Flashcards in Virology term 1 - retroviruses Deck (120):
Simple vs complex retroviruses
Simple = little splicing. Usually 4 genes.
Complex = much splicing.
Endogenous vs exogenous
Endogenous = viral genome integrated. Vertical transmission.
Exogenous = viral particles made. Vertical and horizontal transmission.
Can be markers of speciation.
Endogenous retroviruses; placental development
Endogenous virus important in migration of binucleate cells in ovine placental development.
RNA retrovirus genome structure
Cap - R - U5 - PB - psi - genes - PP - U3 - R - 3' polyA
Retrovirus genome structure: R
Retrovirus genome structure: PB
primer binding site
Retrovirus genome structure: PP
Retrovirus genome structure: psi
RNA element involved in genome packaging.
Retrovirus genome: genes
capsid and nucleocapsid
reverse transcriptase and integrase.
surface glycoprotein and transmembrane glycoprotein.
Who first suggested the existence of reverse transcriptase and why?
Temin because of RSV persistence and heritability, and since actinomycin D (inhibits DNA -->RNA) and cytosine arabinoside (inhibits DNA synthesis) inhibit replication.
How did Temin and Baltimore separately show the existence of an RNA dep DNA pol?
By incubating virions with labelled dTTP; the product was only degraded by DNase, not RNase.
Key features of the retrovirus lifecycle
Entry, reverse transcription, integration, transcription, splicing, export, translation, packaging and maturation
Types of viral genome found in cells infected with retroviruses.
viral RNA genome, linear dsDNA with LTR, circularised DNA with LTRs, integrated provirus.
When discussing history of discovery of reverse transcription, consider...
Types of viral genome.
Linear dsDNA genome structure
U3 R U5 PB gag pol env PP U3 R U5 PB.
Steps in retrotranscription (7)
1) RT activity - making strong stock DNA.
2) RNase H activity
3) First strand switch
4) RT activity, RNase H activity (leaving PP)
5) DNA synthesis, nucleolytic activity of RT
6) 2nd strand switch
7) strand extension.
Different RT forms
monomer/homodimer (MuLV, simple)
heterodimer (avian sarcoma virus, HIV).
Function of RT determined by...
Mechanism of proviral integration in retroviruses.
1) Integrase recognises AAT 5' motif. Context important.
2) Integrase cleaves off TT from 3' end (requires CA at 3')
3) Linear dsDNA translocated to nucleus.
4) Staggered cleavage of host DNA
5) Host DNA undergoes nucleophilic attack by 5' AA.
6) Host cellular machinery repairs the ends and removes the mismatches.
Type of tRNA used for priming
Depends on retrovirus. 4 have been found to be used in mammals: trp, pro, glu and lys.
Requirements for strand switching
R' sequence exposed by RNase H.
CA protein is also critical.
Can occur because genome is diploid.
Integration site choice of retroviruses - hypotheses
1) Depend on virus
2) Cell cycle determines position of integration - but most only integrate into dividing cells; lentiviruses will integrate in either, but no clearly different patterns.
3) Intervention of tethering proteins
Hypothesis for integration site choice: dependence on virus
HIV appears to prefer transcriptional units, MLV promoter regions, but ASLV is almost random.
Hypothesis for integration site choice: cell cycle determines position of integration.
But most only integrate into dividing cells; lentiviruses will integrate in either, but no clearly different patterns.
Hypothesis for integration site choice: intervention of tethering proteins.
Tethering proteins important in integration of yeast transposons.
Processing in integration
Viral att sequences recognised and nicked by IN.
Conserved CA at 3' end is essential.
Upstream sequences affect efficiency.
Joining in integration
Concerted cleavage and ligation.
Staggered cleavage of phosphodiester bonds in host DNA. Joined to processed viral ends.
Consequences of integration
Provides template for transcription of new virus RNA.
Enables vertical transmission.
Can have effects on host cell: promoter insertion, insertional mutagenesis, oncogene insertion.
Efficiency of retrovirus transcription in infected cells
Can be high (10%) but usually lower (1-2%).
Promoters for RNA pol II in provirus
Cis-acting elements in U3 region of LTR. E.g. Sp1.
Can recruit TFs in tissue or hormone dependent manner.
Complex retroviruses encode their own transcriptional activators e.g. HIV-1 rev and HTLV-1 Tax.
Promoter features for host RNA pol II
Can have promoter-proximal sites, or sites far from gene. TATA box at -30. Bound by general transcription factors.
SP1 binding site.
Enhancers and silencers
Sp1 - normal.
Binds GC box. Interacts with TAFii110 and TFIID.
