冠状病毒Coronavirusl论文-1996 [Methods in Enzymology] Viral Polymerases and Related Proteins Volume 275 __ [5] Characterization of coronavirus RN.pdf

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1、68EXPRESSION,PURIFICATION,ANDCHARACTERIZATION[5][5]CharacterizationofCoronavirusRNAPolymeraseGeneProductsByJENSHEROLD,STUARTSIDDELL,andJOHNZIEBUHRIntroductionThecoronavirusRNApolymerasegeneencompassesabout20,000nu-cleotidesandcomprisestwolargeopenreadingf

2、rames(ORFs),ORFlaandORFlb,thatoverlapinthe(-1)readingframebyapproximately40-80nucleotides.Together,thesetwoORFshavethepotentialtoencodepolypeptideswithatotalmolecularmassof750,000-800,000.InvitrostudiessuggestthatthedownstreamORFlbisexpressedbyamechanismi

3、nvolving(-1)ribosomalframeshifting,mediatedbya"slippery"sequenceandatertiarystructure,theRNApseudoknot.TheseelementsarepositionedintheRNApolymerasemRNA(whichisequivalenttotheviralgenomicRNA)intheregionoftheORFla/ORFlboverlap.1-6Geneticanalysisofcoronaviru

4、stemperature-sensitive(ts)mutants,de-fectiveinRNAsynthesisattherestrictivetemperature,hasidentifiedanumberofdistinctviralfunctionsrequiredforthereplicationandtranscrip-tionofgenomicandsubgenomicRNAs.Characterizationofthesemutantsbyrecombinationandsequence

5、analysishasallowedthesefunctionstobelocatedandorderedwithintheRNApolymerasegene.Moreover,becausethecomplementationfrequenciesofthesemutantsareindicativeofin-tergenicratherthanintrageniccomplementation,theyprovideclearevi-dencefortheactivityofproteinasesth

6、atprocesstheprimarytranslationproduct(s)ofthepolymerasegeneintosmaller,functionalpolypeptides.7-1°1M.E.G.Boursnell,T.D.K.Brown,I.J.Foulds,P.F.Green,F.M.Tomley,andM.M.Binns,J.Gen.Virol.68,57(1987).2p.j.Bredenbeek,C.J.Pachuk,A.F.H.Noten,J.Charit6,W.Luytjes,

7、S.R.Weiss,andW.J.M.Spaan,NucleicAcidsRes.18,1825(1990).3H.-J.Lee,C.-K.Shieh,A.E.Gorbalenya,E.V.Koonin,N.LaMonica,J.Tuler,A.Bagdzhadzhyan,andM.M.C.Lai,Virology180,567(1991).4j.Herold,T.Raabe,B.Schelle-Prinz,andS.G.Siddell,Virology195,680(1993).5p.j.Bonilla

8、,A.E.Gorbalenya,andS.R.Weiss,Virology198,736(1994).6J.-F.Eleouet,D.Rasschaert,P.Lambert,L.Levy,P.Vende,andH.Laude,Virology206,817(1995).7M.C.Schaad,S.A.Stohlman,J.Egbert,K.Lum,K.Fu,T.Wei,Jr.,andR.S.Baric,Virology177