齊魯醫(yī)學(xué)HIV發(fā)病機(jī)理

1、HIVCellular Pathogenesis IIBenhur Lee, M.D.2021/10/23 星期六1HIV 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 2021/10/23 星期六2Tat: Transactivator of HIV

2、s LTR PromoterExperimental Observations:Binding of Tat to TAR in vitro does NOT require loop sequences known to be necessary in vivo for functionPre-incubation of nuclear extracts with recombinant Tat depletes a factor necessary for Tat-mediated transcription in vitroTat functions poorly in rodent c

3、ells 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-facto

4、r for Tat activity must exist2 structure of HIV TAR sequence“l(fā)oop”“bulge”2021/10/23 星期六3Predicted 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 “l(fā)oop” and “bulge” residuesTat-Cy

5、cT-Cdk9 complex hyperphosphorylates CTD 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 Tat2021/10/23 星期六4Tat: Transactivator of HIVs LTR PromoterEx

6、perimental Observations Explained:Binding of Tat to TAR in vitro does NOT require loop sequences known to be necessary in vivo for functionPre-incubation of nuclear extracts with recombinant Tat depletes a factor necessary for Tat-mediated transcription in vitroTat functions poorly in rodent cells u

7、nless 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

8、Tat activity must exist: Cyclin T2021/10/23 星期六5RevEssential for nuclear export of unspliced or single spliced viral transcripts2021/10/23 星期六6Importin-bRanGDPArg Rich Domain (ARD)-binds to Importin-b for nuclear import After nuclear import,Ran-GDP is convertedto Ran-GTP, and importin- b dissociates

9、 from Rev “Free” ARD now can bind to RRE, but only in context of Rev multimersRevNuclear Export Signal (NES),leucine-rich domain, binds Exportin-1 (XPO)cooperatively with Ran-GTPRevRevImportin-bRan-GDPRcc1Ran-GTPRan-GDPRan-GTPRanGAPImportin-bRanGTP2021/10/23 星期六7NefMajor determinant of pathogenicity

10、 in vivonef-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 p

11、rogression (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 model2021/10/23 星期六8Pleiotropic Functions of NefN-myristoylation required for Nef activity-implies that membranelocalization of nef is critical for its activi

12、tyMGxxx(S/T)(K/R)(K/R)MGxxx(S)(K)(K/R)100%100%99%50%ConsensusN-myristoylationSignal:HIV sequenceConservation inNef protein:2021/10/23 星期六9Pleiotropic Functions of NefDown-regulates cell surface levels of CD4Down-regulates surface levels of major histocompatibility class I moleculesMediates cellular

13、signaling and activation Enhances viral infectivity 2021/10/23 星期六10I. Down-modulation of surface CD4Down-modulation of surface CD4 via internalization followed by degradation via endosomal/lysosomal pathway Advantages:Prevents disadvantageous super-infection of host cellEnhance viral progeny releas

14、e (by preventing Env-mediated sequestration of CD4 in secretory pathway)Evidence:Nef expression increases number of CD4 containing clathrin-coated pitsNef-induced CD4 down-modulation blocked by inhibitors of clathrin-coated pit-mediated endocytosis (e.g. ikaguramycin)Inhibition of lysosomal acidific

15、ation (e.g. via chloroqine treatment) blocks Nef-induced CD4 degradationExpression of nef alone in T-cell lines can lead to CD4 downregulation (as determined by FACS) CD4Nef-GFP.2021/10/23 星期六11I. Down-modulation of surface CD4Mechanism(s)?Direct interaction with CD4 has not been biochemically demon

16、strable, but NMR analysis suggest a direct interaction with Kd 0.87 mM; yeast two-hybrid assays also suggest an interactionActs as a connector to the host-cell endocytic machineryCo-localizes with AP-2 on inner plasma membraneConserved dileucine based sorting motif (E/DxxxL) in Nef is important for

17、both CD4-down-modulation and AP-2 co-localizationInteracts 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 interacti

18、on, 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 lysosomesNBP-12021/10/23 星期六12II.Down-modulation of MHC Class IAdvantages:Immune evasion; MHC Class I presents antigens to cytotoxic T- lymphoc

