HIV发病机理.pptx

HIVCellular Pathogenesis IIBenhur Lee,M.D.HIV Accessory Genes:TatRevVifVprVpuNefEssential in vitro and in vivoEssential 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 PromoterExperimental 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 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 exist2 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 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 TatTat:Transactivator of HIVs LTR PromoterExperimental Observations Explained: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 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:Cyclin TRevEssential for nuclear export of unspliced or single spliced viral transcriptsImportin-b bRanGDPArg Rich Domain(ARD)-binds to Importin-b b for nuclear import After nuclear import,Ran-GDP is convertedto Ran-GTP,and importin-b b dissociates from Rev“Free”ARD now can bind to RRE,but only in context of Rev multimersRevNuclear Export Signal(NES),leucine-rich domain,binds Exportin-1(XPO)cooperatively with Ran-GTPRevRevImportin-b bRan-GDPRcc1Ran-GTPRan-GDPRan-GTPRanGAPImportin-b bRanGTPNefMajor determinant of pathogenicity in vivonef-deleted SIV is severely attenuated in the rhesus macaque 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,or replicate to as high titers,as wild-type HIV in the SCID-hu mice modelPleiotropic Functions of NefN-myristoylation required for Nef activity-implies that membranelocalization of nef is critical for its activityMGxxx(S/T)(K/R)(K/R)MGxxx(S)(K)(K/R)100%100%99%50%ConsensusN-myristoylationSignal:HIV sequenceConservation inNef protein:Pleiotropic Functions of NefDown-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 CD4Down-modulation 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 number 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 can lead to CD4 downregulation(as determined by FACS)CD4Nef-GFP.I.Down-modulation of surface CD4Mechanism(s)?Direct interaction with CD4 has not been biochemically demonstrable,but NMR analysis suggest a direct interaction with Kd 0.87 m mM;yeast two-hybrid assays also suggest an interactionActs as a connector to the host-cell endocytic machineryCo-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 clathrin-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 b-Cop,a coatamer protein which targets proteins to lysosomesNBP-1II.Down-modulation of MHC Class IAdvantages: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 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 lysisHIVCTLMHC Class IHIV antigen51CrE:T ratio%Lysis1:21:51:101:20100%0%HIV wtHIV D DnefCTLMHC Class IHIV antigenE(Effector Cell)T(Target Cell)III.Mediates Cellular Activation and SignalingNef expression upregulates a transcriptional program that activates the HIV LTR(microarray analysis)III.Mediates Cellular Activation and SignalingNef 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-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures369(days)p24(ng/ml)Advnef supntAdv-GFP supntIII.Mediates Cellular Activation and SignalingNef 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 nefexpressing 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 transcriptionIV.Infectivity EnhancementMagnitude of infectivity enhancement is allele dependentNef mediated 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 particlesVpuCD4 down-modulation16 kDa,membrane spanningBinds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-proteasome pathwayVpu mediated CD4 degradation via ubiquitin-proteasome pathwayEvidence:Vpu activity disrupted by inhibitors of proteasome-mediated proteolysisVpu activity affected by dominant negative mutants of ubiquitin pathwayRemoval of lysine residues (ubiquination targets)in CD4 tail prevents Vpu-mediated degradationVpu binds to b b-TrCP,which in turns binds to the proteasome targeting factor Skp1pOverexpression of b b-TrCP mutant that cannot bind Skp1p inhibits Vpu-mediated CD4 degradationContrast with NefVpu:required for proper maturation and targeting of progeny virions,and for their proper release from the cell surfaceOligomerization of its transmembrane domain results in ion channel activitySimilar to influenza virus M2 protein,an ion channel protein that modulates the 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 pathwayVif:Viral infectivity factor,required for robust replication only in certain cellsHIV-1(D Dvif)HIV-1(PermissiveNon-permissive+replication+replication+replicationno replicationHut78,H9,1 PBLsC8166,293T,HeLaTwo hypotheses:(a)Permissive cells express an activity(factor)that can compensate for vif.(b)Non-permissive cells have an inhibitory activity on viral replication,which is overcomed by vif.See Simon et.al.,Nature Med.4:1397Non-permissivePermissivewtD DvifwtD DvifInfectivity+-+Non-permissive:inhibitory cellular factor overcomed by vifPermissive:compensatory factor similar to vifHeterokaryonwtD DvifWhich phenotype will dominate?Permissive vs Non-Permissive T Cell Line(a)Permissive cells express an activity(factor)that can compensate for vif.(b)Non-permissive cells have an inhibitory activity on viral replication,which is overcomed by vif.Two hypotheses:PermissiveD Denv vs D Denv/D DvifPermissiveNon-PermissiveNon-permissivePermissiveHeterokaryonwtD DvifwtD DvifD DvifInfectivity+-+Non-permissive:inhibitory cellular factor overcomed by vif+-wtExpression of CEM15 inCEM-SS(permissive cells)renders it non-permissiveG2 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 arrestActivates HIV replication in latently infected cellsIncreased HIV replication in macrophagesApoptosis;“bystander”cell killing in lymphoid organs and brain Vpr:Two Independent FunctionsG1M(Mitosis)SDNASynthesisG0G2“Quality Control”phaseMitosis is not initiated untilDNA is free of damageG2 ArrestLTR transcription is more active,i.e.,virus production is more efficient during G2VprPO4-+-G2MWee1-GFPCyc B1-RFPG2 Arrestnuclear herniation due to disruption of nuclear lamin structuremixing of segregated cell cycle regulators may lead to G2 arrest Vpr:G2 Cell Cycle ArrestVpr:Augments Nuclear Import ofPre-Integration ComplexVpr lacks classical NLS sequences-but binds to Importin-a aVpr=Importin-b b analog?Vpr:Augments Nuclear Import ofPre-Integration ComplexImportin-a aImportin-a aTransport of PICs containing Vpr-GFP fusion proteinRed=anti-tubulin antibodiesBlue=anti-tubulinGreen=Vpr-GFPRed=Alexa-dUTPPIC/RTCNuclear Import of PICstalled by:(a)Anti-dynein Mab(b)Nicodazole treatment (nicodazole disrupts microtubles)