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1、VIROLOGY183,91-98(1991)StructuralAnalysisoftheConformationalDomainsInvolvedinNeutralizationofBovineCoronavirusUsingDeletionMutantsoftheSpikeGlycoproteinSlSubunitExpressedbyRecombinantBaculovirusesDONGWANYOO,*MICHAELD.PARKER,*JAEYOUNGSONG*,*GRAHAMJ.COX,*DIRKDE
2、REGT,*s3ANDLORNEA.BABIUK*+*VeterinaryinfectiousDiseaseOrganization,124VeterinaryRoad,andtDepartmentofVeterinaryMicrobiology,UniversityofSaskatchewan,Saskatoon,SaskatchewanS7NOWOCanadaReceivedDecember4,1990;acceptedMarch8,1991Twoconformation-dependentneutraliz
3、ingepitopes,AandB,havebeenmappedtotheSlsubunitoftheSspikeglycoproteinofbovinecoronavirus(BCV).Inordertocharacterizethestructureoftheseantigenicsites,wecon-structedaseriesofcDNAclonesencodingdeletedortruncatedSlderivativesandexpressedthemodifiedgenesininsectce
4、llsusingrecombinantbaculoviruses.MonoclonalantibodiesdirectedagainstepitopesAandBrecognizedonlythemutantSlpolypeptidescontainingaminoacids324-720,asdemonstratedbyimmunoprecipitationandWesternblotanalysisintheabsenceofp-mercaptoethanol.Inaddition,twodomainswit
5、hinthisregionwereidentifiedandonlymutantscontainingbothdomainswereimmunoreactive,indicatingthatbothwerecriticalintheformationoftheantigenicdeterminants.Onedomainwaslocalizedbetweenresidues324and403andtheotheratresidues517-720.Deletionofeitherdomaininhibitedex
6、tracellularsecretionofthemutantproteinswhereasmutantscontain-ingbothornoneofthedomainsweresecretedefficiently.ThisobservationsuggestsavitalfunctionofthenativeconformationoftheSlproteininbothantigenicstructureandintracellulartransport.AntigenicdeterminantsAand
7、Bwerenotdistinguished,butthesedeterminantsappearedtorequirebothdomainsforepitopeformation.OurresultssuggestthattheantigenicdeterminantsformedbytwodomainsarelikelyassociatedwiththeprobablepolymorphicregionoftheBCVSlsubunit.01991AcademicPress,Inc.INTRODUCTIONpo
8、rtionofthepeplomerwhereastheamino-terminalSlsubunitconstitutesthebulbouspartofthepe-Bovinecoronavirus(BCV)isanenteropathogenicplomer.Recently,thenucleotidesequenceoftheBCVcoronavirusthatc
