Reverse Transcribing Viruses Flashcards
Describe some properties of retroviruses
- enveloped
- (+) ssRNA genome
- capped and poly(A) by host enzymes
- genome in virion is not used as an mRNA, it is used to make a DNA copy (provirus)
- provirus is ts by RNAP II to yield more viral gneomes
Describe the steps of a retrovirus replication cycle
- binding
- fusion and entry
- uncoating
- reverse transcription
- trafficking - nuclear entry
- integration
- transcription
- translation
- assembly
- budding
- maturation
How many genomes do retroviruses have, are they the same, why does it have more than one?
Has two genomes (exact same, not chromosomes/segmented)
- thought to ensure a provirus is made
When does retrovirus maturation occur, why does it mature?
Happens only after the virion leaves the cell, necessary for infectivity
What is the role of integrase? Is it prepackaged?
Role:
- inserts the entire provirus by creating a dsDNA break and inserts the ds provirus into the break
Prepackaged in the infecting virion
Where do the ds break by integrase occur in the genome?
Essentially at any NT sequence
- not sequence specific
- but isn’t random
Where does MLV preferentially integrate its genome and what does this effect/cause?
Location: upstream of genes
Effect: can mess up regulation -> e.g. can upregulate proto-oncogenes -> canfcer
Where does HIV preferentially integrate its genome and what does this effect/cause?
Location: within genes
Effect: wrecks that gene -> loss of function, and can be mutagenic
What are some consequences of integration?
- provirus is inherited by all mitotic descendants of the infected cell
- most retroviruses don’t kill the infected cell - provirus can act as an insertional mutagen
- by disrupting protein coding sequence
- by altering regulation of adjacent genes (can contribute to development of cancer)
What enzyme performs reverse transcription, where does it occur, what happens to the infecting RNA genome, and how is the DNA different from the RNA?
Enzyme = reverse transcriptase
Location = infecting virion
Infecting RNA genome = degraded using the RNase H activity of RT
RNA template = 5’cap-R-U5-coding region-U3-R-Poly(A) tail
DNA = U3-R-U5-coding region-U3-R-U5
What do the respective regions of the retrovirus genome stand for: U3, R, U5
U3 = unique 3’
R = repeat
U5 = unique 5’
What does LTR stand for, and what components make up the LTR?
LTR = long terminal repeats
LTR = U3-R-U5
Where is the promoter and poly(A) signal found? Where does transcription start, where does poly(A) start? Describe any nuances
promoter in U3: RNA starts at the 1st NT of R
- ts at the 3’ most U3, if the 5’ most U3 is used then ts is defective
poly(A) signal if R: cleavage at last NT of R
- poly(A) doesn’t happen at the 3’ most R (DNA) because it is too close to the 5’ end of the RNA
When transcribing the mRNA from the inserted provirus, how does one of the U3 and one of the U5 regions get excluded? (i.e provirus DNA contain two U3, two R and two U5, and mRNA contains one U3, two R and one U5, how does this happen?
Transcription starts at the 1st R nucleotide which is upstream of the U3 promoter (excludes one U3 region)
Poly(A) and cleavage occurs after the last R NT, which removes a U5 region
What are the four genes in MLV and what proteins does each ORF encode?
Gag = group specific Ag. (capsid proteins)
Pro = protease
Pol = RT, RNase H, integrase
Env = envelope glycoproteins
How are the different proteins within each gene expressed in MLV?
unspliced mRNA: makes Gag and Gag-Pro-Pol (via suppressible codon), these are further processed via viral protease
spliced mRNA: makes env, which is further processed via cellular protease
How are the different proteins within each gene expressed in HIV?
unspliced RNA: Gag and Gag-Pro-Pol (via ribosomal frameshift)
3 singly spliced mRNAs (one intron is taken out): one for vif, vpr, vpu/env (via alternative initiation)
3 multiply spliced mRNA (multiple introns are taken out): tat, rev, and nef
What is the role of Tat, describe its mechanism? What happens if Tat is non-functional?
Role = RNA-binding transcriptional activator
Mechanism:
1. tat binds nascent HIV RNA, at TAR
2. prevents pausing of ts shortly after initiation
3. interacts with rdRNAP II, increases its processivity
mut where tat is non-functioal = lethal
Describe the temporal regulation during HIV infection (early vs late gene expression)
multiply spliced mRNAs are made early:
- encode regulatory proteins
- e.g. tat activates ts from the HIV LTR
unspliced and singly spliced mRNA are made late
- encode virion structural proteins
- unspliced RNA = the genome
How does HIV switch from early to late gene expression? Describe an enzyme (when/how is it expressed)
Rev (virally encoded RNA binding protein)
- encoded by a multiply spliced mRNA
- binds the rev-responsive element (RRE) in nuclear transcripts (located in the Env coding region)
- block further splicing of the bound RNA, promotes its export to the cytoplasm
What is the role of the rev-response element (RRE)?
bypasses need for transcript to be alternatively spliced in order to be translocated to the cytoplasm
How does rev work?
- rev binds to the RRE on unspliced and singly spliced nuclear RNAs
- rev promotes the accumulation of mRNAs encoding structural proteins at late times
How have retroviruses shaped mammalian genomes?
- form a significant fraction of our genome
- msot are not ts, some are, some encode protein that contribute to mammalian biology
Give an example of how endogenous retrovirus genes have contributed to mammalian biology
Env protein of human HERV-W (syncytin, a fusion protein) likely involved in placental function
