Reverse Transcription and Integration

retrovirus Baltimore classification

\+ RNA virus → - DNA → dsDNA

hepadnavirus Baltimore classification

dsDNA with a gap

hepadnavirus example

hepatitis B

how are retroviruses and hepadnaviruses similar

genomes in RNA and DNA form at different stages in life cycle and rely on reverse transcription

retroviruses OG genome

RNA

hepadnaviruses OG genome

DNA

what were these viruses initially known as?

RNA tumor viruses

how did they know DNA played a critical role in RNA tumor viruses?

infection was inhibited by actinmycin D

actomycin D

inhibitor of host RNA pol II (DDRP)

what suggested that the viral genome was incorporated into host DNA?

stable genetic changes could be transferred to daughter cells after infection

Temin’s theory

provirus theory

provirus theory

postulated the genetic transfer of info from RNA to DNA

what did Temin’s theory require?

presence of RNA-dependent DNA pol or reverse transcriptase

use of RT-PCR

analyzes cellular levels of mRNA and patterns of gene expression

RT-PCR

copy mRNA molecules into complementary cDNA

what does 1 virus generate?

1 provirus

provirus

integrated retroviral genome

is the provirus amplified?

no (amplification is delayed)

Gag protein

core/capsid

Pol protein

enzymes

Env protein

envelope

what can full-length viral RNA in retroviruses serve as?

mRNA for Gag and GagPol or genomic RNA

retrovirus enzyme

RT

retrovirus template

2 copies of viral genomic RNA

retrovirus primer

tRNA

what do retroviruses package?

tRNA, RT, and templates

HIV-1 RT structure

heterodimer of p66 and p51

p66

contains RNase H domain

RT enzymatic activities

RDDP (reverse transcription), DDDP (has proofreading), RNase H

what does RT lack like RDRP?

proofreading ability

RNase H function

destroys RNA template upon copying to DNA

biological significance of high mutation rate

large population of mutants

clinical significance of RT enzyme

drug resistance and immune evasion

retrovirus genomic RNA structure

\+, capped, polyadenylated

what does not happen to retrovirus genomic RNA after entry?

translation to protein

what happens to the two copies of viral genome upon packaging?

they form a dimer via formation of a kissing loop complex

significance of kissing loops

allows recognition of the RNA dimer by Gag

initiation of - DNA synthesis

pbs binds and 5’ end of viral RNA genome degraded by RNase H as - DNA is made

what are complementary during - DNA strand synthesis

R (- DNA) and r (RNA)

retrovirus first template exchange

R-r anneal and RDDP continues

what does RNase H not degrade?

primer ppt

\+ DNA primer

ppt

why is pbs copied twice?

once from RNA genome and another from tRNA primer

what is - DNA equivalent to in retrovirus?

entire genome in permutated order

retrovirus second template exchange

PBS sequences anneal and strand displacement synthesis extends - DNA to + DNA

what creates the long terminal repeats (LTRs)?

duplication of U3-R-U5

reverse transcription net gain

none

destructive replication

1-2 molecules of + DNA → 1 dsDNA

why two copies of RNA

so they can patch together one complete DNA copy from two randomly interrupted or mutated RNA genomes to provide survival value

what triggers recombination?

RT pausing at sequence, structures, or breaks

how does RT finish synthesis as a break?

uses other RNA genome copy

where do internal template exchanges occur?

at regions of homology

copy choice

internal exchanges that take place during RNA-directed DNA synthesis

strand displacement synthesis

exchange of ss ends from one DNA template to another during + strand synthesis

what does integration ensure?

stable association between viral and host DNA

retrovirus amplification step

viral DNA transcribed into viral mRNAs by host RNA pol II (cellular DDRP)

full-length viral mRNAs uses

\+ RNA genome for packaging and viral mRNAs for translated

integration site locations

no strong sequence specificity, different between retroviruses

HIV integration site location

within genes

MLV integration sites location

near transcription start sites

HIV integration site interaction

LEDGF (via IN) and CPSF6 (via CA)

LEDGF

facilitates integration

CPSF6

directs integration to active genes

hepadnavirus full-length viral RNA can serve as

mRNA for capsid and polymerase and genomic RNA

what fills hepadnavirus gap

host repair machinery (also removes primers)

cccDNA

covalently closed circular DNA

what happens to hepadnavirus cccDNA

acquires histone and becomes minichromosome (episome)

what does not occur to hepadnaviruses that occur to retroviruses?

integration and replication along with host DNAs

transcription of + RNA by host RNA pol II creates?

pregenome RNA (pgRNA) and shorter mRNA

pgRNA

encapsidated

what occurs before hepadnavirus exits the cell?

RT creates 1st strand DNA and partially completes the dsDNA

hepadnavirus enzyme

RT (P protein)

hepadnavirus template

pregenomic RNA

hepadnavirus primer

P protein for - DNA and capped RNA fragment for +

hepadnavirus viral genome

full length - DNA and incomplete + DNA (partial dsDNA)

(-) strand hepadnavirus structure

self-priming P protein on 5’ end

(+) strand hepadnavirus structure

capped RNA primer at 5’ end

direct repeats function

used for template exchange

do hepadnaviruses integrate?

no