Enhancer and silencer sequences
Are position independent, orientation independent, and bind regulated TFs.
Enhancers can act from 50kbp away.
Promoter features found in LTRs of retroviruses
Lots of enhancer sequences; these overlap - balance of binding determines initiation.
Enhancer sequences can be hormone responsive, tissue or developmental stage specific.
Hormone responsive enhancer sequence for a retrovirus.
MMTV ( a simple retrovirus) uses the glucocorticoid receptor as a very strong enhancer.
Transcription from downstream LTR
Questions about the PolyA. Why is it not recognised in the upstream LTR?
For some, not far enough from cap. In others snRNPs bind and suppress.
Virus specific regulatory proteins for transcription in retroviruses
Tas and Bel1 in HSRV (spuma)
Tax in HTLV1 and 2
Tat in HIV
Mechanism of Tax in promoting transcription.
Alters initiation and possibly elongation by altering histone deacetylation.
Virus gene expression
Differential splicing and export
Termination codon suppression
RSV uses most of these.
Export of unspliced RNA from nucleus
Needed for export of genomic RNA.
1) Use Nfx1/Tap pathway directly
2) Use Nfx1/Tap pathway indirectly
3) Use the protein pathway.
Retroviruses: using the Nfx1/Tap pathway to export genomic RNA.
Recruit directly using a constitutive transport element in the genomic RNA.
Recruit indirectly by recruiting cellular factors who couple the genomic RNA to the mRNA export pathway.
CTE in genomic export
Loop A and B recruit TAP directly.
Retroviruses: exporting the genomic RNA via the protein pathway: relevant proteins.
HIV Rev protein.
HTLV Rex protein.
MLV recruiting NFX/Tap
Retroviruses using CTEs
MPMV and SRV-1.
Retrovirus coupling RNA to NFX/Tap pathway using proteins.
Gag/Pol frameshift frequency
Most retroviruses, not spuma or gamma. Occurs in 5% of transcriptions.
Gag/Pol frameshift mechanism
Ribosome is delayed on a U or A rich slippery sequence causing it to shift reading frame. Delay caused by a pseudoknot (MMTV) or stem loop (HIV).
Selection of retrovirus genome
Binding of NC to low affinity sites leads to RNA conformational switch allowing dimerisation which allows NC binding to high affinity sites.
Role of NC in genome selection (retroviruses)
Distinguishes viral unspliced RNA from all the other RNAs.
Structural features of NC in genome selection.
Zinc knuckles have CCHC arrays that bind zinc with high affinity.
RNA elements in genome selection (retroviruses)
Psi sites containing stem loops. DIS1&2 (Dimerisation initiation sites 1 and 2). Binding NC --> kissing complex sites exposed --> Kissing complex --> extended duplex.
Exclusion of spliced RNAs from retrovirus genome selection.
Splice donor sites within psi site. HIV2 may have spliced RNAs with psi site, so need a different mechanism.
Summary of particle assembly
Surface and transmembrane protein targetted to membrane.
Gag also targets to membrane, associates with Gag-pol.
NC portion of Gag associates with psi for budding.
tRNA enriched by binding to RT.
Gag and budding
Gag contains PT(S)AP to recruit Tsg101 and hsne others including VPS for ESCRT action.
General retrovirus ESCRT recruitment (except HIV)
PPXY domains bind ubiquitin ligases.
Gag processing after HIV budding.
PRO aspartyl protease released (?) and allows autocatalytic cleavage of Gag and Gag/Pol proteins.
HIV 1 and 2.
HIV1 divides into groups M, N,O and P.
groups divide into clade, with group M being particularly significant.
gag, pol, env,
vif, vpr, rev, tat, vpu and nef
Host restriction factors for HIV
Trim5a, APOBEC1, Tetherin, SAMHD.
Restriction factors in MLV
Fv1; blocks dissassembly of CA and movement of PIC into nucleus.
Ring finger motif, so possibly ubiquitin ligase. But abolishing this does not abolish action, so possibly not.
After core structure destroyed, Trim5a may lead to proteasomal degradation of PIC.
Host cytidine deaminase packaged in virion. Vif triggers ubiquitination and degradation before packaging.
tethers budding virion to the surface. Vpu binds TM domain to prevent action.
Newly discovered restriction factor, possibly has RNase activity.
Prevents BST-2/Tetherin action.
dsDNA, IN, MA, Vpr, RT
Involved in nuclear entry of PIC.
lipid rafts in entry
Chemokine co-receptors found on lipid rafts, probably aids membrane fusion.
