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57 Cards in this Set
- Front
- Back
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What is the difference btwn RVs and Hep B?
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RV: have an RNA genome
Hep B: has only partially ds DNA genome |
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What is RV RNA like?
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- (+) ss RNA
-7-10 kb -Exists as a dimer -> This allows the virus to recombine -> Increases genetic variability |
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What's the difference btw a simple RV and a complex RV?
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Simple: Only has Gag, Pol and Env genes (ex: RSV)
Complex: Has Gag, Pol, Env and regulatory genes (ex: HIV) Complex genes will use more splicing |
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What can regulatory genes be used for?
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-Express ptns that regulate dif stages of the viral life cycle: transcription, RNA processing...
-Express genes needed to fight cell defence systems |
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Wher are the products of the pol gene? What are they?
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In the capsid
-Integrase -Reverse Transcriptase -Viral Protease |
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Where are regulatory seq found? What do they do?
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-Regulatory seq are at the 5' and 3' ends
-They're non-coding seq -Help transcription DNA--> RNA -Help RT from RNA --> DNA |
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What are the structural and pol ptns of HIV-1?
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Gag ptns:
-Matrix: forms a layer on the inner envelope that directs all gag ptns twds the inner side of the cellular mb -Capsid: forsm protective shell for nucleic acid -Nucleocapsid: in direct contact with RNA, implicated in RT Env ptns: -Surface ptn: gp120 (also have gp160 that is cleaved by cellular ptns) -Transmb ptns: gp41 Pol ptns: -Protease: req'd to process precursor ptns to release the fctnal mature ptn (i.e. must cleave itself or the other POL ptns) -RT -Integrase --> Also have Virion ptn R |
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What are the non-structural ptns of HIV1?
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-Viral infectivity factor (Vif): fights cellular APOBEC which restrict the infection of HIV
-Virion ptn unique (Vpu) to HIV: used in release of viral particles -Transactivator of transcription -Regulator of expression of virion ptns -Negative Effector (Nef): vius can live without this ptn |
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How is Nef involved in HIV?
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If replication slows, Nef will be the major ptn that contributes to pathogenesis dev'l
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What are teh 2 accessory ptns that HIV doesn't require?
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-Nef
-Vpr |
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What are the ealry steps in the viral life cycle?
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1) Attchment of enveloped ptns to cell receptor (use gp120 and cell's CD4+ receptors)
2)Fusion of mb, capsid penetrates cytoplasm 3)RT of ssRNA -> ds DNA and capsid is destroyed 4) Proviral DNA is prepared for integration step (makes pre-integration complex and is transported to nucleus, using viral and cell ptns) -Viral Integrase in tnucleus recog ends of the DNA and integrates the proviral DNA randomnly into the host genome Early stages: Attachment-> Integration |
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What are the late stages of viral cycle?
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-Transcription: get viral RNA
-Viral RNA processing by cellular factors (poly A, cap, splice...) and transport to cytoplasm -Translation of mRNA --> ptns in cytoplasm. These ptns can be shuttled back to the nucleus where they can affect transcription and processing -Maturation, assembly and release of viral particles, which can then infect other cells |
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Describe the entry process.
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-gp120 interacts with the CD4+ receptors on the surface of teh CD4+ lymphocytes
-This interaction exposes gp40 -gp40 interacts with coreceptors: CCR5 and CCR4 -Interaction with the coreceptors initiates the fusion process -Conf change and mbs fuse tog -Pore created through which the viral capsid cn pass to get in the cell cytoplasm -In the cell, RT can take place |
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What happens in RT?
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-RT process primed by tRNA (lysine tRNA) that binds to the PBS
-This initiates DNA synthesis and RT, while RNA is degraded (by RNAse H) -ssRNA --> dsDNA |
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Where in the cell does integration take place? Where does proviral DNA usually integrate in the host genome?
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-Takes place in the nucleus
-Integrates into areas that are actively transcribed because the chromatin in these areas are less condensed .: more open and more susceptile to integration |
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How is viral mRNA produced?
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-Virus recruits cell machinery
-RNA pol II binds to transcriptional start site and makes RNA (but not very efficiently, 1/100 will be full RNA) -Processing of RNA: capping and polyadenylation importnatfor determining half-life of the viral RNAs in the nuc and cyto -mRNA transported from nucleus to cytoplasm -mRNA makes TAT ptn: TAT goes back to the nucleus and binds to the 5' Tar region (in the 5' non-translated region) of the viral RNA -TAT ptn recruits a lot of cellular factors, including a cellular kinase -Cellular kinase P the RNA pol, making it better so that it can always generate full length mRNA .: TAT ptns facillitates entire transcription process |
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What is the mechanism of splicing?
