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HIVCellularPathogenesisIIBenhurLee,M.D.第一页，共三十三页。HIVAccessoryGenes:TatRevVifVprVpuNefEssentialinvitroandinvivoEssentialincertaincelltypes(PermissivevsNon-permissivecells)Non-essentialinvitro,butleadstoattenuatedphenotypeinvivo第二页，共三十三页。Tat:TransactivatorofHIV’sLTRPromoter•ExperimentalObservations:–BindingofTattoTARinvitrodoesNOTrequireloopsequencesknowntobenecessaryinvivoforfunction–Pre-incubationofnuclearextractswithrecombinantTatdepletesafactornecessaryforTat-mediatedtranscriptioninvitro–Tatfunctionspoorlyinrodentcellsunlesscomplementedbyfactor(s)presentonChromosome12(radiationhybrids)–TatassociateswithakinasecomplexthathyperphosphorylatesCTDofRNAPII(identifiedthruaninvitrodrugscreenforTatinhibitors)--thiskinaseisCdk9,butCdk9doesNOTbindTat!?–Mysteryhuman-specificco-factorforTatactivitymustexist2ºstructureofHIVTARsequence“loop〞“bulge〞第三页，共三十三页。PredictedandconfirmedpropertiesofTatco-factor:CyclinTBindsdirectlytoTatinacomplexwithCdk9IncreasestheaffinityofTatforTARIncreasesthespecificityofTatfor“loop〞and“bulge〞residuesTat-CycT-Cdk9complexhyperphosphorylatesCTDofRNAPIIandincreasesHIVtranscriptionalprocessivityCycTmapstochromosome12,andpotentiatesTattrans-activationinmurinecells50-to100-foldMurinehomologofhumanCycTdoesNOTbindtoTat第四页，共三十三页。Tat:TransactivatorofHIV’sLTRPromoter•ExperimentalObservationsExplained:–BindingofTattoTARinvitrodoesNOTrequireloopsequencesknowntobenecessaryinvivoforfunction–Pre-incubationofnuclearextractswithrecombinantTatdepletesafactornecessaryforTat-mediatedtranscriptioninvitro–Tatfunctionspoorlyinrodentcellsunlesscomplementedbyfactor(s)presentonChromosome12(radiationhybrids)–TatassociateswithakinasecomplexthathyperphosphorylatesCTDofRNAPII(identifiedthruaninvitrodrugscreenforTatinhibitors)--thiskinaseisCdk9,butCdk9doesNOTbindTat!?–Mysteryhuman-specificco-factorforTatactivitymustexist:CyclinT第五页，共三十三页。Rev•Essentialfornuclearexportofunsplicedorsinglesplicedviraltranscripts第六页，共三十三页。Importin-bRanGDPArgRichDomain(ARD)--bindstoImportin-bfornuclearimportAfternuclearimport,Ran-GDPisconvertedtoRan-GTP,andimportin-bdissociatesfromRev“Free”ARDnowcanbindtoRRE,butonlyincontextofRevmultimersRevNuclearExportSignal(NES),leucine-richdomain,bindsExportin-1(XPO)cooperativelywithRan-GTPRevRevImportin-bRan-GDPRcc1Ran-GTPRan-GDPRan-GTPRanGAPImportin-bRanGTP第七页，共三十三页。Nef•Majordeterminantofpathogenicityinvivo–nef-deletedSIVisseverelyattenuatedintherhesusmacaquemodel–infectionofmacaqueswithrecombinantSIVcarryingaprematureSTOPcodon(pointmutation)resultsinrapidrevertantswiththenefORF–Patientsinfectedwithnef-defectiveHIVhaveadramaticallydecreasedrateofdiseaseprogression(>15years)–nef-deletedHIVdonotdepletethymocytesasmuch,orreplicatetoashightiters,aswild-typeHIVintheSCID-humicemodel第八页，共三十三页。PleiotropicFunctionsofNefN-myristoylationrequiredforNefactivity--impliesthatmembranelocalizationofnefiscriticalforitsactivityMGxxx(S/T)(K/R)(K/R)MGxxx(S)(K)(K/R)100%100%99%~50%ConsensusN-myristoylationSignal:HIVsequenceConservationinNefprotein:第九页，共三十三页。PleiotropicFunctionsofNef•Down-regulatescellsurfacelevelsofCD4•Down-regulatessurfacelevelsofmajorhistocompatibilityclassImolecules•Mediatescellularsignalingandactivation•Enhancesviralinfectivity第十页，共三十三页。I...