《HIV发病机理》PPT课件.ppt

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1、HIVCellular Pathogenesis II,Benhur Lee,M.D.,HIV Accessory Genes:TatRevVifVprVpuNef,Essential in vitro and in vivo,Essential in certain cell types(Permissive vs Non-permissive cells),Non-essential in vitro,but leads to attenuated phenotype in vivo,Tat:Transactivator of HIVs LTR Promoter,Experimental

2、Observations:Binding of Tat to TAR in vitro does NOT require loop sequences known to be necessary in vivo for functionPre-incubation of nuclear extracts with recombinant Tat depletes a factor necessary for Tat-mediated transcription in vitroTat functions poorly in rodent cells unless complemented by

3、 factor(s)present on Chromosome 12(radiation hybrids)Tat associates with a kinase complex that hyperphosphorylates CTD of RNAP II(identified thru an in vitro drug screen for Tat inhibitors)-this kinase is Cdk9,but Cdk9 does NOT bind Tat!?Mystery human-specific co-factor for Tat activity must exist,2

4、 structure of HIV TAR sequence,“loop”,“bulge”,Predicted and confirmed properties of Tat co-factor:Cyclin TBinds directly to Tat in a complex with Cdk9Increases the affinity of Tat for TARIncreases the specificity of Tat for“loop”and“bulge”residuesTat-CycT-Cdk9 complex hyperphosphorylates CTD of RNAP

5、 II and increases HIV transcriptional processivityCycT maps to chromosome 12,and potentiates Tat trans-activation in murine cells 50-to 100-foldMurine homolog of human CycT does NOT bind to Tat,Tat:Transactivator of HIVs LTR Promoter,Experimental Observations Explained:Binding of Tat to TAR in vitro

6、 does NOT require loop sequences known to be necessary in vivo for functionPre-incubation of nuclear extracts with recombinant Tat depletes a factor necessary for Tat-mediated transcription in vitroTat functions poorly in rodent cells unless complemented by factor(s)present on Chromosome 12(radiatio

7、n hybrids)Tat associates with a kinase complex that hyperphosphorylates CTD of RNAP II(identified thru an in vitro drug screen for Tat inhibitors)-this kinase is Cdk9,but Cdk9 does NOT bind Tat!?Mystery human-specific co-factor for Tat activity must exist:Cyclin T,Rev,Essential for nuclear export of

8、 unspliced or single spliced viral transcripts,Arg Rich Domain(ARD)-binds to Importin-b for nuclear import,After nuclear import,Ran-GDP is convertedto Ran-GTP,and importin-b dissociates from Rev,Nef,Major determinant of pathogenicity in vivonef-deleted SIV is severely attenuated in the rhesus macaqu

9、e model infection of macaques with recombinant SIV carrying a premature STOP codon(point mutation)results in rapid revertants with the nef ORF Patients infected with nef-defective HIV have a dramatically decreased rate of disease progression(15 years)nef-deleted HIV do not deplete thymocytes as much

10、,or replicate to as high titers,as wild-type HIV in the SCID-hu mice model,Pleiotropic Functions of Nef,N-myristoylation required for Nef activity-implies that membranelocalization of nef is critical for its activity,MGxxx(S/T)(K/R)(K/R),MGxxx(S)(K)(K/R),100%,100%,99%,50%,ConsensusN-myristoylationSi

11、gnal:,HIV sequenceConservation inNef protein:,Pleiotropic Functions of Nef,Down-regulates cell surface levels of CD4Down-regulates surface levels of major histocompatibility class I moleculesMediates cellular signaling and activation Enhances viral infectivity,I.Down-modulation of surface CD4,Down-m

12、odulation of surface CD4 via internalization followed by degradation via endosomal/lysosomal pathway Advantages:Prevents disadvantageous super-infection of host cellEnhance viral progeny release(by preventing Env-mediated sequestration of CD4 in secretory pathway)Evidence:Nef expression increases nu

13、mber of CD4 containing clathrin-coated pitsNef-induced CD4 down-modulation blocked by inhibitors of clathrin-coated pit-mediated endocytosis(e.g.ikaguramycin)Inhibition of lysosomal acidification(e.g.via chloroqine treatment)blocks Nef-induced CD4 degradationExpression of nef alone in T-cell lines c

14、an lead to CD4 downregulation(as determined by FACS),CD4,Nef-GFP,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.

15、,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.

16、,.,.,.,.,.,.,.,.,.,I.Down-modulation of surface CD4,Mechanism(s)?Direct interaction with CD4 has not been biochemically demonstrable,but NMR analysis suggest a direct interaction with Kd 0.87 mM;yeast two-hybrid assays also suggest an interactionActs as a connector to the host-cell endocytic machine

17、ryCo-localizes with AP-2 on inner plasma membraneConserved dileucine based sorting motif(E/DxxxL)in Nef is important for both CD4-down-modulation and AP-2 co-localizationInteracts with NBP-1(identified through a yeast two-hybrid screen).NBP-1 is part of the vacuolar membrane ATPase complex in clathr

18、in-coated pits(H subunit of vacuolar ATPase-VH1)C-terminal diacidic motif(DD)in Nef is important for NBP-1 interaction,and,at least in SIV Nef,the dileucine motif is also important for NBP-1 interactions?May bind to b-Cop,a coatamer protein which targets proteins to lysosomes,II.Down-modulation of M