Generally enfuvirtide. Peptide based fusion inhibitor fuzeon - homologous domains in gp41 have to interact to promote fusion. Inhibitors mimic one domain and hence disrupt interactions.
Co receptor inhibitors
o Co-receptor CCR5 inhibitors maraviroc – small molecule CCR5 chemokine receptor antagonist. Induces conformational change to CCR5 not recognised by virus envelope.
APOBEC cytodine deaminase action
increase mutation rate above threshold of about inverse to genomic size. High GA mutation rate up to 60% in some strains.
APOBEC other roles
Appears to inhibit even if cytidine deaminase domains are inhibited
Nuclear targetting signals for PIC.
DNA flap – possibly contains nuclear targeting signal? Vpr and IN have non-canonical nuclear-targetting signals. MA also has nuclear targeting signals. Cooperative, or in different cells?
HIV: reasons for becoming latent.
Either latent due to heterochromatin, or absence of activators of HIV LTR enhancer or failure of Tat.
Avoiding transcription of polyA in first LTR.
In some viruses it AAUAAA overlaps with the R cleavage site and U3. Therefore as transcription starts at R it is not transcribed (HTLV)
MoMLV: leaky transcription, inefficient at both poly(A) tails
HIV-1 : encodes a protein that binds and suppresses poly(A) in upstream LTR…. U1 snRNP
Splicing in retroviruses
Exporting unspliced genome
Export of unspliced genome
Direct repeat regions.
Cytoplasmic accumulations signal
Viral transactivating proteins.
CTEs for RNA export.
constitutive transport elements which they can use to recruit host export proteins NXF1 and TAP.
Direct repeat regions for RNA export
RSV has two direct repeat regions which flanking the src oncogene which interact with the cellular mRNA export machinery
Cytoplasmic accumulation signal for RNA export.
MLV recruits NXF1 via a cytoplasmic accumulation signal and uses cellular proteins to couple intron containing RNA to the mRNA export pathway.
Viral transactivating proteins for RNA export.
Complex retroviruses such as HIV and HTLV couple mRNA export to the cellular protein export pathway using viral trans activating proteins Rev and Rex respectively which allow them to use the Crm machinery.
Retroviruses increasing coding capacity.
Frame-shifting and stop-codon read through.
Retroviruses - frameshifting.
Ribosomal frame shift of -1. This depends on a “slippery sequence” in the RNA and a 3’ pseudoknot structure, which causes ribsomal stalling and frameshift in 5% cases. Due to 5’ end dependent nature of eukaryotic translation, Pol can only be expressed as gag-pol which is cleaved after assembly by the viral protease.
Retroviruses stop-codon read through.
Gamma retroviruses use stop-codon readthrough or “termination suppression” to encode gag-pol.
Retroviruses polyprotein processing - env.
Rous Sarcoma Virus Env protein is processed by host protease furin.
Retrovirus polyprotein Gag-pol cleavage.
Gag and gag-pol are cleaved by the viral protease after budding in the maturation step. Gag is cleaved into its 5 consituent subunits: PR, MA, NC and CA. Gag-Pol is cleaved to form Gag and an 100kDa beta reverse transcriptase, which is subsequently cleaved to a 65kDa alpha reverse transcriptase. Integrase is coded at the 3’ end of the pol gene. Sometimes it is cleaved as a separate protein e.g in HIV-1 whereas sometimes it remains part of the pol complex: ASLV.
Simple = have simple splicing (so don’t produce as many proteins) and don’t infect immunocompetent —> archetype = Rous sarcoma virus
Includes the alpha, beta and gamma retroviruses
Have complex splicing (produce many diff proteins) and can combat the immune system more effectively —> archetype = HIV
Includes the delta, epsilon, lenti and spuma retroviruses
1.5m died from AIDs in 2013 and around 35m are living with HIV, deadly in around 10 years without treatment (diagram of genome).
1) Initiated at U3-R border of 5’ LTR and terminates beyond the poly(A) addition site at the R-U5 border of the downstream 3’ LTR
2) U3 site contains promotor elements, enhancer elements and polyA termination signals.
3) Txn from downstream LTR prevent potentially due to promotor occlusion by txn or cis-acting signals.
4) PolyA noticeably leaky in some simple RVs.
Differential mRNA splicing
Simple retroviruses harbour one splice donor and one splice acceptor site - used in formation of env mRNA
for RSV just get products of Gag, gag-pol and src —> exported for nucleus unspliced or already spliced
Complex retroviruses produce additional mRNAs controlled by several splice donor and acceptor sites.
