Reverse Transcription and Integration Flashcards
Reverse transcriptase requires what nuclear material to work with?
Reverse transcriptases (RTs) are enzymes that synthesize DNA from both RNA and DNA templates.
Reverse transcriptase is mainly an RNA-dependent DNA polymerase - converting RNA back in to DNA
But can also work with DNA, as a regular DNA polymerase
The distinct catalytic activities brought into play at various stages in the pathway
of reverse transcription include:
- RNA-directed DNA polymerisation
- DNA-directed DNA polymerization
- DNA unwinding
- the hydrolysis of RNA in RNA-DNA hybrids (RNase H).
Why do steps with reverse transcriptase incur a lot of potential errors?
Reverse transcriptases (RTs) are error-prone, as have no proof-reading activity
Retroviruses have reverse transcriptase. What other enzyme do they posses?
Retroviruses are the only animal viruses with genomes
that encode integrase proteins.
Integrase is a specialized recombinase that mediates
the insertion of retroviral DNA into its host genome,
where it is called a provirus.
Usually integrates into regular cells, but can integrate into germ line cells, and be passed on to offspring. This is evident by analysis of ancient proviral DNA in human genomes. Integration into germ lines often leads to defective replication and possible extinction of the virus
What is a provirus?
When integrase integrates viral genome into host genome, that new genome is called a pro-virus - can potentially replicate from this
Integrase has a wide range of uses in molecular biology
What molecule binding is required before initiation of reverse transcription in retroviruses?
tRNA primer - one particular molecule serves as a primer for initiation of reverse transcription, binding to primer-binding site (pbs) on RNA
The tRNA primer is positioned on the template genome during virus assembly, in a reaction that is facilitated by interactions with the viral polyprotein precursors (Gag and Gag-Pol)
Retroviruses such s HIV have two copies of RNA
Why is this?
Not definitely clear
HIV only needs 1 copy of RNA to fully complete life cycle
Given RT has low fidelity, and mutations occurs frequently, presumably two identical copies of RNA helps from an evolutionary point of view, as mutations in one RNA strand, would still leave a viable strand available for replication
Additionally, having mutations in one strand, can allow recombination between strands, and create a new combination of sequences which may be beneficial to the virus
What are the main steps in reverse transcription?
Initiation of (-) strand DNA synthesis
First template exchange
Initiation of (+) strand DNA synthesis
Second template exchange
Integration into host genome
During reverse transcription,
What occurs during initiation of (-) strand DNA synthesis
Our understanding of DNA synthesis indicates that the simplest way of copying an RNA template to produce full-length complementary DNA would be to start at its 3’ end and finish at its 5’ end.
However reverse transcription starts near the 5’ end, but quickly runs out of nucleotides as RNaseH degrades the RNA as it is copying.
This short section of DNA produced, acts as a key components for transcription signalling - termed (-) strong-stop DNA
During reverse transcription,
What occurs during first template exchange
(-) strong-stop DNA binds to 3’ end of RNA sequence. Once engaged, the RNA-dependent DNA polymerase activity of RT can continue copying until the 5’ end, with the RNaseH activity digesting the RNA template in it wake
Which part of the retroviral genome encodes RT?
pol genes
What are the steps of pro-viral DNA integration
integrase cuts open host DNA, and pro-viral DNA inserted
this damage promotes recruitment of proteins of the DNA damage-sensing pathway, and they inadvertently repair the combined DNA
What is unique about HBV viral genome?
Incomplete circular DNA
one full length (-) strand
one incomplete complementary (+) strand
gapped DNA of virus particle enters cell, and moves to nucleus where it is repaired to produce covalently closed circular molecule (ccDNA). Host cell recognises that the genetic material is “damaged”, so host DNA repair pathways produce the closed circular DNA
Does HBV integrate into host genome, like HIV?
HBV DNA does not integrate into host genome - as does not contain an integrase.
But the host cell can occasionally mistakenly repair the DNA and integrate it into host DNA. But this is not its primary method of survival
HBV ccDNA is only produced via reverse transcription. In contrast to HIV provirus which is copied when cellular DNA copies
Both HIV and HBV are maintained in infected cells for life, but in different ways
How does the end product of HIV and HBV reverse transcription final product differ?
HIV - double-stranded DNA
HBV - covalently closed episomal DNA