19、HC Class I,Advantages:Immune evasion;MHC Class I presents antigens to cytotoxic T-lymphocytes;alerts innate and adaptive immune system to virally infected cellsEvidence:Nef expression reduces susceptibility of HIV-infected cells to CTL mediated lysis in vitroselectively down-regulates only HLA-A and

20、 HLA-B,which presents antigens to CTLs;does NOT down-regulate HLA-C and HLA-E,which inhibit NK-cell mediated cell lysis Thus,efficiency of CTL-mediated lysis(adaptive immunity)is reduced without increasing increasing susceptibility to NK cell lysis,HIV,51Cr,E:T ratio,%Lysis,1:2,1:5,1:10,1:20,100%,0%

21、,HIV wt,HIV Dnef,E(Effector Cell),T(Target Cell),III.Mediates Cellular Activation and Signaling,Nef expression upregulates a transcriptional program that activates the HIV LTR(microarray analysis),III.Mediates Cellular Activation and Signaling,Nef expression upregulates a transcriptional program tha

22、t activates the HIV LTR(microarray analysis)Nef can induce secretion of paracrine factors that enhance viral replication;macrophage supernatants from cells transduced with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures,3,6,9,(days),p24(ng/ml),Adv-nef sup

23、nt,Adv-GFP supnt,III.Mediates Cellular Activation and Signaling,Nef expression upregulates a transcriptional program that activates the HIV LTR(microarray analysis)Nef can induce secretion of paracrine factors that enhance viral replication;macrophage supernatants from cells transduced with nef-expr

24、essing adenoviral vector can facilitate HIV replication in resting lymphoid culturesNef interacts with Pak2(p21 activated kinase 2)and Nef/Pak2 complex may regulate many of Nefs effect on gene transcription,IV.Infectivity Enhancement,Magnitude of infectivity enhancement is allele dependentNef mediat

25、ed enhancement can be provided in transreporter gene(e.g.GFP or luciferase)expression under control of the LTR promoter can be enhanced when nef expression vector is co-transfectedMechanisms:Increased RT activity;increased proviral DNA synthesisIncreased cytoplasmic delivery of viral particles,Vpu:C

26、D4 down-modulation,16 kDa,membrane spanningBinds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-proteasome pathway,Vpu mediated CD4 degradation via ubiquitin-proteasome pathway,Evidence:Vpu activity disrupted by inhibitors of proteasome-mediated proteolysisVpu activity affected by domi

27、nant negative mutants of ubiquitin pathwayRemoval of lysine residues(ubiquination targets)in CD4 tail prevents Vpu-mediated degradationVpu binds to b-TrCP,which in turns binds to the proteasome targeting factor Skp1pOverexpression of b-TrCP mutant that cannot bind Skp1p inhibits Vpu-mediated CD4 deg

28、radation,Contrast with Nef,Vpu:required for proper maturation and targeting of progeny virions,and for their proper release from the cell surface,Oligomerization of its transmembrane domain results in ion channel activitySimilar to influenza virus M2 protein,an ion channel protein that modulates the

29、 pH in the Golgi compartmentIon channel activity of Vpu may be required for proper virion maturation and assembly by protecting newly formed Env protein from premature conformational changes in the secretory pathway,Vif:Viral infectivity factor,required for robust replication only in certain cells,H

30、IV-1(Dvif),HIV-1(,PermissiveNon-permissive,+replication,+replication,+replication,no replication,Hut78,H9,1 PBLs,C8166,293T,HeLa,Two hypotheses:,Permissive cells express an activity(factor)that can compensate for vif.Non-permissive cells have an inhibitory activity on viral replication,which is over

31、comed by vif.,See Simon et.al.,Nature Med.4:1397,Non-permissive,Permissive,Infectivity,Non-permissive:inhibitory cellular factor overcomed by vifPermissive:compensatory factor similar to vif,Permissive vs Non-Permissive T Cell Line,Permissive cells express an activity(factor)that can compensate for

32、vif.Non-permissive cells have an inhibitory activity on viral replication,which is overcomed by vif.,Two hypotheses:,Permissive,Denv vs Denv/Dvif,Permissive,Non-Permissive,Non-permissive,Permissive,Heterokaryon,wt,Dvif,wt,Dvif,Dvif,Infectivity,+,+,Non-permissive:inhibitory cellular factor overcomed

33、by vif,wt,G2 ArrestLTR transcription,i.e.,virus production is more efficient during G2Augments Nuclear Import of Pre-Integration ComplexExtracellular vpr(from decaying virions,or cytosolic leakage from infected apoptotic cells)re-capitulates intracellular vpr function Induces cell cycle arrestActiva

34、tes HIV replication in latently infected cellsIncreased HIV replication in macrophagesApoptosis;“bystander”cell killing in lymphoid organs and brain,Vpr:Two Independent Functions,G0,G2 ArrestLTR transcription is more active,i.e.,virus production is more efficient during G2,Vpr:G2 Cell Cycle Arrest,V

35、pr:Augments Nuclear Import ofPre-Integration Complex,Vpr lacks classical NLS sequences-but binds to Importin-a,Vpr:Augments Nuclear Import ofPre-Integration Complex,Transport of PICs containing Vpr-GFP fusion protein,Red=anti-tubulin antibodies,Blue=anti-tubulinGreen=Vpr-GFPRed=Alexa-dUTP,PIC/RTC,Nuclear Import of PICstalled by:(a)Anti-dynein Mab(b)Nicodazole treatment(nicodazole disrupts microtubles),