retroviral integration Flashcards
what is formed after reverse transcirption and used for nuclear import?
pre-integration complex: DNA; integrase and other proteins
When in the infection cycle does entry take place?
around an hour
When does reverse transcription take place during hte infection cycle?
around 3-4 hours
When does integration take place during hte retroviral cycle?
around 7 hours
When does maturation take place during hte infection cycle?
more than 10 hours
What is the function of time-of-addition experiments?
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)
What mechanisms may play a role in favoured integration targeting?
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
What was the first cellular protein shown to have a role in directing HIV DNA integration?
LEDGF/p75
What is the counterpart of HIV’s LEDGF for MLV IN ?
BET proteins
What is the first action of IN?
3’ processing
what happens in 3’ processing?
typicall cleavage of a dinucletodie from both 3’ endso the viral DNA to expose 3’ hydroxyls attached to invariant CA dinucleotides
What happens upon the intasome entering the nucleus?
binds to target DNA forming hte target capture complex
What happens once the target capture complex is made?
strand transfer ensues forming hte strand tranfer complex
What happens to the gapped intermediate DNA?
repaired to produce the stable provirus
What metals do integrases contain?
divalent Mg and Mn cations
What is hte long-lived intermediate containing viral DNA with processed 3’ ends known as ?
pre-integration complex
what is foudn in the intasome?
pair of viral DNA ends and a tetramer of IN
what are the integrase catalytic activities in vitro?
3’ processing; strand transfer or integration and disintegration(doesnt happen in vivo)
What are the 3 domains of HIV-1 IN?
N-terminal domain; catalyitc core domain (active site) and C-terminal domain
Why is the integration reaction irreversible?
structural change in the active site
When do integrase inhibitors bind to intasome?
after 3’ processing and prevent binding to target DNA
Where are most HIV-1 proviruses found in the genome?
transcription units
Where are most MLV proviruses found in the genome?
promoter regions
What is the function of LEDGF?
tightly binds HIV-1 integrase protein and tethers it to hcromatin
what happens in depletion of knockout of cellular LEDGF?
reduction of HIV integration, residual integration events are essentially random
What domain LEDGF binds to chromatin?
PWWP domain
Which domain of LEDGF binds to lentiviral INs and transcription factors??
IBD domain
What is the function of targeting LEDGF?
can tagret gene therapy into specific place that is safe- by changing PWWP to a specific targeting domain whilst keeping the IBD
What is the problem with targeting?
the PIC can still bind to other non-tagretted LEDGFs in the genome
What is the solution to targeting LEDGF?
specific targeting; where the PIC and IBD charges are altered so that PIC can only bind to the targeted iBDs
What is one of the functions of extreme flexibility of the core gp120 structure?
allows extreme conformational change upon CD4 engagement without destabilising the interaction with gp41`
What are the 3’ hydroxyl groups at the end of viral DNA joined to?
trget DNA 5’ phosphates
How does the intasome accomodate target DNA?
within a cleft between ufnctional active sites in a severely bent conformatio- allowing intasome active sites to access their target phopsphodiester bonds
What does IN binding domain of LEDGF consist of?
HEAT repeats
What do IN strand transfer inhibitors consist of?
heteroatoms that chelate the active site metal atoms and benzyl groups which ejects the 3’ processed viral DNA responsible for strand transfer
What effect does intasome binding haveo n hte tDNA?
induces severe bending
What are LEDGINs?
mimic the LEDGFR-IN interaction and inihibit protein-protein binding
What transition in replication cycle does integration mark?
the transition from the early to late phase of HIV-1 replication
Where does transcription of HIV start from?
U3 promoter within the upstream LTR
What is required for efficient elongation during vrial transcrption?
Tat transactivator
Where is each end of HIV-1 DNA cleaved?
in the LTR adjacent to the invariant dinucelotide sequence CA
What is the MOA of raltegravir?
inhibts DNA strand transfer activity
Why is the development of allosteric HIV IN inhibitors such as LEDGINs desirable?
the INSTI binding at the active site is highly conserved and there is a high likelihood of cross-resistance amond INSTIs
What do the unspliced and singly spliced mRNAs of HIV require to exit the nucleus?
action of Rev
What does Rev act as?
an adaptor protein
What does Rev bind to?
Rev-response element located wtihin the mRNA env coding region and CRM1- a nuclear export factor
What does Tat recruit to the stalled transcripts?
elongation factor P-TEFb
What does P-TEFb consist of?
Cdk9 kinase and cyclin T1
Where does P-TEFb bind?
the viral trans-activation response (TAR) element
What is the function of P-TEFb?
Cdk9 kinase phosphorylates residues on RNA polymerase stimulating transcirptional elongation upon Tat binding
What suggests that it might be possible to devleop anti-HIV agents directed against P-TEFb with limited SE on its normal cellular function?
Tat binding induces conformational changes in the substrate-binding surface
What allows gag to associate preferentially with the plasma membrane rather than intracellular membranes?
