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IMIN 324 > Reverse Transcribing Viruses > Flashcards

Reverse Transcribing Viruses Flashcards

1
Q

Describe some properties of retroviruses

A
  • 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
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2
Q

Describe the steps of a retrovirus replication cycle

A
  1. binding
  2. fusion and entry
  3. uncoating
    - reverse transcription
    - trafficking
  4. nuclear entry
  5. integration
  6. transcription
  7. translation
  8. assembly
  9. budding
  10. maturation
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3
Q

How many genomes do retroviruses have, are they the same, why does it have more than one?

A

Has two genomes (exact same, not chromosomes/segmented)
- thought to ensure a provirus is made

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4
Q

When does retrovirus maturation occur, why does it mature?

A

Happens only after the virion leaves the cell, necessary for infectivity

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5
Q

What is the role of integrase? Is it prepackaged?

A

Role:
- inserts the entire provirus by creating a dsDNA break and inserts the ds provirus into the break

Prepackaged in the infecting virion

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6
Q

Where do the ds break by integrase occur in the genome?

A

Essentially at any NT sequence
- not sequence specific
- but isn’t random

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7
Q

Where does MLV preferentially integrate its genome and what does this effect/cause?

A

Location: upstream of genes
Effect: can mess up regulation -> e.g. can upregulate proto-oncogenes -> canfcer

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8
Q

Where does HIV preferentially integrate its genome and what does this effect/cause?

A

Location: within genes
Effect: wrecks that gene -> loss of function, and can be mutagenic

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9
Q

What are some consequences of integration?

A
  1. provirus is inherited by all mitotic descendants of the infected cell
    - most retroviruses don’t kill the infected cell
  2. provirus can act as an insertional mutagen
    - by disrupting protein coding sequence
    - by altering regulation of adjacent genes (can contribute to development of cancer)
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10
Q

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?

A

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

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11
Q

What do the respective regions of the retrovirus genome stand for: U3, R, U5

A

U3 = unique 3’
R = repeat
U5 = unique 5’

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12
Q

What does LTR stand for, and what components make up the LTR?

A

LTR = long terminal repeats
LTR = U3-R-U5

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13
Q

Where is the promoter and poly(A) signal found? Where does transcription start, where does poly(A) start? Describe any nuances

A

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

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14
Q

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?

A

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

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15
Q

What are the four genes in MLV and what proteins does each ORF encode?

A

Gag = group specific Ag. (capsid proteins)
Pro = protease
Pol = RT, RNase H, integrase
Env = envelope glycoproteins

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16
Q

How are the different proteins within each gene expressed in MLV?

A

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

17
Q

How are the different proteins within each gene expressed in HIV?

A

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

18
Q

What is the role of Tat, describe its mechanism? What happens if Tat is non-functional?

A

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

19
Q

Describe the temporal regulation during HIV infection (early vs late gene expression)

A

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

20
Q

How does HIV switch from early to late gene expression? Describe an enzyme (when/how is it expressed)

A

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

21
Q

What is the role of the rev-response element (RRE)?

A

bypasses need for transcript to be alternatively spliced in order to be translocated to the cytoplasm

22
Q

How does rev work?

A
  • rev binds to the RRE on unspliced and singly spliced nuclear RNAs
  • rev promotes the accumulation of mRNAs encoding structural proteins at late times
23
Q

How have retroviruses shaped mammalian genomes?

A
  • form a significant fraction of our genome
  • msot are not ts, some are, some encode protein that contribute to mammalian biology
24
Q

Give an example of how endogenous retrovirus genes have contributed to mammalian biology

A

Env protein of human HERV-W (syncytin, a fusion protein) likely involved in placental function

25
Q

How have retroviruses contributed to our knowledge of biology?

A
  1. 1st delineation of the genetic basis of cancer
    - some encode proteins that convert cells into tumor cells -> acutely transforming which is efficient and fast
    - some downregulate the expression of cellular genes that control cell growth -> inefficient, delayed onset
  2. vectors for efficient gene delivery
    - include genes for gene therapy of genetic diseases in humans, especially in cases where the disease can be cured by transforming hematopoietic SCs
26
Q

How have retroviruses been used to treat X-linked severe combined immunodeficiency? What is a drawback?

A

MLV carrying gammaC cytokine receptor
- some patients developed leukemia because MLV preferentially integrates upstream of genes which can deregulate cellular genes

27
Q

How have retroviruses been used to treat Metachromatic leukodystrophy and Wiscott-Aldrich syndrome? What is a drawback?

A

Lentivirus (HIV-I derived) carrying arylysufatase A or WASP gene, respectively.
- no concerns so far, HIV preferentially integrates within genes

28
Q

What are retrotransposons? Give two examples. How do they transpose?

A

Retrotransposons = almost viruses

Example = yeast Ty element and drosophila copia elemts

Transpose through an RNA intermediate

29
Q

Why has it been suggested that retrotransposons may be degenerate retroviruses or cellular progenitors of retroviruses?

A
  • transcribed gene is packaged into a viral particle (no env) and the RNA is then integrated into the host genome somewhere (transposable elements move)
  • can’t move from one cell to another
30
Q

Describe the structure of the yeast Ty retrotransposon

A

5’-LTR-gag-pro-pol-LTR-3’

31
Q

What are hepadnaviruses? Name an example

A

hepadnavirus = small DNA viruses that cause hepatitis

example = HepB

32
Q

Describe hepadnaviruses genome structure. What key protein is packaged into the virion?

A

genome = partially ds circular DNA genome (one complete strand, one incomplete strand)

protein = DNAP (both DNA and RNA dep)

33
Q

Describe how hepadnavirus replicate their genome. Why does it use this strategy?

A
  1. nuclear import of DNA
  2. repair fully to dsDNA
  3. ts into pre-genomic ssRNA
    - > genome length
    - contains repeat at the ends
  4. pre-genomic RNA -> progeny genome (reverse transcription)
    - accomplished in cytoplasmic virions

Why:
- we don’t know, but it works

IMIN 324 (14 decks)

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