嗜神经病毒躲避宿主先天性免疫反应的机制及应用 ppt课件

嗜神经病毒躲避宿主先天性免疫反应的机制及对策赵凌教授华中农业大学10-15-2013Rabiesvirus180nmx75nmRobertHurt-USCRabiespathogenesisPatientsdieofcirculatoryinsufficiency,cardiacarrestandrespiratoryfailure.*RabiesinfectionandinnateimmunityWangetal.JournalofVirology,2005*Zhaoetal.JournalofVirology,2009 重组狂犬病病毒构建*ExpressionofMIP-1αattenuatedRABVpathogenicity,whileexpressionofRANTESorIP-10increasedRABVpathogencityZhaoetal.JournalofVirology,2009*ControlrHEPHEP-MIP1aHEP-RANTESHEP-IP10D3D6D9HEstainingofmousebrainsZhaoetal.JournalofVirology,2009ExpressionofMIP-1αenhancesVNAproductionandprotectionZhaoetal.JournalofVirology,2010Zhaoetal.JournalofVirology,2010外周过量表达MIP-1α会吸引更多的树突状细胞和B细胞CoronavirusescausediseasesinhumansanddomesticanimalsAdaptedfromHolmesandLai,FieldsVirology AntigenicGroup Virus Host Disease I 229ENL63 humanhuman respiratoryinfectionrespiratoryinfection,croup TGEV pig respiratoryandentericinfection CCV dog entericinfection FECV cat entericinfection FIPV cat respiratory,enteric,hepatitisandneurologicalinfection II OC43 human Respiratoryandpossiblyentericinfection SARS-CoVHKU1 humanhuman RespiratoryRespiratory MHV mouse Respiratory,enteric,neurologicinfection HEV pig Respiratory,enteric,neurologicinfection BCV cow Entericinfection TCV turkey Respiratoryandentericinfection III IBV chicken Respiratoryandentericinfection,hepatitis*Coronavirusesinfectanumberofdifferentspeciesincludinghumans,domesticmammalsandbirdsandinduceavarietyofdiseasesfromrespiratorytoentericinfections-Furthermore,theseverityofdiseaseishighlyvarianle-Toillustratethis,letmementionthatcoronavirusescauseasignificantpercentageofthecommoncold,afterrhinovirus-fairlymilddisease,whereasSARSinducessevererespiratorydisease.Coronavirusesaredividedintothreeantigenicgroups-MHVcausesavarietyofdifferentdiseaseesaswell,butwhatIhavefocusedonistheCNSdisease.WhyMHV? MHVproducesabroadspectrumofdiseaseinthemouse-pneumonia(MHV-1)-hepatitis(MHV-A59)-encephalitis(MHV-A59/JHM)-demylination(MHV-A59) Itprovidesexcellentsmallanimalmodelsforhepatitis,forSARS,andformultiplesclerosisPartI:MHVns2interferestypeIinterferonresponsesMutationofns2confersattenuationofhepatitisbutnotCNSdiseaseIC500PFUIH500PFURoth-Cross,J.K.etal,JVI,2009,83(8):3743-3753.brainliverNs2isanorganspecificvirulencefactor2Hphosphodiesterase;2predictedcatalyticHis-x-Thr/Sermotifs(Mazumdereta.,2002;Snijderetal.,2003)(Mazumbderetal.,2002Snijderetal.,2003;Roth-Cross,2009)*Mutationofns2confersattenuationofreplicationinmacrophagesandmicrogliabutnotinothercelltypesZhao,L.etal,JVI,2011.Oct;85(19):10058-10068.MOI1MOI0.01ns2mutantsrecovertheabilitytoreplicateefficientlyinmacrophagesandmicrogliafromIFNARknockoutmiceMOI1Zhao,L.etal,JVI,2011.Oct;85(19):10058-10068.Type1interferoninductionandsignalingpathwaysIFN-**WhenavirusentersthecellitsRNAisrecognizedasforeignbypatternrecognitionreceptorseitherToll-likereceptorslocatedintheendosomalmemebranesorcytoplasmichelicasesRIG-IandMDA5.ThesemoleculestheninitiateasignalingcascadethatleadstophosphorylationandactivationofIRF3andNfKBwhichtranslocatetothenucleusalongwithATF-2andtogetherwithCBPbindtothepromoterofIFN-Btostimulateproductionofinterferon.Italsobeenfoundthatvirusescandirectlystimulateanumberofothergenescalledinterferonstimulatedgenes,calledthisbecausetheyarestimulatedbyinterferon.Severalgroupshaveshownthattheinterferon-stimulatedresponseelementcanalsobeinduceddirectlybyviralinfectionthroughthissamepathway.Asubsetofinterferonstimulatedgenesareproducedintheinfectedcellsandarethoughttohavedirectantiviraleffects.Theinterferonproducedservestoamplifytheimmuneresponseandprotectneghboringcellsbybindingtotheinterferonreceptor,leadingtophosphorylationandactivationofSTATproteins,whichcombinewithinterferonresponsefactors(IRFs)toactivateISGs.ThemostwellstudiedtranscriptonalactivationcomplexiscomposedofaheterodimerofSTAT1,STAT2andIRF9,whichiscalledtheISGF3complex.ItbindstheISREandactivatestranscriptionofanumberofISGsmanyofwhichhavedirectantiviraleffects.IwillfirstsummarizeourdataonIFNproductioninresponsetoMHVandtellyouaboutfindingsthatMHVisabletoblockIFNsignalling.ns2mutantsarenotdefectiveininductionofIFN-α/mRNABothwtRA59andns2mutantsinduceminimalamountsofIFN-α,βmRNAinL2cellsandastrocyteswtRA59andns2mutantsinducesimilarlevelsofIFN-α,βmRNAinBMMfrombothB6andIFNAR-/-miceZhao,L.etal,JVI,2011.Oct;85(19):10058-10068.BMMBMMMicroglians2mutantsaremoresensitivetotheantiviraleffectsofIFN-α/thanwtA59inmacrophagesandmicrogliaViralreplicationandIFNsensitivityinthehepatocytesNoIFNIFNInvivomacrophagedepletionLiposomes,encapsulatingtheClodronatemolecules(squares),areingestedbymacrophagesviaendocytosis.Afterfusionwithlysosomes(L)containingphospholipases(arrowheads),thelatterdisruptthebilayersoftheliposomes.Themoreconcentricbilayersaredisrupted,thegreateristheClodronatereleasewithinthecell.ThecellsarekilledbyClodronatethroughapoptosis.ns2mutantsreplicateandinducehepatitisinmacrophagedepletedmice500foldvs10foldModel:Kupffercellsprovideabarriertotheliverparenchymatoviruses=rA59LSECKCHepatocytesinusoidparenchyma=ns2-H126A*PartI:conclusions ns2isanorganspecificvirulencefactorandantagonizesIFNsignaling ns2isrequiredforreplicationinmacrophages;depletionofmacrophagesinvivopromotesns2mutantvirusreplication wesuggestthatMHVhastoreplicateinKupffercellsintheliversinusoidsinordertoreachtheliverparenchymaandinducehepatitis Type1interferoninductionandsignalingpathwaysIFN-**WhenavirusentersthecellitsRNAisrecognizedasforeignbypatternrecognitionreceptorseitherToll-likereceptorslocatedintheendosomalmemebranesorcytoplasmichelicasesRIG-IandMDA5.ThesemoleculestheninitiateasignalingcascadethatleadstophosphorylationandactivationofIRF3andNfKBwhichtranslocatetothenucleusalongwithATF-2andtogetherwithCBPbindtothepromoterofIFN-Btostimulateproductionofinterferon.Italsobeenfoundthatvirusescandirectlystimulateanumberofothergenescalledinterferonstimulatedgenes,calledthisbecausetheyarestimulatedbyinterferon.Severalgroupshaveshownthattheinterferon-stimulatedresponseelementcanalsobeinduceddirectlybyviralinfectionthroughthissamepathway.Asubsetofinterferonstimulatedgenesareproducedintheinfectedcellsandarethoughttohavedirectantiviraleffects.Theinterferonproducedservestoamplifytheimmuneresponseandprotectneghboringcellsbybindingtotheinterferonreceptor,leadingtophosphorylationandactivationofSTATproteins,whichcombinewithinterferonresponsefactors(IRFs)toactivateISGs.ThemostwellstudiedtranscriptonalactivationcomplexiscomposedofaheterodimerofSTAT1,STAT2andIRF9,whichiscalledtheISGF3complex.ItbindstheISREandactivatestranscriptionofanumberofISGsmanyofwhichhavedirectantiviraleffects.IwillfirstsummarizeourdataonIFNproductioninresponsetoMHVandtellyouaboutfindingsthatMHVisabletoblockIFNsignalling.NDV-bioassayinVerocellspCAGGS-IFNNS2a-IFNSV5V-IFNpCAGGS-noIFNTansfectionofpCAGGSorpCAGGS-ns2orpCAGGS-SV5VinVerocellsTreatmentwithorw/o1000U/mlfor16hInfectionofNDV-GFP24h12hGFPISGscreeninginKOBMMISG15IFIT1IFIT2PKRRNaseLPartII:MHVns2antagonizesOAS-RNaseLpathwayInterferonsignalingmodelViraldsRNAMDA5*,RIG-I*IFNAntiviralISGsOAS*2-5ARNaseLCellularandviralRNASmallRNAs2’-PDEns2?MainpathwayOAS=2’,5’-oliogoadenylatesynthetase2’-PDE=2’phosphodiesterase2-5A=2’,5’oligoadenylate**WildtypeA59replicationinBMMwasnotaffectedbytheOAS-RNaseLsystemDefectivereplicationofns2mutantisrestoredinRNaseL-/-BMMButnotinPKR-/-BMMZhao,Letal.CellHost&Microbe,2012,(11)607–616.ns2expressionin293TcellspCAGGS-ns2pCAGGSpCAGGS-ns2-H126RZhao,Letal.CellHost&Microbe,2012,(11)607–616.ns2preventsrRNAcleavageand2-5AproductioninBMMZhao,Letal.CellHost&Microbe,2012,(11)607–616.Overexpressionofns2in293TcellspreventsrRNAcleavageand2-5AproductioninducedbypolyI:CZhao,Letal.CellHost&Microbe,2012,(11)607–616.Ns2cleaves2-5AintoATPandAMPZhao,Letal.CellHost&Microbe,2012,(11)607–616.Ns2enhancesvirusreplicationintheliverZhao,Letal.CellHost&Microbe,2012,(11)607–616.Ns2facilitatestheinductionofviralhepatitisZhao,Letal.CellHost&Microbe,2012,(11)607–616.PartII:conclusionsMousehepatitisvirusns2proteininhibitstheIFN-inducedOAS-RNaseLpathwayns2reducestheintracellularlevelof2’,5’-oligoadenylate(2-5A)ns2hasa2’,5’-phosphodiesterasetodirectlycleave2-5Ans2mutantvirusreplicationandhepatitis-inductionarerescuedinRNaseLdeficientmice 狂犬病病毒如何逃逸中枢神经系统先天性免疫反应？狂犬病病毒如何限制血脑屏障的打开？如何打开血脑屏障清除狂犬病病毒？ 研究方向*谢谢！*Coronavirusesinfectanumberofdifferentspeciesincludinghumans,domesticmammalsandbirdsandinduceavarietyofdiseasesfromrespiratorytoentericinfections-Furthermore,theseverityofdiseaseishighlyvarianle-Toillustratethis,letmementionthatcoronavirusescauseasignificantpercentageofthecommoncold,afterrhinovirus-fairlymilddisease,whereasSARSinducessevererespiratorydisease.Coronavirusesaredividedintothreeantigenicgroups-MHVcausesavarietyofdifferentdiseaseesaswell,butwhatIhavefocusedonistheCNSdisease.***WhenavirusentersthecellitsRNAisrecognizedasforeignbypatternrecognitionreceptorseitherToll-likereceptorslocatedintheendosomalmemebranesorcytoplasmichelicasesRIG-IandMDA5.ThesemoleculestheninitiateasignalingcascadethatleadstophosphorylationandactivationofIRF3andNfKBwhichtranslocatetothenucleusalongwithATF-2andtogetherwithCBPbindtothepromoterofIFN-Btostimulateproductionofinterferon.Italsobeenfoundthatvirusescandirectlystimulateanumberofothergenescalledinterferonstimulatedgenes,calledthisbecausetheyarestimulatedbyinterferon.Severalgroupshaveshownthattheinterferon-stimulatedresponseelementcanalsobeinduceddirectlybyviralinfectionthroughthissamepathway.Asubsetofinterferonstimulatedgenesareproducedintheinfectedcellsandarethoughttohavedirectantiviraleffects.Theinterferonproducedservestoamplifytheimmuneresponseandprotectneghboringcellsbybindingtotheinterferonreceptor,leadingtophosphorylationandactivationofSTATproteins,whichcombinewithinterferonresponsefactors(IRFs)toactivateISGs.ThemostwellstudiedtranscriptonalactivationcomplexiscomposedofaheterodimerofSTAT1,STAT2andIRF9,whichiscalledtheISGF3complex.ItbindstheISREandactivatestranscriptionofanumberofISGsmanyofwhichhavedirectantiviraleffects.IwillfirstsummarizeourdataonIFNproductioninresponsetoMHVandtellyouaboutfindingsthatMHVisabletoblockIFNsignalling.***WhenavirusentersthecellitsRNAisrecognizedasforeignbypatternrecognitionreceptorseitherToll-likereceptorslocatedintheendosomalmemebranesorcytoplasmichelicasesRIG-IandMDA5.ThesemoleculestheninitiateasignalingcascadethatleadstophosphorylationandactivationofIRF3andNfKBwhichtranslocatetothenucleusalongwithATF-2andtogetherwithCBPbindtothepromoterofIFN-Btostimulateproductionofinterferon.Italsobeenfoundthatvirusescandirectlystimulateanumberofothergenescalledinterferonstimulatedgenes,calledthisbecausetheyarestimulatedbyinterferon.Severalgroupshaveshownthattheinterferon-stimulatedresponseelementcanalsobeinduceddirectlybyviralinfectionthroughthissamepathway.Asubsetofinterferonstimulatedgenesareproducedintheinfectedcellsandarethoughttohavedirectantiviraleffects.Theinterferonproducedservestoamplifytheimmuneresponseandprotectneghboringcellsbybindingtotheinterferonreceptor,leadingtophosphorylationandactivationofSTATproteins,whichcombinewithinterferonresponsefactors(IRFs)toactivateISGs.ThemostwellstudiedtranscriptonalactivationcomplexiscomposedofaheterodimerofSTAT1,STAT2andIRF9,whichiscalledtheISGF3complex.ItbindstheISREandactivatestranscriptionofanumberofISGsmanyofwhichhavedirectantiviraleffects.IwillfirstsummarizeourdataonIFNproductioninresponsetoMHVandtellyouaboutfindingsthatMHVisabletoblockIFNsignalling.**