hiv發(fā)病機(jī)理

1、HIVCellular Pathogenesis II,Benhur Lee, M.D.,,,,,,,,HIV Accessory Genes:TatRevVifVprVpuNef,Essential in vitro and in vivo,Essential in certain cell types(Permissive vs Non-permissive cells),Non-essential in vit

2、ro, but leads to attenuated phenotype in vivo,,Tat: Transactivator of HIV’s LTR Promoter,Experimental Observations:Binding of Tat to TAR in vitro does NOT require loop sequences known to be necessary in vivo for functi

3、onPre-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) present on Chromosome

4、 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!?Myste

5、ry human-specific co-factor for Tat activity must exist,2º structure of HIV TAR sequence,“l(fā)oop”,“bulge”,Predicted and confirmed properties of Tat co-factor: Cyclin TBinds directly to Tat in a complex with Cdk9Incr

6、eases the affinity of Tat for TARIncreases the specificity of Tat for “l(fā)oop” and “bulge” residuesTat-CycT-Cdk9 complex hyperphosphorylates CTD of RNAP II and increases HIV transcriptional processivityCycT maps to

7、chromosome 12, and potentiates Tat trans-activation in murine cells 50- to 100- foldMurine homolog of human CycT does NOT bind to Tat,Tat: Transactivator of HIV’s LTR Promoter,Experimental Observations Expl

8、ained: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 trans

9、cription in vitroTat 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 (identif

10、ied 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 unsplic

11、ed or single spliced viral transcripts,,Arg 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,,,,Nef,Major determin

12、ant of pathogenicity 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 wit

13、h 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 wil

14、d-type HIV in the SCID-hu mice model,,Pleiotropic Functions of Nef,N-myristoylation required for Nef activity--implies that membranelocalization of nef is critical for its activity,MGxxx(S/T)(K/R)(K/R),MGxxx(S)(K)(K/R

15、),,,,,,100%,100%,99%,~50%,ConsensusN-myristoylationSignal:,HIV sequenceConservation inNef protein:,Pleiotropic Functions of Nef,Down-regulates cell surface levels of CD4Down-regulates surface levels of major histoc

16、ompatibility class I moleculesMediates cellular signaling and activation Enhances viral infectivity,I. Down-modulation of surface CD4,Down-modulation of surface CD4 via internalization followed by degradation via end

17、osomal/lysosomal pathway Advantages:Prevents disadvantageous super-infection of host cellEnhance viral progeny release (by preventing Env-mediated sequestration of CD4 in secretory pathway)Evidence:Nef expression

18、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.

19、via chloroqine treatment) blocks Nef-induced CD4 degradationExpression of nef alone in T-cell lines can lead to CD4 downregulation (as determined by FACS),,,CD4,Nef-GFP,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,

20、.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,

21、.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,

22、.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,,,I. Down-modulation of surface CD4,Mechanism(s)?Direct interaction with CD4 has not been biochemically demonstrable, but NMR analysis suggest a direct int

23、eraction 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

24、motif (E/DxxxL?) in Nef is important for 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 cla

25、thrin-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 ?? M

26、ay 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

27、 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 CT

28、Ls; 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,

29、,,,,,,,,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

30、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 indu

31、ce 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,(d

32、ays),,,,,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 sec

33、retion 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 w

34、ith Pak2 (p21 activated kinase 2) and Nef/Pak2 complex may regulate many of Nef’s effect on gene transcription,IV. Infectivity Enhancement,Magnitude of infectivity enhancement is allele dependentNef mediated enhancement

35、 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 prov

36、iral DNA synthesisIncreased cytoplasmic delivery of viral particles,,Vpu: CD4 down-modulation,16 kDa, membrane spanningBinds CD4 tail in the ER Targets CD4 for proteolysis via ubiquitin-proteasome pathway,Vpu medi

37、ated CD4 degradation via ubiquitin-proteasome pathway,Evidence:Vpu activity disrupted by inhibitors of proteasome- mediated proteolysisVpu activity affected by dominant negative mutants of ubiquitin pathw

38、ayRemoval 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-TrC

39、P 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 t

40、he cell surface,Oligomerization 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

41、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

42、factor, required for robust replication only in certain cells,HIV-1 (Dvif),HIV-1 (???,PermissiveNon-permissive,+++ replication,+++ replication,+++ replication,no replication,Hut78, H9, 1º PBLs,C8166, 293T

43、, 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 Sim

44、on et. al., Nature Med. 4: 1397,,,,,Non-permissive,Permissive,Infectivity,,Non-permissive: inhibitory cellular factor overcomed by vifPermissive: compensatory factor similar to vif,,,Permissive vs Non-Permissive T C

45、ell 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.,

46、Two hypotheses:,Permissive,Denv vs Denv/Dvif,,,,Permissive,Non-Permissive,,,,,,,,,,Non-permissive,Permissive,Heterokaryon,wt,Dvif,,,wt,Dvif,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Dvif,,Infectivity,,++,

47、++,Non-permissive: inhibitory cellular factor overcomed by vif,wt,,,G2 ArrestLTR transcription, i.e.,virus production is more efficient during G2Augments Nuclear Import of Pre-Integration ComplexExtracellular vpr (

48、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 replicat

49、ion in macrophagesApoptosis; “bystander” cell killing in lymphoid organs and brain,Vpr: Two Independent Functions,G0,G2 ArrestLTR transcription is more active, i.e., virus production is more efficient during G2,V

50、pr: G2 Cell Cycle Arrest,,Vpr:Augments Nuclear Import ofPre-Integration Complex,Vpr lacks classical NLS sequences--but binds to Importin-a????????,Vpr:Augments Nuclear Import ofPre-Integration Complex,Transport of P

51、ICs containing Vpr-GFP fusion protein,Red=anti-tubulin antibodies,,,,Blue=anti-tubulinGreen=Vpr-GFPRed=Alexa-dUTP,PIC/RTC,,,Nuclear Import of PICstalled by:(a)Anti-dynein Mab(b)Nicodazole treatment (nicodazole d