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94 Cards in this Set
- Front
- Back
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What genes are in all RV?
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Gag
Pol Env |
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What ptns do HIV have?
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Gag, Pol, Env
Also have Tat and Rev |
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Describe the HIV-1 replication cycle
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Virus binds receptor CD4+ and coreceptor (CXCR4, CCR5)
Envelope stays outside, pore enters Genomic RNA remains in the core, use RT Nuclear localization and integration: Form pre-integration complex Provirus goes to nucleus where it integrates into genome using integrase Get T and splicing Only RNA that is doubly spliced can get out of the nucleus => This will become Tat, Rev and Nef -Tat and Rev go to nucleus and start T Unspliced and singly spliced mRNA get out of the nucleus using Rev These get Tl to become Gag, Pol and Env Start assembly Get budding Immature particle formation and then maturation |
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What is the order of ptns that are made?
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1) Tat, Rev, Nef
2) Then Gag, Pol, Env |
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What are the regulatory ptns of HIV?
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Tat
Rev |
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Whhat is Tat?
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Trans-activator of HIV promoter
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What is Rev?
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Nuclear export of late, unspliced RNA to cytoplasm
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What are the accessory ptns of HIV?
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VPR
NEF VIF VPU |
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What is Vpr?
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Induces G2 cycle arrest and nuclear import or the pre-integration complex
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What is Nef?
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Downregulation of cell surface CD4 and MHC-1
Enhances virion infectivity |
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What is Vif?
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Virion infectivity factor
Counteracts APOBEC |
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What is Vpu?
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Enhancement of virion release and CD4 degradation by targeting to the proteasome
Counteracts tetherin -->Does this differently than how Nef works |
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TRANSCRIPTION AND CHROMATIN
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TRANSCRIPTION AND CHROMATIN
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When does T start?
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When proviral DNA is integrated into chromatin
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What is the 1st step of transcription
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T starts with TAR RNA (Tat binds Tar)
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Why is Tat different from other transactivators?
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It can bind an RNA target
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Describe Tar RNA
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Present at the 5' end of all viral RNA (spliced or not)
Mediates Tat transactivation Has a role in Tl |
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Where is the HIV-1 provirus integrated?
Where is it structured in? |
Integrated into cellular chromatin
Structured in nucleosomes |
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Describe nucleosomes
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146 bp DNA wrapped around a central histone octamer that constitutes the nucleosome core
Nucleosomes need to be unfolded to allow transcription initiation |
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Where does T start on the HIV genome?
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At +1
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Why can't the virus be transcribed if it is in a nucleosome form?
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RNA pol doesn't have easy access to the template
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What are teh 2 main nucleosomes?
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Nuc 0
Nuc 1 |
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What is needed to get transactivation by the Tat ptn?
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Tat has an RNA target, but the promoter is DNA
1st need to activate chromatin modelling complexes Need to unfold chromatin so its DNA is open and TF and pol can bind |
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What mediates chromatin remodelling?
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Cellular activation
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What are the complexes involved in HIV-1 remodelling?
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ATP-dependent chromatin complex
2) Complexes that modify histone acetylaton (HAT, CBP, hGCN5, PCAF, HDAC) |
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Describe the ATP-dependent chromatin remodelling complex
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SWI-SNF with ATPase subunits
Roles: Alter histone-DNA interaction |
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Describe the complexes that modify histone acetylation
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-Histone acetyl transferase (HAT):adds an acetyl to histones, which unfolds the nucleosome
ex: p300/CREB binding ptn (CBP); hGCN5; p300/CBP ass't factor (PCAF) -Histone deacetylases (HDAC): remove an acetyl gp from histones and allow nucleosomes structure to take place |
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What do HATs allow?
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Allow transcription
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What do HDACs do?
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Repress T
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How can T be activated?
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Cellular activation: NF-kB activation, important for activation
External stimulus: Tetradecanoyl phorbyl acetate (TPA) or Phorbyl Myristate acetate (PMA) -->These activate HATs |
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What happens once transcription is activated?
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Unfold nucleosomes
Get cellular remodelling 1st step to get T -->This is how you get basal T (can get a few transcripts made) |
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What does the basal T make?
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Tat
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What happens once Tat is made?
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Can go to nucleuse, act on nucleosome, unfold it and get increased T
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MECHANISMS OF TAT ACTIVITY
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MECHANISMS OF TAT ACTIVITY
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Describe T cycle
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Get formation of PIC
Get initiation complex, in which factor A/D is hypoP Pol is P at its CTD Get promoter clearance and hyperP (by pTEFb) Elongation Termination of elongation Phosphatase used to get to re-initiation |
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What is required to get hyper-P of the CTD of Polymerase?
