03-病毒学virus巴斯德研究所

VirusEntryandUncoatingPaulZhouInstitutPasteurofShanghaiGeneralconsideration•Envelopedviruses–nucleus•Envelopedviruses–cytosol•Non-envelopedviruses–nucleus•Non-envelopedviruses-cytosolGeneralmethodstoidentifyreceptorsandco-receptorsforviralentry•Permissibleandnon-permissiblecells•Non-permissiblecellsthatblockearlystageofviralreplication•IndicatorcellssuchasMagi•Cell-cellfusionassay•Pseudotypes•Geneticapproaches–animalcross;somaticcellhybrids;transfectants(genomicDNAorcDNAlibrary)•Receptor-interferenceapproaches–differentviruses;differentserotypes;receptordown-regulation;solubleviralattachmentproteins;specificmonoclonalantibodies•Carbohydrate-destroyingenzyme–neuraminidase;heparinaseandheparitinase;antibodiesagainstgalactosylceramideAdditionalcomplicationsforviralentry•AttachmentmoleculesthatcaptureandtransfervirustotargetcellssuchasDC-SIGN•Additionalproteolyticprocess•Antibody-dependentenhancementofviralentry•Receptor-mediatedendocytosisandreceptor-associatedsignaltransductionAntibody-dependentenhancementofviralentry•Denguevirus,WestNilevirus,influenzaAvirus,HIV-1,HIV-2andEBV•ThroughFcreceptor•Altercelltropism•CanoccurinmucosalsurfacethroughpolymericIgA•InsuboptimaldosesofantibodyconcentrationReceptor-mediatedendocytosisinviralentry•pH-dependentandindependent–carboxylicionophoresandbafilomycinA1•Clathrin-dependentandindependent–dominantnegativemutantsofcellularguanosinetriphosphatase(GTPase)dynaminandcaveoline•Clathrin-dependent:SemlikiForestvirus,Sindbisvirus,Influenzavirus,rhinovirus14andadnoviruses•Clathrin-independent:poliovirusandMMLV•Caveolae-dependent:SV40pH-dependentpH-independent__________________________InfluenzaAvirusRetrovirusesReovirusesHerpesSimplexAdnovirusesvirusesSemlikiForestvirusVacciniavirusVesicularStomatitisRotavirusesvirusParamyxovirusesFilovirusesTransportofviralgenomesintothecellnucleus•Thedoublemembranousnuclearenvelope•Impotins–NLSinviralproteinsandfibrilsextendingfromtheNPC•TranslocationthroughnuclearporealsoinvolvesacomplexoftheRanGTP-bindingproteinandp10orNTF2•HIV-1-PIC:Vpr,MA,integraseandcPPTinfluenzavirus–RNPs:viralRNAsegmentsandPA,PB1andPB2adenovirus–capsidproteinbindstoNPCanddsDNAtranslocatesintonucleusHSV–nucleocapsidproteinbindstomicrotubulesandtoNPCanddsDNAtranslocatesintonucleusPoliovirusentry•Receptorforpoliovirus:Pvrwithunknownfunction;identificationandtransgenicmice•Attachment–domain1ofPvr,CD155andcanyonofcapsidproteins•InsertionoftheexposedlipophilicN-terminusofVP1intothecellmembrane•Formationofaporecomplex•ViralRNAentersthecytoplasmParamxyovirusesentry•Twointegralmembraneproteins–one(HN,H,andG)forattachmentandtheother(F)formediatedpHindependentfusion•HNbindstosialicacid-containingcellsurfacemoleculesandalsomediatesneuraminidaseactivity•Structureoffusionprotein:activationthroughcleavagebytwocellularenzymesfurinandcarboxypeptidaseOrthomyxovirusentry•HA,NAandM2proteins•Sialicacidlinkedtogalactosebyα2,3orα2,6linkages(avianintestineα2,3;humantracheaα2,6;andpigtracheaα2,3andα2,6)•Receptormediatedendocytosisthroughclathrin-coatedmembrane-boundvesicles•FunctionofM2•M1dissociation•RNPsenterintonucleusAdenovirusentry•Knobdomainoffiberprotein•ReceptorsCARorCD46dependentonserotypesofadenoviruses(A,C,D,E,FversusB)•Integrinsαvβ3andαvβ5havebeenimplicatedascoreceptorsthatinteractwithRGDmotifofpentonbaseproteins•Receptor-mediatedendocytosis•Disruptearlyendosome•Microtubuletransport•Asequentialdisassemblyofvirionoccurs(uncoating)HSVentry•AttachmentofheparansulfateoncellsurfacebyviralenvelopegBandgC•InteractionofviralenvelopegDwithoneofthreereceptorfamiliesHveA,HveB,andHveC•Virus-cellfusioninvolvesgDandgH-gLheterodimerwhichreleasesthecapsid-tegumentstructureintothecytoplasm•Thecapsid-tegumentstructureistransportedtonuclearporethroughmicrotubulenetwork•AtthenuclearporethenucleocapsidreleasesitsDNAintothenucleusandleavesanemptycapsidatthecytoplasmicsideoftheNPCEntryofPoxviruses•FourviralproteinsL1R,A27L,D8RandH3Lhavebeenimplicatedtomediatecellentryandpenetrationofpoxvirus•Heparinsulfatehasbeenimplicatedasreceptor•Virus-cellfusiontakesplaceincytoplasmmembrane•AsequentialuncoatingprocesstakesplacebeforeandafterearlygeneexpressionincytosolViralentryinhibitors•Neutralizingantibodiesandvaccinedevelopement•Membrane-boundantibodies•Solublereceptors•Peptideinhibitors•Ligand-basedinhibitors•SmallmoleculeinhibitorsInhibittransductionofHIV-1envelope-pseudotypedlentivectorsbyGPI-anchoredscFvPseudotypeCEMss-EGFPCEMss-scFv/DAFpValuepDOL15,352±12881926±2130.0001Q16827,774±2480117±740.0001VSV-G4463,480±2374004364,720±1318450.5mock226252Summaryofthelectureonviralentryanduncoating•Ourknowledgeinviralentryanduncoatingisincompleteandfragmented•Viralentryanduncoatinginvolveintimateinteractionsbetweenviralandhostfactors•Understandingtheseinteractionswillshedlightonbiologicalprocessesofcells•Understandingtheseinteractionswillhelptodevelopvaccineandtherapeuticinterventionstrategies