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1、VIROLOGY245,303±312(1998)ARTICLENO.VY989164CharacterizationoftheTwoOverlappingPapain-likeProteinaseDomainsEncodedinGene1oftheCoronavirusInfectiousBronchitisVirusandDeterminationoftheC-TerminalCleavageSiteofan87-kDaProtein1K.P.LimandD.X.LiuInstituteofMolecularAgrobiology,TheNationalUniversityo
2、fSingapore,1ResearchLink,Singapore117604ReceivedDecember10,1997;returnedtoauthorforrevisionJanuary16,1998;acceptedMarch26,1998Inapreviousreport,weshowedthatproteolyticprocessingofan87-kDamatureviralproteinfromthecoronavirusinfectiousbronchitisvirus(IBV)1aand1a/1bpolyproteinswasmediatedbytwopu
3、tativeoverlappingpapain-likeproteinasedomains(PLPDs)encodedwithintheregionfromnucleotides4243to5553ofORF1a(Liuetal.,1995).Inthisstudy,wedemonstratethatonlythefirstdomain,PLPD-1,isresponsibleforthiscleavage,asdeletionoftheseconddomaindidnotaffecttheformationofthe87-kDaprotein.Site-directedmuta
4、genesisstudiesfurthershowedthatapreviouslypredicted12741437nucleophiliccysteineresidue(Cys)andahistidineresidue(His)wereessentialfortheproteinaseactivity,indicatingthattheymaybeimportantcomponentsofthecatalyticcenteroftheproteinase.Meanwhile,expressionofaseriesofdeletionmutantsrevealedthatthe
5、87-kDaproteinwasencodedbythe59-most2.6kbofORF1a.Deletionandaminoacid673674substitutionmutationstudiesdemonstratedthattheGly±GlydipeptidebondwasmostlikelythecleavagesiteresponsibleforreleasingtheC-terminusofthe87-kDaproteinfromthe1aand1a/1bpolyproteins.©1998AcademicPressINTRODUCTIONviralorcell
6、ularproteinasestoproducefunctionalprod-uctsassociatedwithviralreplication.Avianinfectiousbronchitisvirus(IBV)istheproto-Recently,productsencodedbythesetwoORFshavetypeofCoronaviridae,afamilyofenvelopedvirusesbeenidentified.Theseinclude87-,10-,and100-kDawhichpossessalargecontinuouspositive-stra
7、ndedpolypeptides(Liuetal.,1994,1995,1997;LiuandBrown,RNAgenome.ThegenomicRNAis27.6kbinlength1995).ThreeORF1a-encodingproteinasedomains,i.e.,andcontainsatleast10distinctopenreadingframestwooverlappingpapain-likeproteinasedomains(PLPDs)(ORFs)(B