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-Have an intron in btw 2 exons
-Nuc attack of 2'OH of intron on PO4 group of Exon1 -Exon 1 leaves - then 3' OH of Exon 1 ttacks the junction btw the intron and Exon 2 -The 2 exons are fused together and the intron leaves as a lariat group (have 2 transesterification steps, including 2 nuc attacks) |
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Why would the virus want to diminish splicing?
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-Want to generate full length mRNAs that code for the structural ptns
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How does the virus diminsh splicing?
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-Rev element ptn
-After being made in the cyto, Rev element ptn goes into the nucleus and binds RRE (Rev Response Element) -This binding diminishes splicing and favours the generation of unspliced/ singly-spliced mRNA |
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What are the early ptns that are made? What are the later ptns made?
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-Early ptns are highly spliced: Tat, Rev
-Late ptns, less spliced: Env , Vpr, Gag, Pol |
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What is Vpr for?
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Needed for packaging
Once the virus infects a new cell, Vpr is req'd for pre-integration process |
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How are the Gag ptns translated?
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-Gag ptns are all on 1 polyptn (all in one ORF)
-This polyptn gets cleaved later on -The polyptn has 4 ptns and some spacer peptides |
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What are the Gag ptns? (4)
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-MA (matrix): p17
-CA (capsid): p24 -NC (nucleocapsid): p7 -p6 |
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What role does the Matrix ptn play in the assembly process?
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Matrix ptns are on the inner side of the virion mb
-MA forms a mb bound layer -N-term myristyl group is needed for mb binding -> MA ptn modified, myristylated so that it can attach to the mb -this attachment is req'd so tht all the other compnents can be directed to the inner side of the mb where assembly takes place -MA domain targets the precursor pt to the mb |
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What role does the Capsid ptn play in the assembly process?
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Particle assembly
-CA forms a conial core: makes hexamers with a hole in the center that lets nt's penetrate -CA conical structure req'd when virus infects a new cell. When CA is released and penetrates the cell, it allows diffusion of nt into the capsid structure, which fuels RT process. After DNA made. don't need capsid anymore so it is degraded and just left with the Pre-Integration Complex |
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What role does the NC ptn play in the assembly process?
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-The highly basic N-term and zince fingers bind HIV RNA (acts as a chperone)
-NC unwinds tRNA primer (used for RT to make -DNA) -NC helps tRNA bind PBS -NC facilitated processive DNA synthesis (RT) (stabilizes RT enz so it doesn't diss't) -Nc interacts with Vpr, which is involved in the Pre-Int. Comp |
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How do you get Gag-Pol ptns?
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Gag and Pol ptns located in different reading frames
-Need ribosomal frameshift of -1 to get Gag-Pol polyptn -Ribosome must slip 1 nt upstream ('backwards) to get in the right frame to produce Pol ptns |
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What are the Pol ptns?
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-Integrase
-Protease -RT: heterodimer of p51 and p66 -RNAse H: C-term of p66 subunit |
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What is the frequency of making Gag-Pol polyptn?
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~5%
usually ribosome only makes the Gag ptn, hits a stop codon and falls of, instead of frame shifting |
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What process, after entry, requires host cell enzymes?
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Transport from cytoplasm to nucleus
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What are the 2 steps to viral integration into the host cell genome?
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1) 3' processing (cytoplasm)
2) Strand transfer (Nucleus) |
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What happens in 3' processing?
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IN binds the U5 and U3 LTRS
IN removes the terminal G T nt -> this is catalyzed by H2O which acts as a nuc. Get exposure of a new hydroxyl OH group at the viral 3' ends -This prepares the viral DNA for strand transfer |
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What happens in strand transfer stage of integration?
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The new 3' OH acts as a nuc amd attacks the dsDNA host genome at an internucleotide phosphodiester bond of the target DNA
-This covalntly links the ends of the viral DNA into the host genome |
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How is the gap after strand transfer fixed?
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Cellular repair enzymes
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What does the current integrase inhibitor target?
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Only the strand transfer process
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What is another way to target the integration process (without dealing with integrase)
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Target the Pre-Integration Complex (PIC)
-This blocks the transport of viral dsDNA into the nucleus |
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What do cellular factors in the integration process do?
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1) Directly interact with IN
2) Bind to the viral DNA 3) Participate in gap repair |
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How is the Proviral DNA transported directly to the chromosome?
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Need host cell factor: LEDGF/p75
(Not solely due to IN, since can't be reproduced in vitro) |
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What is LEDGF/p75?
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-LEDGF is part of the hepatoma-derived Growth Factor family
-Dominant cell factor req'd for integrase fct -might be a transcriptional regulator of stress related anti-apoptotic ptns -p75: Transcriptional co- activator |
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Where does LEDGF bind?
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Interface btw 2 IN monomers is a groove where part of LEDGF can bind. The part that binds is the Integrase Binding Domain (IBD)
-IBD can contact IN at both sides of the IN dimer |
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How does LEDGF/p75 help integration?