N-terminal myristic acid on the structural protein matrix, is differentially exposed through a process known as myristyl switch which allows this preferential association
What can activate the myristyl switch?
phospholipid phosphatidylinositol which is concentrated on the inner leaflet of the PM and interacts directly with MA
What orchestrates retroviral budding?
interactions between proline-rich motifs in gag known as late domains and Vps proteins
What does Vps in Vps proteins stand for?
vacuole protein sorting
What is the function of hte interaction between late domains and Vps proteins?
required to form the nascent particle and sever it from the plasma memrbane
What are the functionso f Vps proteins?
formation of multi-vesicular bodies; most function as subunits of endosomal sorting complexes required for transcport
What is the function of ESCRT-1 and ESCRT-2?
function during membrane budding
whati s hte unfciotn of ESCRT-3?
membrnae scission
What is the function of tetherin?
inhibits the release of budding particles by retaining them on the plasma membrane of hte virus producer cell
What HIV protein counteracts the restriction of viral egress by tetherin?
Vpu
How are immature particles converted to infectious virions?
via proteolysis of gag and gag-pol precursor polypeptides
What does proteolysis of gag and gag-pol precurosor polypeptides yield?
Matrix; capsid; nucleocapsid and the protease; reverse transcriptase and integrase enymes
What is the function of TRIM5a?
HIV-1 restriction factor isolated from rhesus macaques recognises the assembled CA structure to accelerate uncoating and activate innate immune signalling pathways
What is HIV-1 RT composed of?
p66 and p51 subunits
What are the 2 functional active sites on the p66 subunit of Rt?
N-terminal RAN and DNA-dependent DNA polymerase and a C-terminal RNase
How do NRTIs act as chain terminators?
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
What is the mechanism of resistance to AZT?
the mutant RT incorporates AZT into viral DNA but was able to excise the drug from the primer strand
How do NNRTIs work?
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
What cellular resitriction factor inhibits reverse transcription?
APOBEC3G
What is the MOA of APOBEC3G?
virion-incorporated cytidine deaminase that impedes elongation and converts nascent cytidines in viral cDNA to uracils
What HIV protein antagonises APOBEC3G?
Vif protein by binding and inducing its degradation
What is the formation of the integrase within the retroviral intasome?
tetramer
What is the N-terminal domain of IN stabilised by binding to?
zinc atom
What are the pair of divalent metal cations in IN thought to be coordinated by?
3 carboxylates of the invariant DDE motif within the catalytic core domain
What is the intasome?
minimal functional complex involving viral DNA and IN
Where in the intasome does IN bind to target DNA?
cleft bewteen IN dimers
What is thought to be a function of the NTDs and CTDs of the IN?
involved in target DNA capture
What atoms in INSTIs are responsible for metal chellation of the IN?
oxygen atoms
How many nucleotides does IN remove in 3’ processing?
di- or trinucleotides
How does IN carry out strand transfer?
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
In which complex do the reactions catalysed by IN proceed in ?
preintegration complex consisting of IN; viral DNA and other viral and cellular components
What is the minimal function unit within the PIC?
IN tetramer assmebled on the viral DNA ends- intasome
what other name does the intasome have
?
stable synaptic complex
What hlds the viral ends in the active sites of hte catalytic core domain of the IN?
NTD and CTD of the sam IN subunit
What forms the retroviral IN active site?
3 acidic residues forming the DDXE motif form hte basis of hte active iste, through coordination of a pair of essential divalent metal cations
What is the MOA of INSTIs?
bind to the catalytic metal cations, inactivating the intasome by blocking the active site and dislocating hte terminal 3’ nucleotide of the viral DNA
What retrovirus has been used as a convewnient proxy for structural studies of HIV INSTIs?
PFV (prototype foamy virus)
Why are Mn atoms often used in experiments using x-rays to look at the structure of the intasome?
produce stronger electron density peaks than Mg, allowing more precise refinement of metal positions
Why did 2 patients with SCID who had gene therapy develop a leukaemia-like illness?
due to insertion of a therapeutic retroviral vector in or near the LMO2 proto-oncogene
What type of genes are favoured for integration?
active genes; especially those active after infection wti hteh HIV-based vector
How does integration site selection differ between retroviruses?
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
Where does MLV tend to insert itself?
near transcription start sites; CpG islands
What are CpG islands?
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
Why would active genes favour integration?
open chromatin at active genes favrous integration
Why is there a role suggested for locally bound proteins in directing integration?
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
Why will integration differ from tissue to tissue?
because of cell-type specific transcription- integration is favoured in active genes
Why is the magnitude of the tissue-specific biases on integration modest?
most of the cellular transcriptional program appears to be common among cell types
In addition to gene density or gene activity, hwat other mechanisms may play a role in determining integration frequency?
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
Why is ASLV though to be a good prospect for use in gene therapy?
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
What is ASLV?
avian sarcoma-leukosis virus
What protein accounts for the characteristic preference of lentiviruses to integrate within active transcription units?
LEDGF
what flanks the provirus following gap repair?
short suplicatiosn of the target RNA sequences
How does LEDGF affect iN?
stimualtes its enzymatic activity in vitro
What is thought to be the function of LEDGF?
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
What are the function of LEDGF in the context of HIV?
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)
What are the characteristic features of lentiviral integration?
propensity to integrate within active trancription units of hte host cell genoma and their bias against insertion into promoters nad CpG islands
What is the advatnge for lentiviruses for this integration preference?
integration into transcriptionally-acitve genomic loci improves the efficiency of lentiviral gene expression
Which domain of IN interacts with LEDGF?
CCD
What domain of IN is reqruied for high-affinity binding of LEDGF to IN?
NTD
