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1、HIVCellular Pathogenesis IIBenhur Lee,M.D.第一页，共三十三页。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 第二页，共三十三页。Tat:Transactivator of HIVs LTR Promote
2、rExperimental 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 cells unless c
3、omplemented 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 activit
4、y must exist2 structure of HIV TAR sequence“loop”“bulge”第三页，共三十三页。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 hyperphosph
5、orylates 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 Tat第四页，共三十三页。Tat:Transactivator of HIVs LTR PromoterExperimental Observations Explained:Bi
6、nding 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 unless complemented by factor(s)prese
7、nt 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第五页，共三十三
8、页。RevEssential for nuclear export of unspliced or single spliced viral transcripts第六页，共三十三页。Importin-b bRanGDPArg Rich Domain(ARD)-binds to Importin-b b for nuclear import After nuclear import,Ran-GDP is convertedto Ran-GTP,and importin-b b dissociates from Rev“Free”ARD now can bind to RRE,but only
9、in context of Rev multimersRevNuclear Export Signal(NES),leucine-rich domain,binds Exportin-1(XPO)cooperatively with Ran-GTPRevRevImportin-b bRan-GDPRcc1Ran-GTPRan-GDPRan-GTPRanGAPImportin-b bRanGTP第七页，共三十三页。NefMajor determinant of pathogenicity in vivonef-deleted SIV is severely attenuated in the r
10、hesus 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 thymoc
11、ytes as much,or replicate to as high titers,as wild-type HIV in the SCID-hu mice model第八页，共三十三页。Pleiotropic 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-myr
12、istoylationSignal:HIV sequenceConservation inNef protein:第九页，共三十三页。Pleiotropic Functions of NefDown-regulates cell surface levels of CD4Down-regulates surface levels of major histocompatibility class I moleculesMediates cellular signaling and activation Enhances viral infectivity 第十页，共三十三页。I.Down-mo
13、dulation 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 release(by preventing Env-mediated sequestration of CD4 in secretory pathway)Evidenc
14、e: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 acidification(e.g.via chloroqine treatment)blocks Nef-induced CD4 degradationExpression
15、of nef alone in T-cell lines can lead to CD4 downregulation(as determined by FACS)CD4Nef-GFP.第十一页，共三十三页。I.Down-modulation of surface CD4Mechanism(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 as
16、says 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 both CD4-down-modulation and AP-2 co-localizationInteracts with NBP-1(identified through a ye
17、ast 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 t
18、o b b-Cop,a coatamer protein which targets proteins to lysosomesNBP-1第十二页，共三十三页。II.Down-modulation of MHC Class IAdvantages:Immune evasion;MHC Class I presents antigens to cytotoxic T-lymphocytes;alerts innate and adaptive immune system to virally infected cellsEvidence:Nef expression reduces suscep
19、tibility 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 inhibit NK-cell mediated cell lysis Thus,efficiency of CTL-mediated lysis(adaptive immunity)is reduced without inc
20、reasing increasing susceptibility to NK cell lysisHIVCTLMHC Class IHIV antigen51Cr第十三页，共三十三页。E:T ratio%Lysis1:21:51:101:20100%0%HIV wtHIV D DnefCTLMHC Class IHIV antigenE(Effector Cell)T(Target Cell)第十四页，共三十三页。III.Mediates Cellular Activation and SignalingNef expression upregulates a transcriptional
21、 program that activates the HIV LTR(microarray analysis)第十五页，共三十三页。III.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
22、 supernatants from cells transduced with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures369(days)p24(ng/ml)Adv-nef supntAdv-GFP supnt第十六页，共三十三页。III.Mediates Cellular Activation and SignalingNef expression upregulates a transcriptional program that activat
23、es 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 culturesNef interacts with Pak2(p21 activated kinase
24、2)and Nef/Pak2 complex may regulate many of Nefs effect on gene transcription第十七页，共三十三页。IV.Infectivity EnhancementMagnitude of infectivity enhancement is allele dependentNef mediated enhancement can be provided in transreporter gene(e.g.GFP or luciferase)expression under control of the LTR promoter