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Need pTEFb (T elongation factor), made of 2 ptns
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What does pTEFb do?
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Maintains P throughout T elongation
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What role does Tat play here?
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Increases hyperP of CTD of pol
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What do T activators do?
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Bind DNA
Recruit TFIID/B & H |
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What are the targ IIets of T activators? What is the result of activator-GTF interaction?
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TFIID: Enhances recruitment of TFIID to nucleosomal templates
TFIIB: Enhances recruitment of TFIIB to initiation complex; this allows subsequent loading of RNA Pol II TFIIH: Ehances efficiency of promotoer clearance, allowing multiple RNA Pol IIs. to initiate T pTEFb: Enhances efficiency of promoter clearance and elongation, allowing multiple RNA Pol IIs to initiate T and efficient elongation (Tat binds pTEFb) |
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What is the result of these things?
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Increase T
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Describe the Tat gene
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2 exons
Acdic -- Cystein-- Core-- RNA binding domain |
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What part is absolutely required for Tat fct?
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Cysteins and Core
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How is Tat synthesized?
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From multiply spliced RNA
Takes parts from exons 1 and 2 Need 1-58 for transcription (up to RNA binding domain) 1-48 is the activation domain (acid, core + cysteines) The rest is required for other fcts, but not transactivation |
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What is the RNA target of Tat?
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TAR RNA
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Describe Tar RNA:
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Forms a stable stem-bulge-loop structure
The sequence in the loop and the bulge are required for Tat fct |
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Where does Tat bind on Tar?
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Binds directly to the bulge of Tar RNA
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What is required for Tat fct?
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Cellular factors
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How can Tat activity be measured/.
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Use reporter genes
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How can the RNA amount of Tat be measured by S1 nuclease protection?
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Isolate RNA
Incubate with probe See what happens -->In the absence of Tat, don't have any cleavage, no bars are shown on the gel ->In the presence of Tat, get cleavage, have one band |
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How are reporter genes used?
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Transfect promoter and reporter gene with the vector
Plasmid that expresses this is put in the cell Measure ptn or T lvl |
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How is Tat activity measured at the ptn level?
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Measure activity of Cat or Luc genes
--> Avg activation ~100 fold (btw 10-1000 depending on conditions) -->If Tat is present, get CAT activates CA, so you get Acetylation of Tat at 2 places -->If use Luc, get light emittance if Tat present |
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Describe the mechanism of Tat activity
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-Tat Acts by a complex mechanism of T activation/reinitiation and elongation
-Tat promotes the formation of a competent transcription initiation complex by recruiting a kinase that P RNA Pol's CTD -TAR RNA/Tat/Cyclin T1/CDK9 is the fctnal complex for Tat-TAR interaction and polymerase CTD P -HATs regulate the binding of Tat/CycT/CDK9 complex by Tat Acetylation |
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What kind of complex is formed with TAR RNA?
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Tat/Cyclin T1/CDK9 complex
-->Need all of them to form a complex |
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How do we know this is the complex that forms?
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Used an RNA mobility shift
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How is this complex formed? (what binds what)
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Ptn-ptn interactions are used
-->Verified using the yeast-2-hybrid method Tat Binds CycT1 CDK9 binds Cyc T1 (Tat does NOT bind CDK9) Tat--CycT1 -- CDK9 |
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Describe the in vivo trans-activation by Tat
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Multi-step mechanism
1) Tat binds HAT 2) HAT A Tat on Lys-28 3) After A, has inc affinity for CycT1-CDK9 (usually stored as inactive complex in the cell) 4) Get 3ry complex (Tat-CycT1-CDK9), which has high affinity for TAR RNA 5)After binds, get Tat A, nut from dif points (Lys 50 &51) Once A here, get removed from TAR 6) Tat will localize on promoter and recruit all the other factors, TBP to TATA box to form active complext ->With TAT, complex becomes very active |
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Describe how initiation and then elongation occurs
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1st have activity of CDK7 on TFIIH
Once complex forms, CDK9 can hyperP CTD of Pol Then get rapid departure and processive elongation |
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What mediates the initiall P of the RNA Pol II CTD?
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TFIIH
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What does Tat acetylation by HATs do?
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Increases TAR-Tat-CycT1 binding and a 2nd A favor the release of Tat-CycT-CDK9 from the TAR RNA
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What is the functional complex of this?
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Tat/CycT/CDK9
-->CDK9 hyperP CTD of RNAPII |
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What does P of RNAPII CTD promote?
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Competent T initiation complex
Polymerase departure T elongation Rapid initiation |
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How does HIV-11 Tat and HTLV-1 Tax compare in their trans-activation mechanisms?
