1、HIV Cellular Pathogenesis II Benhur Lee,M.D.HIV Accessory Genes:Tat Rev Vif Vpr Vpu Nef 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 Experimen
2、tal Observations: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 complemen
3、ted 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 e
4、xist 2 structure of HIV TAR sequence“loop”“bulge”Predicted and confirmed properties of Tat co-factor:Cyclin T Binds directly to Tat in a complex with Cdk9 Increases the affinity of Tat for TAR Increases the specificity of Tat for“loop”and“bulge”residues Tat-CycT-Cdk9 complex hyperphosphorylates CTD
5、of RNAP II and increases HIV transcriptional processivity CycT maps to chromosome 12,and potentiates Tat trans-activation in murine cells 50-to 100-fold Murine homolog of human CycT does NOT bind to Tat Tat:Transactivator of HIVs LTR Promoter Experimental Observations Explained:Binding of Tat to TAR
6、 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 Chromosome
7、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 T Rev Essential for nuclea
8、r export of unspliced or single spliced viral transcripts Importin-b b Ran GDP Arg Rich Domain(ARD)-binds to Importin-b b for nuclear import After nuclear import,Ran-GDP is converted to Ran-GTP,and importin-b b dissociates from Rev “Free”ARD now can bind to RRE,but only in context of Rev multimers R
9、ev Nuclear Export Signal(NES),leucine-rich domain,binds Exportin-1(XPO)cooperatively with Ran-GTP Rev Rev Importin-b b Ran-GDP Rcc1 Ran-GTP Ran-GDP Ran-GTP RanGAP Importin-b b Ran GTP Nef Major determinant of pathogenicity in vivo nef-deleted SIV is severely attenuated in the rhesus macaque model in
10、fection 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 replic
11、ate 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 membrane localization of nef is critical for its activity MGxxx(S/T)(K/R)(K/R)MGxxx(S)(K)(K/R)100%100%99%50%Consensus N-myristoylation Signal:HIV sequ
12、ence Conservation in Nef 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 I.Down-modulation of surface CD4 Down-modulation o
13、f surface CD4 via internalization followed by degradation via endosomal/lysosomal pathway Advantages:Prevents disadvantageous super-infection of host cell Enhance viral progeny release(by preventing Env-mediated sequestration of CD4 in secretory pathway)Evidence:Nef expression increases number of CD
14、4 containing clathrin-coated pits Nef-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 degradation Expression of nef alone in T-cell lines can lead
15、 to CD4 downregulation(as determined by FACS)CD4 Nef-GFP.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 m mM;yeast two-hybrid assays also suggest an interaction Acts as a con
16、nector to the host-cell endocytic machinery Co-localizes with AP-2 on inner plasma membrane Conserved dileucine based sorting motif(E/DxxxL)in Nef is important for both CD4-down-modulation and AP-2 co-localization Interacts 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