2022年醫(yī)學專題-HIV發(fā)病機理

1、HIVCellular Pathogenesis IIBenhur Lee, M.D.第一頁，共三十三頁。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 第二頁，共三十三頁。Tat: Transactivator of HIVs LTR Prom

2、oterExperimental 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 unles

3、s 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

4、activity must exist2 structure of HIV TAR sequence“l(fā)oop”“bulge”第三頁，共三十三頁。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 “l(fā)oop” and “bulge” residuesTat-CycT-Cdk9 complex

5、 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 Tat第四頁，共三十三頁。Tat: Transactivator of HIVs LTR PromoterExperimental Observati

6、ons 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 unless complemented b

7、y 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 ex

8、ist: Cyclin T第五頁，共三十三頁。RevEssential for nuclear export of unspliced or single spliced viral transcripts第六頁，共三十三頁。Importin-bRanGDPArg Rich Domain (ARD)-binds to Importin-b for nuclear import After nuclear import,Ran-GDP is convertedto Ran-GTP, and importin- b dissociates from Rev “Free” ARD now can b

9、ind 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-bRanGTP第七頁，共三十三頁。NefMajor determinant of pathogenicity in vivonef-deleted SIV is severely

10、 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 H

11、IV do not deplete thymocytes as much, or replicate to as high titers, as wild-type HIV in the SCID-hu mice model第八頁，共三十三頁。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)10

12、0%100%99%50%ConsensusN-myristoylationSignal:HIV sequenceConservation inNef protein:第九頁，共三十三頁。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 infec

13、tivity 第十頁，共三十三頁。I. 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 release (by preventing Env-mediated sequestration of CD4

14、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 acidification (e.g. via chloroqine treatment) blocks Nef-in

15、duced CD4 degradationExpression of nef alone in T-cell lines can lead to CD4 downregulation (as determined by FACS) CD4Nef-GFP.第十一頁，共三十三頁。I. Down-modulation of surface CD4Mechanism(s)?Direct interaction with CD4 has not been biochemically demonstrable, but NMR analysis suggest a direct interaction w

16、ith 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 both CD4-down-modulation and AP-2 co-localizationInterac

17、ts 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 als

18、o important for NBP-1 interactions ? May bind to b-Cop, a coatamer protein which targets proteins to lysosomesNBP-1第十二頁，共三十三頁。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 inf

19、ected 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 inhibit NK-cell mediated cell lysis Thus, efficiency of CTL-me

20、diated lysis (adaptive immunity) is reduced without increasing increasing susceptibility to NK cell lysisHIVCTLMHC Class IHIV antigen51Cr第十三頁，共三十三頁。E:T ratio% Lysis1:21:51:101:20100%0%HIV wtHIV DnefCTLMHC Class IHIV antigenE (Effector Cell)T (Target Cell)第十四頁，共三十三頁。III. Mediates Cellular Activation

21、and SignalingNef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis)第十五頁，共三十三頁。III. Mediates Cellular Activation and SignalingNef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis)Nef can induce secretion of

22、 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 supntAdv-GFP supnt第十六頁，共三十三頁。III. Mediates Cellular Activation and SignalingNef

23、 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 restin

24、g lymphoid culturesNef interacts with Pak2 (p21 activated kinase 2) and Nef/Pak2 complex may regulate many of Nefs effect on gene transcription第十七頁，共三十三頁。IV. Infectivity EnhancementMagnitude of infectivity enhancement is allele dependentNef mediated enhancement can be provided in transreporter gene

25、(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 cytoplasmic delivery of viral particles第十八頁，共三十三頁。Vpu: CD4 down-modulation16 kDa, membrane spa

26、nningBinds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-proteasome pathway第十九頁，共三十三頁。Vpu mediated CD4 degradation via ubiquitin-proteasome pathwayEvidence:Vpu activity disrupted by inhibitors of proteasome- mediated proteolysisVpu activity affected by dominant negative mutants of ubi

27、quitin 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 Skp1pOverexpression of b-TrCP mutant that cannot bind Skp1p inhibits Vpu-mediated CD4 degradationContrast with Nef

28、第二十頁，共三十三頁。Vpu: 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 activitySimilar to influenza virus M2 protein, an ion channel protein that modulates the pH in the Gol

29、gi 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 pathway第二十一頁，共三十三頁。Vif: Viral infectivity factor, required for robust replication only in certain cellsHIV

30、-1 (Dvif)HIV-1 (PermissiveNon-permissive+ replication+ replication+ replicationno replicationHut78, H9, 1 PBLsC8166, 293T, HeLaTwo hypotheses:Permissive cells express an activity (factor) that can compensate for vif.Non-permissive cells have an inhibitory activity on viral replication, which is over

31、comed by vif.See Simon et. al., Nature Med. 4: 1397第二十二頁，共三十三頁。Non-permissivePermissivewtDvifwtDvifInfectivity+-+Non-permissive: inhibitory cellular factor overcomed by vifPermissive: compensatory factor similar to vifHeterokaryonwtDvifWhich phenotype will dominate?第二十三頁，共三十三頁。Permissive vs Non-Perm

32、issive 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:PermissiveDenv vs Denv/DvifPermissiveNon-Permissive第二十四頁，共三十三頁。Non-permissivePermissiveHeteroka

33、ryonwtDvifwtDvifDvifInfectivity+-+Non-permissive: inhibitory cellular factor overcomed by vif+-wt第二十五頁，共三十三頁。Expression of CEM15 inCEM-SS (permissive cells)renders it non-permissive第二十六頁，共三十三頁。G2 ArrestLTR transcription, i.e.,virus production is more efficient during G2Augments Nuclear Import of Pre

34、-Integration ComplexExtracellular vpr (from decaying virions, 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 Independ