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They have the same fct, but they use a different mechanism
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What does HTLV-1 Tax do?
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DNA binding ptn
Some elements on the promoter of DNA that mediate Tax activation (Tax responsive elements) ->21 bp repeats CREB binds TxRE Tax binds CREB Other factors bind, cause activation directly on the promoter and PIC ->Just get increased initiation (no elongation effect) -->Build directly on the promoter |
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MECHANISM OF REV ACTIVITY
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MECHANISM OOF REV ACTIVITY
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What is Rev used for?
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Nuclear export of late, unspliced RNA to cytoplasm
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What is Rev required for?
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Cytoplasmic expression of unspliced and partially spliced HIV-1 RNA
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What are the transcripts of WT virus that make it to the cytoplasm?
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9 kb RNA
4 kb RNA 2 kb RNA |
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What happens if the virus is deficient in Rev?
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Only the 2 kb RNA transcript ends up in the cytoplasm
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Is Rev involved in splicing?
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No, it works on the export of unspliced mRNA
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Describe HIV transcription
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Get T of the whole DNA
-->Get 9 kb RNA -1 element in it has a strong structure -Starts to be spliced ==> 4 kb -Gets doubly spliced ==> 2 kb 9 and 4 kb can't leave the nucleus -2kb goes to the cyto, makes Tat, Rev, Nef Tat: Inc T Rev: goes to nucleas |
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What does Rev do oince it gets to the nucleus?
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starts the late phase of replication
Rev binds Rev RE (on the 9 and 4 kb RNA) |
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What pathway does Rev use to transport through?
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CRM-1 path
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What is exported once Rev goes back to nucleus?
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Export unspliced (9kb) and singly-spliced (4 kb) RNA
-->Stop making the 2 kb RNA |
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Describe the REV structure
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NLS & RRE binding domain (have an arginine-rich region): mediates RNA binding
Activation domain/Nuc export domain |
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Describe the mechanism of action of REV
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In the nucleus, get RNA
REV synthesized in the cytoplasm, has NLS and NES Goes through nuc pore complex, goes to nucleus -Binds RRE Then binds CRM-1 -->binds GTP -Entire complex goes through nuclear pore -In the cytoplasm, RNA is released, ptns can be made Requires GTP-GDP -Rev recycled and all HIV RNA Tl or packaged into virions -HIV RNA can go back to nucleus to be used again |
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Compare viral mRNA export mechanism used by HIV-1 Rev and HTLV-1 Rex
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Same function and same mechanism for both
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What does Rex do?
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Exports un/singly spliced RNA
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What helicase is used by both HIV and HTLV for this export?
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DDX3
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What pathway do other viruses use for export?
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Use different system (Tap/Nxt)
Bind directly or (for Herpes), bind to ICP27, which binds onto another ptn, that binds Tap/Nxt Brings RNA to cytoplasm and use the rest of the ptns as shuttle ptns btw the cyto and nuc |
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What does Rev export?
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Un/singly spliced HIV RNA
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What does Rev bind?
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RRE RNA in the envelope gene, through its RNA binding motif (Arg rich motif, which is the NLS)
Also binds CRM-1 and nuclear export machinery |
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Where does Rev go?
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Shuttles btw cytoplasm and nucleus
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REGULATION OF HIV-1 TRANSLATION
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REGULATION OF HIV-1 TRANSLATION
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What does HIV-1 Tl depend on?
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Cap-depended
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What inhibits HIV-1 Tl?
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TAR RNA structure
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What role does Tar play in inhibiting Tl?
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mRNA with TAR has weaker Tl then mRNA without TAR
TAR RNA is a block to HIV Tl |
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If this is true, how can the virus Tl?
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Tar can activate PKR (IFN stimulated, gets activated by dsRNA)
When PKR is P, can P eIF2a and get inhibition of Tl Structure of TAR and P/activation of PKR causes Tl inhibition |
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What happens to PKR during virus replication?
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PKR isn't active
=>TRBP: Binds PKR and stops its P, thereby releasing the block of Tl due to TAR |
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What else can TRBP bind?
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Can bind PACT so that PACT can't P PKR
.: get T; agaom |
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When is PKR activated?
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PKR is only transiently activated after HIV infection and is deactivated during high viral replication
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Where does HIV replicate? Why?
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Replicates in cells that produce a lot of TRBP so that there is a release of the block of Tl due to Tar and counteract PKR activation
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What does HIV infection induce to inhibit PKR?
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Induces production of ADAR1
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What does TRBP do?
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Neutralizes PACT (PKR activator) so can't activate PKR
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