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1、VIROLOGY190,278-287(1992)LocationandCharacteroftheCellularEnzymeThatCleavestheHemagglutininofaVirulentAvianInfluenzaVirusJOHNA.WALKER,*TAKEMASASAKAGUCHI,-tYOSHIKOMATSUDA,+TETSUYAYOSHIDA,tANDYOSHIHIROKAWAOKA”,’*DepartmentofVirologyandMolecularBiology,St.JudeChildren’sResearchHospital,332NorthLauderda
2、le,P.O.Box318,Memphis,Tennessee38101;tDepartmentofBacteriology,HiroshimaUniversitySchoolofMedicine,l-2-3Kasumi,Minami-ku,Hiroshima734,Japan;and*DepartmentofBiologicalScienceandTechnology,FacultyofEngineering,UniversityofTokushima,2-1Minamijosanjima-Mishima,Tokushima770,JapanReceivedApril15,1992;revi
3、sedJune12,1992H.-D.Klenk,W.Garten,andR.Rott(1984,EMBOJ.3,2911-2915)havereportedthathemagglutinin(HA)cleavageofvirulentavianinfluenzavirusesoccursinlaterstepsofitsintracellulartransportandthatthecleavageenzymeiscalciumdependentandhasaneutralpHoptimum.ThepreciseintracellularlocationoftheHAcleavage,how
4、ever,hasneverbeenestablished.Furthermore,becauseKlenketa/.usedthewholecelllysatetoexaminethecleavageactivityandtheaminoacidsequencingofthecleavedproductwasnotdone,theidentityofthecleavageenzymeremainstobeestablished.WethereforeattemptedtosystematicallycharacterizetheHAcleavageofthevirulentavianvirus
5、A/tern/SouthAfrica161(H5N3).Usinganinhibitorofglycoproteintransport(BrefeldinA)andtemporalmarkersofglycoproteinprocessing,wefoundthattheendoproteaseresponsiblefortheHAcleavageactsaftertheacquisitionofendo-//-ace-tylglucosaminidaseHresistancebutbeforetheadditionofgalactosetothemolecule,andthusisloca
6、tedinthemedialand/ortransGolgi.ThisobservationwasdirectlyconfirmedbyinvitroexperimentsusingratliversubcellularmembranefractionscontainingGolgicomplex.Afractionrichingalactosyltransferase(atransGolgimarker)demonstratedthehighestHAcleavageactivity.TheendoproteaseinthisfractioncleavedonlytheHAoftheviru
7、lentavianinfluenzavirusbutnotthatofanavirulentvirus.Throughamino-terminalsequencingoftheHA2producedbydigestionwiththeendoproteaseintheratGolgifraction,weestablishedthatHAcleavagebytheproteaseoccursatt
