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1、VIROLOGY214,453–463(1995)MutationalAnalysisoftheMurineCoronavirusSpikeProtein:EffectonCell-to-CellFusion1EVELYNEC.W.BOS,LEOHEIJNEN,WILLEMLUYTJES,andWILLYJ.M.SPAANDepartmentofVirology,InstituteofMedicalMicrobiology,FacultyofMedicine,LeidenUniversity,2300AHLeiden,TheNetherland
2、sReceivedJuly5,1995;acceptedOctober4,1995Thespike(S)proteinofmurinecoronavirusstrainA59(MHV-A59)isatypeImembraneproteinthatinducesmembranefusion.InthisstudywehaveanalyzedtheroleoftwodomainsintheSproteinonfusion.The180-kDamatureSproteinispartiallycleavedintotwo90-kDasubunitsd
3、uringtransporttotheplasmamembrane.WehaveidentifiedseveralaminoacidsthatareimportantforcleavageofS,andweshowthatcleavageisnotstrictlyrequiredforfusion.However,thelevelofcleavageseemstoinfluencethefusionkinetics.AfterintroductionofanarginineatpositionP2tomimicktheMHV-JHMcleava
4、gesite,fullcleavageofthespikeproteinwasobtained.Further,weanalyzedtheeffectofmutationsinthetransmem-brane(TM)domainoftheSprotein.Maturationandcellsurfaceexpressionofthemutantproteinswerenotaffected,andallproteinsbecameacylated.Themutantinwhichthepredictedtransmembranedomainw
5、asshorteneddidnotinducesyncytia.FromagroupofmutantsinwhichseveralconservedcysteinesintheTMdomainhadbeenreplacedbyserines,onewasunabletoinducesyncytia,anothershoweddelayedsyncytiaformation,andthethirdmutantinducedsyncytiaasdidthewild-typeprotein.Thepotentialroleofthetransmemb
6、ranedomaininfusionisdiscussed.q1995AcademicPress,Inc.INTRODUCTIONandmodified,givingrisetoanalmostendo-H-resistant180-to200-kDaprotein.AdistinctivefeatureamongviralMembranefusionisakeyeventinthereplicationcyclefusionproteinsisthat,irrespectiveofthepHoptimum,ofenvelopedviruses
7、.Duringpenetrationofthehostcell,somemustundergocleavageactivation(e.g.,hemagglu-theviralmembranefuseswitheithertheplasmamem-tininofinfluenzavirus,gp160ofHIV),whereasothersarebraneorendosomalmembraneresultinginthereleasefusogenicinanuncleavedform(e.g.,Gproteinofrhab-ofthevira
8、lgenomeintothecytosoloftheinfectedcell.doviruses)(reviewedbyWhite,1990).Whe
