transcription in HIV-1
- PPT’ provides a primer for the synthesis of ds CDNA (+), complimentary to the (-)cDNA
- RNase H activity removes all remaining RNA template, allowing for the second jump, with PBS from the second
strand complimenting the PBS’ of the 5’ end of the (-)cDNA template - Result is dsDNA with long terminal repeats (LTRs) at both ends, containing U3, R and U5
Comparison of HIV RNA genome and proviral DNA
- 5’ end of the genome differs in sequence from the 5’ LTR
- The 3’ end of the genome is poly-adenylated, while in
the provirus there is another copy of the LTR. - The provirus is flanked by host DNA
HIV-1 cDNA integration into host cell DNA by viral integrase
- One DNA produced from 2 genomic RNAs by viral RT
- Viral LTR (U3, R and UF domains) is promoter
- Proviral DNA (ie. Integrated viral DNA) directs the host transcription machinery to synthesise many copies of viral mRNA
- Viral mRNA is translated into viral proteins OR encapsidated into new viral particles
- No mechanism for excision of integrated provirus
LTRs
- Essential for transcription
- Many sequences in LTR that interact with host TFs
- Core promoter in composed of TATA box and many transcription binding sites
- The 5’ and 3’ LTRs are identical, but serve different functions
3’ LTR
Regulates 3’ end processing of the pre mRNA affecting polyadenylation and transcription termination
HIV-1 accessory TAT protein
Promotes elongation through interaction with a stem-loop structure called the Tar element, found in all HIV-1 mRNA
Event following Tar binding to Tat
- The whole complex then binds to host proteins including a kinase
- The kinase phosphorylates Pol 2 which promotes elongation
Five non enzymatic structural subunits expressed by all retroviruses
- SU
- TM
- MA
- CA
- NC
SU
The surface protein for receptor binding
TM
Transmembrane protein found in viral envelope
MA
MAtrix protein, between capsid and envelope
CA
CApsomer
NC
Nucleoprotein, associated with RNA genome
Three enzymes encoded for by all retroviruses
- Reverse transcriptase
- Integrase
- Protease
Protease
Processes three seperate polyproteins:
- Gag
- Gag-pol
- Env
Hepadnaviruses
- Enveloped viruses,
- Circular genomes (3.3kb) with both DNA and RNA-
- dsDNa with gap of ssDNA
- The longer DNA strand has terminal protein (P) attached at 5’ end
- Shorter DNA is a DNA-RNA chimera, small number of RNA nts at 5’ end
- Triplex DNA region
- Use reverse transcription to amplify their genomes, even though their genomes are predominately DNA
- No genome integration
Example of hepadnavirus
Hepatitis B