25、can be enhanced when nef expression vector is co-transfectedMechanisms:Increased RT activity;increased proviral DNA synthesisIncreased cytoplasmic delivery of viral particles第十八页，共三十三页。Vpu:CD4 down-modulation16 kDa,membrane spanningBinds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-p
26、roteasome pathway第十九页，共三十三页。Vpu mediated CD4 degradation via ubiquitin-proteasome pathwayEvidence:Vpu activity disrupted by inhibitors of proteasome-mediated proteolysisVpu activity affected by dominant negative mutants of ubiquitin pathwayRemoval of lysine residues (ubiquination targets)in CD4 tail
27、 prevents Vpu-mediated degradationVpu binds to b b-TrCP,which in turns binds to the proteasome targeting factor Skp1pOverexpression of b b-TrCP mutant that cannot bind Skp1p inhibits Vpu-mediated CD4 degradationContrast with Nef第二十页，共三十三页。Vpu:required for proper maturation and targeting of progeny v
28、irions,and for their proper release from the cell surfaceOligomerization of its transmembrane domain results in ion channel activitySimilar 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
29、 maturation and assembly by protecting newly formed Env protein from premature conformational changes in the secretory pathway第二十一页，共三十三页。Vif:Viral infectivity factor,required for robust replication only in certain cellsHIV-1(D Dvif)HIV-1(PermissiveNon-permissive+replication+replication+replicationn
30、o replicationHut78,H9,1 PBLsC8166,293T,HeLaTwo hypotheses:(a)Permissive cells express an activity(factor)that can(b)compensate for vif.(b)Non-permissive cells have an inhibitory activity on(c)viral replication,which is overcomed by vif.See Simon et.al.,Nature Med.4:1397第二十二页，共三十三页。Non-permissivePerm
31、issivewtD DvifwtD DvifInfectivity+-+Non-permissive:inhibitory cellular factor overcomed by vifPermissive:compensatory factor similar to vifHeterokaryonwtD DvifWhich phenotype will dominate?第二十三页，共三十三页。Permissive vs Non-Permissive T Cell Line(a)Permissive cells express an(b)activity(factor)that can(c
32、)compensate for vif.(b)Non-permissive cells have an(c)inhibitory activity on(d)viral replication,(e)which is overcomed by vif.Two hypotheses:PermissiveD Denv vs D Denv/D DvifPermissiveNon-Permissive第二十四页，共三十三页。Non-permissivePermissiveHeterokaryonwtD DvifwtD DvifD DvifInfectivity+-+Non-permissive:inh
33、ibitory cellular factor overcomed by vif+-wt第二十五页，共三十三页。Expression of CEM15 inCEM-SS(permissive cells)renders it non-permissive第二十六页，共三十三页。G2 ArrestLTR transcription,i.e.,virus production is more efficient during G2Augments Nuclear Import of Pre-Integration ComplexExtracellular vpr(from decaying vir
34、ions,or cytosolic leakage from infected apoptotic cells)re-capitulates intracellular vpr function Induces cell cycle arrestActivates HIV replication in latently infected cellsIncreased HIV replication in macrophagesApoptosis;“bystander”cell killing in lymphoid organs and brain Vpr:Two Independent Fu
35、nctions第二十七页，共三十三页。G1M(Mitosis)SDNASynthesisG0G2“Quality Control”phaseMitosis is not initiated untilDNA is free of damageG2 ArrestLTR transcription is more active,i.e.,virus production is more efficient during G2VprPO4-+-G2MWee1-GFPCyc B1-RFPG2 Arrestnuclear herniation due to disruption of nuclear l
36、amin structuremixing of segregated cell cycle regulators may lead to G2 arrest Vpr:G2 Cell Cycle Arrest第二十八页，共三十三页。Vpr:Augments Nuclear Import ofPre-Integration ComplexVpr lacks classical NLS sequences-but binds to Importin-Vpr=Importin-b b analog?第二十九页，共三十三页。Vpr:Augments Nuclear Import ofPre-Integr
37、ation ComplexImportin-Importin-第三十页，共三十三页。Transport of PICs containing Vpr-GFP fusion proteinRed=anti-tubulin antibodies第三十一页，共三十三页。Blue=anti-tubulinGreen=Vpr-GFPRed=Alexa-dUTPPIC/RTCNuclear Import of PICstalled by:(a)Anti-dynein Mab(b)Nicodazole treatment (nicodazole disrupts microtubles)第三十二页，共三十三页。内容(nirng)总结HIVCellular Pathogenesis II。HIVCellular Pathogenesis II。to Ran-GTP,and。Vpu:CD4 down-modulation。C8166,293T,。Non-permissive:inhibitory cellular。Denv vs Denv/Dvif。CEM-SS(permissive cells)。microtubles)第三十三页，共三十三页。