19、ytes; alerts innate and adaptive immune system to virally infected cellsEvidence:Nef expression reduces susceptibility of HIV-infected cells to CTL mediated lysis in vitroselectively down-regulates only HLA-A and HLA-B, which presents antigens to CTLs; does NOT down-regulate HLA-C and HLA-E, which i

20、nhibit NK-cell mediated cell lysis Thus, efficiency of CTL-mediated lysis (adaptive immunity) is reduced without increasing increasing susceptibility to NK cell lysisHIVCTLMHC Class IHIV antigen51Cr2021/10/23 星期六13E:T ratio% Lysis1:21:51:101:20100%0%HIV wtHIV DnefCTLMHC Class IHIV antigenE (Effector

21、 Cell)T (Target Cell)2021/10/23 星期六14III. Mediates Cellular Activation and SignalingNef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis)2021/10/23 星期六15III. Mediates Cellular Activation and SignalingNef expression upregulates a transcriptional program

22、 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 cultures369(days)p24(ng/ml)Adv-nef sup

23、ntAdv-GFP supnt2021/10/23 星期六16III. Mediates Cellular Activation and SignalingNef 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 transduc

24、ed with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid culturesNef interacts with Pak2 (p21 activated kinase 2) and Nef/Pak2 complex may regulate many of Nefs effect on gene transcription2021/10/23 星期六17IV. Infectivity EnhancementMagnitude of infectivity enhancem

25、ent is allele dependentNef mediated enhancement can be provided in transreporter gene (e.g. GFP or luciferase) expression under control of the LTR promoter can be enhanced when nef expression vector is co-transfectedMechanisms:Increased RT activity; increased proviral DNA synthesisIncreased cytoplas

26、mic delivery of viral particles2021/10/23 星期六18Vpu: CD4 down-modulation16 kDa, membrane spanningBinds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-proteasome pathway2021/10/23 星期六19Vpu mediated CD4 degradation via ubiquitin-proteasome pathwayEvidence:Vpu activity disrupted by inhibit

27、ors of proteasome- mediated proteolysisVpu activity affected by dominant negative mutants of ubiquitin pathwayRemoval of lysine residues (ubiquination targets) in CD4 tail prevents Vpu-mediated degradationVpu binds to b-TrCP, which in turns binds to the proteasome targeting factor Skp1pOverexpressio

28、n of b-TrCP mutant that cannot bind Skp1p inhibits Vpu-mediated CD4 degradationContrast with Nef2021/10/23 星期六20Vpu: required for proper maturation and targeting of progeny virions, and for their proper release from the cell surfaceOligomerization of its transmembrane domain results in ion channel a

29、ctivitySimilar to influenza virus M2 protein, an ion channel protein that modulates the pH in the Golgi compartmentIon 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 pathw

30、ay2021/10/23 星期六21Vif: Viral infectivity factor, required for robust replication only in certain cellsHIV-1 (Dvif)HIV-1 (PermissiveNon-permissive+ replication+ replication+ replicationno replicationHut78, H9, 1 PBLsC8166, 293T, HeLaTwo hypotheses:Permissive cells express an activity (factor) that ca

31、n 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: 13972021/10/23 星期六22Non-permissivePermissivewtDvifwtDvifInfectivity+-+Non-permissive: inhibitory cellular factor overcomed by vifPermissive: compens

32、atory factor similar to vifHeterokaryonwtDvifWhich phenotype will dominate?2021/10/23 星期六23Permissive 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 v

33、if.Two hypotheses:PermissiveDenv vs Denv/DvifPermissiveNon-Permissive2021/10/23 星期六24Non-permissivePermissiveHeterokaryonwtDvifwtDvifDvifInfectivity+-+Non-permissive: inhibitory cellular factor overcomed by vif+-wt2021/10/23 星期六25Expression of CEM15 inCEM-SS (permissive cells)renders it non-permissive2021/10/23 星期六26G2 ArrestLTR transcription, i.e.,virus production is more efficient during G2Augments Nuclear Import of Pre-Integration ComplexExtracellular vpr (from decaying virions, or cytosolic leakage from infected apoptotic cells) re-capitulates intracellular vpr function Induces