In these cases, the pattern of viral RNAs in the cytoplasm may be regulated in a temporal manner by a viral protein —> late and early mRNAs(e.g. in HIV get many proteins such as Nef, Vpr and Vpu)
In HIV, Rev is required for the production of late mRNAs
some splicing only partial (e.g. for structural proteins) —> difficulty in exporting as cellular mRNAs almost all fully spiced to have to avoid usual checks —> e.g. HIV-Rev protein links mRNA export to protein export pathway
Key feature differentiating complex viruses.
Presence of a set of accessory genes whose products are involved in the regulation of transcription, RNA transport, gene expression, assembly and immune evasion
RNA-binding protein that promotes late phase gene expression. It is also important for the transport of the unspliced or singly-spliced mRNAs, which encode viral structural proteins, from the nucleus to the cytoplasm.
RNA-binding protein that enhances transcription.
Binds TAR in presence of cyclin T1 and cdk9 enhances RNA pol II elongation
promotes down regulation of MHC I and CD4
Enhances the release of the virus from the cell surface to the cytoplasm during entry —> blocks BST-2/tetherin that tethers budding virions to host PM
Promotes G2 cell cycle arrest and facilitates infection on macrophages
Necessary for replication of lentiviruses due to its ability to down regulate the host’s antiviral response —> blocked APOBEC 3F/G cytidine deaminases
Key points about elongation of HIV transcription.
P-TEFb is a key host factor for Tat-dependent HIV-1 transcription.
P-TEFb is maintained in a functional equilibrium.
Tat assembles the super elongation complex to promote HIV-1 transcription.
Brd4-P-TEFb interaction and its effect on HIV-1 transcription and latency.
Factors that contribute to HIV latency.
Initiation of transcription: chromatin modifications, transcriptional interference, Limited availability of transcription factors.
Elongation of transcription: limited elongation factors, insufficient Tat activity.
Post-transcriptional mechanisms: blocking mRNA export.
Subdivisions of pol II mediated transcription.
Pre-initiation, initiation, promoter clearance, elongation and termination. Previously thought that first 2 steps were most important, then discovered that elongation a key rate limiting step in HIV-1 gene expression.
Tat-dependent HIV-1 transcription.
• Tat is absolutely essential for activating transcriptional elongation from the viral LTR. In its absence, only aborted transcripts are made, in its presence, full length ones are made.
• Tat acts by interacting with transactivation response RNA element (TAR) in a stem-loop structure at 5’ end of nascent viral transcripts, synthesised before pol II pausing.
• Tat-TAR alone are not sufficient to stimulate full length transcription; requires host co-factors.
• P-TEFb is a heterodimer composed of CDK9 and CycT1. Recruited to HIV-1 LTR through interactions with Tat-TAR.
Able to phosphorylate, directly or indirectly Ctd of largest subunit of Pol II on serines on heptapeptide repeats. Other phosphorylation events also occur to antagonise inhibitory effects of negative elongation regulators.
P-TEFb can be temporarily and reversibly sequestered in the 7SK snRNP (kinase inactive complex). Otherwise is often bound to Brd4, which competes with Tat.
Super elongation complex.
Tat assembles the super elongation complex to promote HIV-1 transcription.
Interactes with P-TEFb through small region near cyclin box in CycT1. Tat triggers release of P-TEFb from 7SK snRNP. Tat then assembles Tat-TAR-P-TEFb trio, but anecdotal evidence suggests it may also recruit additional factors. They are recruited by some mechanism however to give a multi-subunit complex called SEC.
Brd4-P-TEFb interaction and its effect on HIV-1 transcription and latency.
• Brd4 releases Pol II from pausing at celluular promoters, but competes with Tat, so is a potent inhibitor of Tat-transactivation as it competes with Tat for binding to P-TEFb. Contributes significantly to the establishment of latency.
• BET bromodomain inhibitor efficiently reactivates latent HIV-1 in several models, and co-operates well with other activators in reactivating latently infected T cells.
• Possible use of ‘shock and kill’ strategy?
HIV latency: chromatin modification.
Proviral silencing appears to be mediated by H3K9 methyltransferase Suv39H1. Also DNA methylation inhibits transcription factor binding.
HIV latency: transcriptional interference.
o Proviruses from latently infected cells tend to be integrated into highly expressed genes.
o A process whereby transcription that originates at one promoter can interfere with transcription at another.
HIV latency: limited availability of transcription factors.
Limited availability of transcription factors, including NFkB and NFAT. Establishment appears to be regulated by AP-1 binding site in LTR.
HIV latency: elongation of transcription.
• Limited availability of elongation factors e.g. P-TEFb.
• Insufficient Tat activity – e.g. mutants with attenuated activity are more likely to be latent.