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Module 2 > retroviral integration > Flashcards

retroviral integration Flashcards

1
Q

what is formed after reverse transcirption and used for nuclear import?

A

pre-integration complex: DNA; integrase and other proteins

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2
Q

When in the infection cycle does entry take place?

A

around an hour

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3
Q

When does reverse transcription take place during hte infection cycle?

A

around 3-4 hours

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4
Q

When does integration take place during hte retroviral cycle?

A

around 7 hours

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5
Q

When does maturation take place during hte infection cycle?

A

more than 10 hours

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6
Q

What is the function of time-of-addition experiments?

A

to determine at what point during viral replication that ertain drug causes a block (if know at the times of events in the replication cycle)

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7
Q

What mechanisms may play a role in favoured integration targeting?

A

open chromatin may be preferentially accessible for viral DNA integration; DNA replication during cell division might facilitate access of integration complexes to favoured sites; cellular proteins bound to the host chromosme might tether integration complexes to favroured regiosn

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8
Q

What was the first cellular protein shown to have a role in directing HIV DNA integration?

A

LEDGF/p75

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9
Q

What is the counterpart of HIV’s LEDGF for MLV IN ?

A

BET proteins

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10
Q

What is the first action of IN?

A

3’ processing

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11
Q

what happens in 3’ processing?

A

typicall cleavage of a dinucletodie from both 3’ endso the viral DNA to expose 3’ hydroxyls attached to invariant CA dinucleotides

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12
Q

What happens upon the intasome entering the nucleus?

A

binds to target DNA forming hte target capture complex

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13
Q

What happens once the target capture complex is made?

A

strand transfer ensues forming hte strand tranfer complex

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14
Q

What happens to the gapped intermediate DNA?

A

repaired to produce the stable provirus

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15
Q

What metals do integrases contain?

A

divalent Mg and Mn cations

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16
Q

What is hte long-lived intermediate containing viral DNA with processed 3’ ends known as ?

A

pre-integration complex

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17
Q

what is foudn in the intasome?

A

pair of viral DNA ends and a tetramer of IN

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18
Q

what are the integrase catalytic activities in vitro?

A

3’ processing; strand transfer or integration and disintegration(doesnt happen in vivo)

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19
Q

What are the 3 domains of HIV-1 IN?

A

N-terminal domain; catalyitc core domain (active site) and C-terminal domain

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20
Q

Why is the integration reaction irreversible?

A

structural change in the active site

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21
Q

When do integrase inhibitors bind to intasome?

A

after 3’ processing and prevent binding to target DNA

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22
Q

Where are most HIV-1 proviruses found in the genome?

A

transcription units

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23
Q

Where are most MLV proviruses found in the genome?

A

promoter regions

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24
Q

What is the function of LEDGF?

A

tightly binds HIV-1 integrase protein and tethers it to hcromatin

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25
Q

what happens in depletion of knockout of cellular LEDGF?

A

reduction of HIV integration, residual integration events are essentially random

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26
Q

What domain LEDGF binds to chromatin?

A

PWWP domain

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27
Q

Which domain of LEDGF binds to lentiviral INs and transcription factors??

A

IBD domain

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28
Q

What is the function of targeting LEDGF?

A

can tagret gene therapy into specific place that is safe- by changing PWWP to a specific targeting domain whilst keeping the IBD

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29
Q

What is the problem with targeting?

A

the PIC can still bind to other non-tagretted LEDGFs in the genome

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30
Q

What is the solution to targeting LEDGF?

A

specific targeting; where the PIC and IBD charges are altered so that PIC can only bind to the targeted iBDs

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31
Q

What is one of the functions of extreme flexibility of the core gp120 structure?

A

allows extreme conformational change upon CD4 engagement without destabilising the interaction with gp41`

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32
Q

What are the 3’ hydroxyl groups at the end of viral DNA joined to?

A

trget DNA 5’ phosphates

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33
Q

How does the intasome accomodate target DNA?

A

within a cleft between ufnctional active sites in a severely bent conformatio- allowing intasome active sites to access their target phopsphodiester bonds

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34
Q

What does IN binding domain of LEDGF consist of?

A

HEAT repeats

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35
Q

What do IN strand transfer inhibitors consist of?

A

heteroatoms that chelate the active site metal atoms and benzyl groups which ejects the 3’ processed viral DNA responsible for strand transfer

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36
Q

What effect does intasome binding haveo n hte tDNA?

A

induces severe bending

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37
Q

What are LEDGINs?

A

mimic the LEDGFR-IN interaction and inihibit protein-protein binding

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38
Q

What transition in replication cycle does integration mark?

A

the transition from the early to late phase of HIV-1 replication

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39
Q

Where does transcription of HIV start from?

A

U3 promoter within the upstream LTR

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40
Q

What is required for efficient elongation during vrial transcrption?

A

Tat transactivator

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41
Q

Where is each end of HIV-1 DNA cleaved?

A

in the LTR adjacent to the invariant dinucelotide sequence CA

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42
Q

What is the MOA of raltegravir?

A

inhibts DNA strand transfer activity

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43
Q

Why is the development of allosteric HIV IN inhibitors such as LEDGINs desirable?

A

the INSTI binding at the active site is highly conserved and there is a high likelihood of cross-resistance amond INSTIs

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44
Q

What do the unspliced and singly spliced mRNAs of HIV require to exit the nucleus?

A

action of Rev

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45
Q

What does Rev act as?

A

an adaptor protein

46
Q

What does Rev bind to?

A

Rev-response element located wtihin the mRNA env coding region and CRM1- a nuclear export factor

47
Q

What does Tat recruit to the stalled transcripts?

A

elongation factor P-TEFb

48
Q

What does P-TEFb consist of?

A

Cdk9 kinase and cyclin T1

49
Q

Where does P-TEFb bind?

A

the viral trans-activation response (TAR) element

50
Q

What is the function of P-TEFb?

A

Cdk9 kinase phosphorylates residues on RNA polymerase stimulating transcirptional elongation upon Tat binding

51
Q

What suggests that it might be possible to devleop anti-HIV agents directed against P-TEFb with limited SE on its normal cellular function?

A

Tat binding induces conformational changes in the substrate-binding surface

52
Q

What allows gag to associate preferentially with the plasma membrane rather than intracellular membranes?

A

N-terminal myristic acid on the structural protein matrix, is differentially exposed through a process known as myristyl switch which allows this preferential association

53
Q

What can activate the myristyl switch?

A

phospholipid phosphatidylinositol which is concentrated on the inner leaflet of the PM and interacts directly with MA

54
Q

What orchestrates retroviral budding?

A

interactions between proline-rich motifs in gag known as late domains and Vps proteins

55
Q

What does Vps in Vps proteins stand for?

A

vacuole protein sorting

56
Q

What is the function of hte interaction between late domains and Vps proteins?

A

required to form the nascent particle and sever it from the plasma memrbane

57
Q

What are the functionso f Vps proteins?

A

formation of multi-vesicular bodies; most function as subunits of endosomal sorting complexes required for transcport

58
Q

What is the function of ESCRT-1 and ESCRT-2?

A

function during membrane budding

59
Q

whati s hte unfciotn of ESCRT-3?

A

membrnae scission

60
Q

What is the function of tetherin?

A

inhibits the release of budding particles by retaining them on the plasma membrane of hte virus producer cell

61
Q

What HIV protein counteracts the restriction of viral egress by tetherin?

A

Vpu

62
Q

How are immature particles converted to infectious virions?

A

via proteolysis of gag and gag-pol precursor polypeptides

63
Q

What does proteolysis of gag and gag-pol precurosor polypeptides yield?

A

Matrix; capsid; nucleocapsid and the protease; reverse transcriptase and integrase enymes

64
Q

What is the function of TRIM5a?

A

HIV-1 restriction factor isolated from rhesus macaques recognises the assembled CA structure to accelerate uncoating and activate innate immune signalling pathways

65
Q

What is HIV-1 RT composed of?

A

p66 and p51 subunits

66
Q

What are the 2 functional active sites on the p66 subunit of Rt?

A

N-terminal RAN and DNA-dependent DNA polymerase and a C-terminal RNase

67
Q

How do NRTIs act as chain terminators?

A

mimic natural nucleoside triphosphates and are incorporated into viral DNA by RT but lack the 3-‘OH group needed for incorporation of the subsequent nucleotide

68
Q

What is the mechanism of resistance to AZT?

A

the mutant RT incorporates AZT into viral DNA but was able to excise the drug from the primer strand

69
Q

How do NNRTIs work?

A

allosteric inhibitors that induce the formation of a flexible binding pocket thorugh conformation changes maybe displaicing the primer grip or the sheet that contains the catatlytic triad

70
Q

What cellular resitriction factor inhibits reverse transcription?

A

APOBEC3G

71
Q

What is the MOA of APOBEC3G?

A

virion-incorporated cytidine deaminase that impedes elongation and converts nascent cytidines in viral cDNA to uracils

72
Q

What HIV protein antagonises APOBEC3G?

A

Vif protein by binding and inducing its degradation

73
Q

What is the formation of the integrase within the retroviral intasome?

A

tetramer

74
Q

What is the N-terminal domain of IN stabilised by binding to?

A

zinc atom

75
Q

What are the pair of divalent metal cations in IN thought to be coordinated by?

A

3 carboxylates of the invariant DDE motif within the catalytic core domain

76
Q

What is the intasome?

A

minimal functional complex involving viral DNA and IN

77
Q

Where in the intasome does IN bind to target DNA?

A

cleft bewteen IN dimers

78
Q

What is thought to be a function of the NTDs and CTDs of the IN?

A

involved in target DNA capture

79
Q

What atoms in INSTIs are responsible for metal chellation of the IN?

