1、 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 Promoter Experimental O
2、bservations: Binding of Tat to TAR in vitro does NOT require loop sequences known to be necessary in vivo for function Pre-incubation of nuclear extracts with recombinant Tat depletes a factor necessary for Tat-mediated transcription in vitro Tat functions poorly in rodent cells unless complemented
3、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
4、 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 CT
5、D 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 Promoter Experimental Observations Explained: Binding of Tat to
6、 TAR in vitro does NOT require loop sequences known to be necessary in vivo for function Pre-incubation of nuclear extracts with recombinant Tat depletes a factor necessary for Tat-mediated transcription in vitro Tat functions poorly in rodent cells unless complemented by factor(s) present on Chromo
7、some 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 TRev Essential
8、for nuclear export of unspliced or single spliced viral transcriptsImportin-RanGDPArg Rich Domain (ARD)-binds to Importin- for nuclear import After nuclear import,Ran-GDP is convertedto Ran-GTP, and importin- dissociates from Rev “Free” ARD now can bind to RRE, but only in context of Rev multimersRe
9、vNuclear Export Signal (NES),leucine-rich domain, binds Exportin-1 (XPO)cooperatively with Ran-GTPRevRevImportin-Ran-GDPRcc1Ran-GTPRan-GDPRan-GTPRanGAPImportin-RanGTPNef Major determinant of pathogenicity in vivo nef-deleted SIV is severely attenuated in the rhesus macaque model infection of macaque
10、s 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 hig
11、h 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 Nef Down-regulates cell surface levels of CD4 Down-regulates surface levels of major histocompatibility class I molecules Mediates cellular signaling and activation Enhances viral infectivity