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-IBD and N-terminal PWWP domain/AT-hook DNA binding motif mediate chromatin binding, which is essential for HIV-1 infection
**-N-term seq of LEDGF/p75 tether HIV-1 PIC to chromatin for integration. The domain can interact directly with the host chromosome. -Basically, entire LEDGF acts as an anchor, attaches ciral DNA on one side and the host CHR on the other and brings them close to each other so that they can interact -LEDGF protrects and stabilizes IN, keeps it from getting degraded |
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What is the only current IN inhibitor?
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Raltegravir
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Why is LEDGF an ideal target for drugs?
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-Req'd for viral replication
-Block it get more degradation of IN and block poviral DNA integration by disruting the chromatn that tethers IN -Would be harder for the virus to dev'l resistance to this cuz cell factors have much lower genetic variability |
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What are host restriction factors?
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Host factors that fight the virus
-Intrinsic/innate immunity (i.e IFN response) -TRIM-5alpha and Human APOBEC3GL 2 imp cellular factors not ass't with IFN |
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What is TRIM-5alpha?
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-Member of Tripartite motif ptn family
-Prevents lung cells from being infected with HIV (even if all receptor requirements aer met) (blocks infection of rhesus macaque lung fibroblasts) |
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What is TRIM-5alpha thought to do?
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-Has domain that determine ubiquitin-ligase substrate specificity
-It attracts ptns of ptn degradation path -directs virus to a proteosome -Virus still penetrates and does RT, but then TRIM-5alpha binds (known) and directs the viral DNA to proteosome (unknown, theoretical) |
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What are other possible mechanisms of action of TRIM-5alpha?
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-It triggers innate immunity when it binds the viral capsid
-Coats viral particle and disrupts core rearrangement/uncoating -May mediate disruptive rapid uncoating of the viral capsid -Proteosome independent paths -Circular viral DNA remains coated with TRIM-5alpha which may inhibit nuclear import Multiple anti-viral mechanisms may be involved |
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What happens when the proteosome degradation path is artificially blocked?
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-Virus no longer degraded
-RT resumes, but virus is no longer infectious .: probably multiple anti-viral mechanisms involved |
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How is TRIM-5a in humans?
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Doesn't block HIV
-Rhesus macaque orthologs can protect human cell lines vs HIV, so its the family of TRIM that determnes the tropism of the virus -HIV may have evolved a way to bypass human TRIM-5 |
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How was Human APOBE3G discovered?
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By working on Vif (Virion infectivity factor)
-Vif expression is necessary in virus producing cells -w/out Vif, virus only grew in some cells, .: in the cells that virus didn't grow, there was probably a ptn that inhibits HIV (cells that don't need Vif probably don't make the restricitive ptn that stops HIV) |
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What is the relationship btw Vif and APOBEC3G?
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Vif may counteract the antiviral activity of APOBEC3G
-APOBEC3G somehow edits viral RNA, diminishing viral replication -APOBEC3G is a potent inhibitor of Vif-Deficient HIV replication |
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What does APOBEC3G do to APOBEC (+) cells?
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-Genome has exclusively G--> A mutations
-End up with a T in the (-) DNA strnd and get an A instead of a C when the (+) strand is synthesized -It was proven that this G--> A mutation was not due to RT, but actually to APOBEC3G -APOBEC3G also has deaminase activity that makes C--> U, end up again with an A when the second strand is synthesized, so this is another way of getting a G--> A mutation, in addition to the RT |
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What is important about APOBEC3G?
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-Its incorporated into viral particles
-Its located in teh cytoplasm (where RT also takes place) -It catalytic site is important for Vif-defective replication (no catalytic site, Vif-defective cells can replicate) -it diminishes DNA replication in general -When Vif binds to it, it is degraded |
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How is APOBEC3G so destructive to HIV?
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It can change C-> U
1) The U can bp with A (end up with a G-> A switch, cuz should have had a G there). These G--> A mutation will eventually end up in critical areas and the enz drives the virus to catastrophy 2) The U's can be recognized by cellular enz, which will chop the base off the ssDNA and thencell endonucleases will see this and degrade the DNA |
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What is the role of Vif?
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-Looks for a viral producer cell and target cell
-Targets APOBEC3G for degradation (or APOBEC3G can be incorporated into the viral particles) |
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What are teh restriction factors involved in HIV infection?
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-TRIM-5alpha: early post-entry block by blocking steps before RT and PIC entry
-Tetherin/CD317: Restricts HIV at very late stage. Mb-bound ptn. Vpu from HIV counteracts it. |
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What is Vpu req'd for?
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-Particle release of virus in some cells (Hela) but not others (293T)
-IfVpu is rly req'd, might fight something that is only in that type of cell (same principle as APOBEC3G/Vif) -Vpu targets tetherin for degradation -Single mutation in Tetherin can render the restriction factor insensitive to Vpu downregulation (If tetherin not expressed in some cells, virus doesn't need to express Vpu) |
