Reverse transcription and integration

Why are retroviral genomes called pseudodiploid? Why does the presence of two genomes in the capsid contribute to recombination?

• Pseudodiploid because only one of the two copies will be reverse transcribed and integrated

• Presence of 2 genomes contributes to recombination because the two genomes can exchange genetic information with each other through copy choice or strand displacement synthesis

What is used to prime the first RT reaction? Where does this primer come from? What would happen if it wasn’t available?

Retroviruses use tRNA packaged from host cells to prime the first reverse transcriptase reaction

• If the tRNA wasn’t available, reverse transcription would not occur

What does the RNase H activity of RT do? What would happen if the HIV RT had a mutation that removed the RNase H activity?

• RNase activity degrades RNA of the template strand after the tRNA primer is used to synthesize the first short segment of DNA

• If HIV RT had a mutation that removed the RNase H activity, only a small segment of the genome would be reverse transcribed and then RT would halt, resulting in a failure to replicate

What is template exchange?

Template exchange is a part of reverse transcription where the newly synthesized DNA anneals to the RNA template at the 3’ end, and the newly synthesized DNA is used as a primer for the rest of synthesis to occur

Why must integrase be present in the viral capsid?

Integrase is not present in host cells, and the virus cannot successfully replicate without integrase since the viral genome would not be integrated into the host’s genome

What types of interactions account for the variation in integration target site seen for different retroviruses?

• Cell transcription factors/coactivators and other cell proteins interact with the viral DNA and integrase

Reverse transcriptase

An enzyme that makes DNA from RNA

How are reverse transcriptases similar to polymerases?

• Shares sequence motifs with other polymerases

• Primers are needed, base pairing occurs to direct synthesis

How many copies of the genome do retroviruses carry?

2

How are retrovirus genomes arranged in the capsid?

The two genomes are base paired together, secondary structure present

What do retroviruses use as a primer for reverse transcriptase?

tRNA

When does reverse transcription begin?

Shortly after viral entry into the cell, as soon as dNTPs are available

3 Major functions of reverse transcriptases

• RNA/DNA- directed DNA polymerization

• DNA unwinding

• RNase H hydrolysis of RNA in DNA/RNA hybrids

How many molecules of RT are there in retroviruses?

50 to 100

How fast and error prone are reverse transcriptases compared to other polymerases? Is there proofreading?

Slow (1/10 speed) and very error prone, with no proofreading

Can reverse transcriptase be found in cells?

Yes, it can be found in many cells, including bacteria archaea, eukaryotes and even DNA viruses. Telomerase is an example of a reverse transcriptase used by eukaryotes

Where does initiation of reverse transcription begin?

5’ end

What are the steps of reverse transcription?

• tRNA primer binds to template strand

• Reverse transcriptase uses the primer to begin transcription at the 5’ end

• After a small segment of DNA is transcribed, RNase H degrades the RNA template paired to the newly synthesized DNA

• The newly synthesized DNA anneals to the 3’ end of the RNA template and is used as a primer for synthesis to begin at the 3’ end as a part of a template exchange

• RNase H degrades the genome as the genome is copied

• When the other strand is synthesized, RNase H makes a RNA primer before synthesis begins.

What is the final product of reverse transcription?

A linear dsDNA that has long terminal repeat ends that are needed for integration into the host genome

Would a circular dsDNA product be viable for retroviruses? Why?

No, strand displacement is essential for an essential linear dsDNA product

What are the steps of retroviral integration?

• Several nucleotides are removed from the ends of the viral DNA

• Viral DNA joined to target DNA using a cleavage-ligation reaction with gaps in intermediate

• Host cell repair mechanisms repair the gaps in the DNA

Integrase structure

3 domains

• Catalytic core domain with enzymatic function

• Integrase forms a tetramer in order to do the integration reaction