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HIVCellularPathogenesisIIBenhurLee,M.D.HIVAccessoryGenes:TatRevVifVprVpuNefEssentialinvitroandinvivoEssentialincertaincelltypes(PermissivevsNon-permissivecells)Non-essentialinvitro,butleadstoattenuatedphenotypeinvivoTat:TransactivatorofHIV’sLTRPromoter•ExperimentalObservations:–BindingofTattoTARinvitrodoesNOTrequireloopsequencesknowntobenecessaryinvivoforfunction–Pre-incubationofnuclearextractswithrecombinantTatdepletesafactornecessaryforTat-mediatedtranscriptioninvitro–Tatfunctionspoorlyinrodentcellsunlesscomplementedbyfactor(s)presentonChromosome12(radiationhybrids)–TatassociateswithakinasecomplexthathyperphosphorylatesCTDofRNAPII(identifiedthruaninvitrodrugscreenforTatinhibitors)--thiskinaseisCdk9,butCdk9doesNOTbindTat!?–Mysteryhuman-specificco-factorforTatactivitymustexist2ºstructureofHIVTARsequence“loop”“bulge”PredictedandconfirmedpropertiesofTatco-factor:CyclinTBindsdirectlytoTatinacomplexwithCdk9IncreasestheaffinityofTatforTARIncreasesthespecificityofTatfor“loop”and“bulge”residuesTat-CycT-Cdk9complexhyperphosphorylatesCTDofRNAPIIandincreasesHIVtranscriptionalprocessivityCycTmapstochromosome12,andpotentiatesTattrans-activationinmurinecells50-to100-foldMurinehomologofhumanCycTdoesNOTbindtoTatTat:TransactivatorofHIV’sLTRPromoter•ExperimentalObservationsExplained:–BindingofTattoTARinvitrodoesNOTrequireloopsequencesknowntobenecessaryinvivoforfunction–Pre-incubationofnuclearextractswithrecombinantTatdepletesafactornecessaryforTat-mediatedtranscriptioninvitro–Tatfunctionspoorlyinrodentcellsunlesscomplementedbyfactor(s)presentonChromosome12(radiationhybrids)–TatassociateswithakinasecomplexthathyperphosphorylatesCTDofRNAPII(identifiedthruaninvitrodrugscreenforTatinhibitors)--thiskinaseisCdk9,butCdk9doesNOTbindTat!?–Mysteryhuman-specificco-factorforTatactivitymustexist:CyclinTRev•EssentialfornuclearexportofunsplicedorsinglesplicedviraltranscriptsImportin-RanGDPArgRichDomain(ARD)--bindstoImportin-fornuclearimportAfternuclearimport,Ran-GDPisconvertedtoRan-GTP,andimportin-dissociatesfromRev“Free”ARDnowcanbindtoRRE,butonlyincontextofRevmultimersRevNuclearExportSignal(NES),leucine-richdomain,bindsExportin-1(XPO)cooperativelywithRan-GTPRevRevImportin-Ran-GDPRcc1Ran-GTPRan-GDPRan-GTPRanGAPImportin-RanGTPNef•Majordeterminantofpathogenicityinvivo–nef-deletedSIVisseverelyattenuatedintherhesusmacaquemodel–infectionofmacaqueswithrecombinantSIVcarryingaprematureSTOPcodon(pointmutation)resultsinrapidrevertantswiththenefORF–Patientsinfectedwithnef-defectiveHIVhaveadramaticallydecreasedrateofdiseaseprogression(>15years)–nef-deletedHIVdonotdepletethymocytesasmuch,orreplicatetoashightiters,aswild-typeHIVintheSCID-humicemodelPleiotropicFunctionsofNefN-myristoylationrequiredforNefactivity--impliesthatmembranelocalizationofnefiscriticalforitsactivityMGxxx(S/T)(K/R)(K/R)MGxxx(S)(K)(K/R)100%100%99%~50%ConsensusN-myristoylationSignal:HIVsequenceConservationinNefprotein:PleiotropicFunctionsofNef•Down-regulatescellsurfacelevelsofCD4•Down-regulatessurfacelevelsofmajorhistocompatibilityclassImolecules•Mediatescellularsignalingandactivation•EnhancesviralinfectivityI.Down-modulationofsurfaceCD4•Down-modulationofsurfaceCD4viainternalizationfollowedbydegradationviaendosomal/lysosomalpathway•Advantages:–Preventsdisadvantageoussuper-infectionofhos...