A

oxygen atoms

80
Q

How many nucleotides does IN remove in 3’ processing?

A

di- or trinucleotides

81
Q

How does IN carry out strand transfer?

A

uses the 3’ hydroxyls for nucelophilic attacks at a pair of phophodiester bonds on opposing strands of chromosmal DNA results in transesterficiation and subsequent joining of hte 3’ ends of viral DNA to the chromsome

82
Q

In which complex do the reactions catalysed by IN proceed in ?

A

preintegration complex consisting of IN; viral DNA and other viral and cellular components

83
Q

What is the minimal function unit within the PIC?

A

IN tetramer assmebled on the viral DNA ends- intasome

84
Q

what other name does the intasome have

?

A

stable synaptic complex

85
Q

What hlds the viral ends in the active sites of hte catalytic core domain of the IN?

A

NTD and CTD of the sam IN subunit

86
Q

What forms the retroviral IN active site?

A

3 acidic residues forming the DDXE motif form hte basis of hte active iste, through coordination of a pair of essential divalent metal cations

87
Q

What is the MOA of INSTIs?

A

bind to the catalytic metal cations, inactivating the intasome by blocking the active site and dislocating hte terminal 3’ nucleotide of the viral DNA

88
Q

What retrovirus has been used as a convewnient proxy for structural studies of HIV INSTIs?

A

PFV (prototype foamy virus)

89
Q

Why are Mn atoms often used in experiments using x-rays to look at the structure of the intasome?

A

produce stronger electron density peaks than Mg, allowing more precise refinement of metal positions

90
Q

Why did 2 patients with SCID who had gene therapy develop a leukaemia-like illness?

A

due to insertion of a therapeutic retroviral vector in or near the LMO2 proto-oncogene

91
Q

What type of genes are favoured for integration?

A

active genes; especially those active after infection wti hteh HIV-based vector

92
Q

How does integration site selection differ between retroviruses?

A

each retrovirus has a unique pattern of integration site selection within the human genome- suggesting there may be local recognition of chromosomal features unique to each virus

93
Q

Where does MLV tend to insert itself?

A

near transcription start sites; CpG islands

94
Q

What are CpG islands?

A

chromosomal regions enriched in the rare CpG dinucleotide which commonly correspond to gene regulatory regions containing clustered transcription factor binding sites– CpG islands are more frequent in gene-rich regions

95
Q

Why would active genes favour integration?

A

open chromatin at active genes favrous integration

96
Q

Why is there a role suggested for locally bound proteins in directing integration?

A

a distinctive set of sequence specific DNA-binding proteins bound at or near CpG islands disfavour HIV integration while proteins bound in active transcription units are favrouable, whilst for MLV, the proteins boudn at CpG islands instead afvour integration

97
Q

Why will integration differ from tissue to tissue?

A

because of cell-type specific transcription- integration is favoured in active genes

98
Q

Why is the magnitude of the tissue-specific biases on integration modest?

A

most of the cellular transcriptional program appears to be common among cell types

99
Q

In addition to gene density or gene activity, hwat other mechanisms may play a role in determining integration frequency?

A

intranuclear position of chromosomes- relatively fixed for cells of speciic types but differs among cell types- thought as biases in frequency not explained by gene desnity or activity

100
Q

Why is ASLV though to be a good prospect for use in gene therapy?

A

shows only wek preference of integration in active genes; no favouring of integration near transcription sites; is able to infect a variety of human cell types; can transduce nondividing cells

101
Q

What is ASLV?

A

avian sarcoma-leukosis virus

102
Q

What protein accounts for the characteristic preference of lentiviruses to integrate within active transcription units?

A

LEDGF

103
Q

what flanks the provirus following gap repair?

A

short suplicatiosn of the target RNA sequences

104
Q

How does LEDGF affect iN?

A

stimualtes its enzymatic activity in vitro

105
Q

What is thought to be the function of LEDGF?

A

binds to transcription factors and regulators, likely to play a role in regulation of gene expression and/or as an adaptor protein tethering a plethora of cellular proteins to chromatin

106
Q

What are the function of LEDGF in the context of HIV?

A

tethers IN to host cell chromatin and may also be invovled in nuclear import and protection from proteasomal degradation ( suggested by chromatin binding activity depdence on LEDGF and nuclear accumulation and stability of IN impaired in LEDGF depletion)

107
Q

What are the characteristic features of lentiviral integration?

A

propensity to integrate within active trancription units of hte host cell genoma and their bias against insertion into promoters nad CpG islands

108
Q

What is the advatnge for lentiviruses for this integration preference?

A

integration into transcriptionally-acitve genomic loci improves the efficiency of lentiviral gene expression

109
Q

Which domain of IN interacts with LEDGF?

A

CCD

110
Q

What domain of IN is reqruied for high-affinity binding of LEDGF to IN?

A

NTD

Module 2 (38 decks)

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