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1、_ArchivesArchVirol(1992)125:141-160Virology©Springer-Verlag1992PrintedinAustriaLocalizationofanRNA-bindingdomaininthenucleocapsidproteinofthecoronavirusmousehepatitisvirusP.S.MastersWadsworthCenterforLaboratoriesandResearch,NewYorkStateDepartmentofHealth,Albany,an
2、dDepartmentofBiomedicalSciences,SchoolofPublicHealth,StateUniversityofNewYorkatAlbany,Albany,NewYork,U.S.A.AcceptedDecember11,1991Summary.Theinteractionbetweenthenucleocapsid(N)proteinofmousehep-atitisVirus(MHV)andRNAwasstudiedinanefforttodefineportionsoftheNmolec
3、ulethatparticipateinbindingtoRNA.NmRNAstranscribedfromSP6andT7vectorsweretranslatedinarabbitreticulocytelysate.AnalysisofsynthesizedNproteininanondenaturinggelsystemshowedthatitboundinvitrotoanendogenousRNAinthereticulocytelysatebutnottoitsownmRNA.Asetofdeletionmu
4、tantswasconstructedinordertolocalizetheRNA-bindingactivityoftheNprotein.Itwasfoundthatremovalofasmuchas135amino-terminalor57carboxy-terminalaminoacidsfromthemoleculehadlittleornoeffectonRNAbinding.Moreover,deletionmutantslackingbothterministillretainedRNA-bindinga
5、bility.Bycontrast,internaldeletionsortruncationsextendingbeyondthesetwolimitseffectivelyabolishedRNAbindingbyNprotein.Thus,theRNA-bindingregionofNhasbeenmappedtothesecond(central)ofthethreestructuraldomainsofthemolecule.IntroductionCoronavirusesconstituteafamilyof
6、viruseswhosesingle-stranded,positive-senseRNAgenomeshavethelargestcodingcapacitiesamongtheRNAviruses[27,31].Paradoxically,coronaviruseshaverelativelyfewstructuralproteins.Theirlarge,roughlysphericalvirionsareenclosedbyamembraneenvelopecontainingtwo(or,insomecases,
7、three)typesofmembrane-boundglycopro-teins.Internaltothis,multiplemonomersofanucleocapsidprotein(N)en-capsidatetheviralgenome,which,inthecaseoftheprototypemousehepatitisvirus(MHV),issome31,000nucleotidesinlength[10,19].Thenucleocapsidsofcoronavirusesarehelicallysym
8、metric[12],afeatureseeninonlyoneothergroupofpositive-strandedRNAviruses,thoseoftheproposedfamilyToro-142P.S.Mastersviridae[25].Consequently,thesestructu
