HIV发病机理.ppt

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,Experimental 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 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,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 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 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 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 nuclear export of unspliced or single spliced viral transcripts,Arg Rich Domain (ARD) -binds to Importin-b for nuclear import,After nuclear import, Ran-GDP is converted to Ran-GTP, and importin- b dissociates from Rev,Nef,Major determinant of pathogenicity in vivo nef-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 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 sequence 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 of 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 CD4 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 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 mM; yeast two-hybrid assays also suggest an interaction Acts as a connector 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 motif is also important for NBP-1 interactions ? May bind to b-Cop, a coatamer protein which targets proteins to lysosomes,II.Down-modulation of MHC Class I,Advantages: Immune evasion; MHC Class I presents antigens to cytotoxic T- lymphocytes; alerts innate and adaptive immune system to virally infected cells Evidence: Nef expression reduces susceptibility of HIV-infected cells to CTL mediated lysis in vitro selectively 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 lysis,HIV,51Cr,E:T ratio,% Lysis,1:2,1:5,1:10,1:20,100%,0%,HIV wt,HIV Dnef,E (Effector Cell),T (Target Cell),III. Mediates Cellular Activation and Signaling,Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis),III. Mediates Cellular Activation and Signaling,Nef 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 cultures,3,6,9,(days),p24 (ng/ml),Adv-nef supnt,Adv-GFP supnt,III. Mediates Cellular Activation and Signaling,Nef 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 cultures Nef interacts with Pak2 (p21 activated kinase 2) and Nef/Pak2 complex may regulate many of Nefs effect on gene transcription,IV. Infectivity Enhancement,Magnitude of infectivity enhancement is allele dependent Nef mediated enhancement can be provided in trans reporter gene (e.g. GFP or luciferase) expression under control of the LTR promoter can be enhanced when nef expression vector is co-transfected Mechanisms: Increased RT activity; increased proviral DNA synthesis Increased cytoplasmic delivery of viral particles,Vpu: CD4 down-modulation,16 kDa, membrane spanning Binds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-proteasome pathway,Vpu mediated CD4 degradation via ubiquitin-proteasome pathway,Evidence: Vpu activity disrupted by inhibitors of proteasome- mediated proteolysis Vpu activity affected by dominant negative mutants of ubiquitin pathway Removal of lysine residues (ubiquination targets) in CD4 tail prevents Vpu-mediated degradation Vpu binds to b-TrCP, which in turns binds to the proteasome targeting factor Skp1p Overexpression of b-TrCP mutant that cannot bind Skp1p inhibits Vpu-mediated CD4 degradation,Contrast with Nef,Vpu: required for proper maturation and targeting of progeny virions, and for their proper release from the cell surface,Oligomerization of its transmembrane domain results in ion channel activity Similar to influenza virus M2 protein, an ion channel protein that modulates the pH in the Golgi compartment Ion 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 pathway,Vif: Viral infectivity factor, required for robust replication only in certain cells,HIV-1 (Dvif),HIV-1 (,Permissive Non-permissive,+ replication,+ replication,+ replication,no replication,Hut78, H9, 1 PBLs,C8166, 293T, HeLa,Two hypotheses:,Permissive cells express an activity (factor) that can compensate for vif. Non-permissive cells have an inhibitory activity on viral replication, which is overcomed by vif.,See Simon et. al., Nature Med. 4: 1397,Non-permissive,Permissive,Infectivity,Non-permissive: inhibitory cellular factor overcomed by vif Permissive: compensatory factor similar to vif,Permissive vs Non-Permissive T Cell Line,Permissive cells express an activity (factor) that can compensate for vif. Non-permissive cells have an inhibitory activity on viral replication, which is overcomed by vif.,Two hypotheses:,Permissive,Denv vs Denv/Dvif,Permissive,Non-Permissive,Non-permissive,Permissive,Heterokaryon,wt,Dvif,wt,Dvif,Dvif,Infectivity,+,+,Non-