In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation.docx

InVitroReconstitutionofSARS-CoronavirusmRNACapMethylation1nVitroReconstitutionofSARS-CoronavirusmRNACapMethylationMickaelBouvet1,ClaireDebarnot1 ,IsabelleImbert1,BarbaraSelisko1,EricJ.Snijder2 ,BrunoCanard1*,EtienneDecroly1*IArchitectureetEonctiondesMacromoleculesBiologiques,CNRSandUniversitesdAi-MarseiHeIetII,UMR6098,ESI1.Case925,Marsei11e,France,2Mo1ecu1arViro1Ogy1.aboratory,DepartnientofMedicalMicrobiology,CenteroflnfectiousDiseases,1.eidenUniversityMedicalCenter,1.eiden,TheNetherlandsAbstractSAKS-coronavirus(SARS-CoV)genomeexpressiondependsonthesynthesisofasetofmRNAs,whichpresumablyarecappedattheir59endanddirectthesynthesisofalIviraIproteinSintheinfectedcell.Sixteenviralnon-Structuralproteins(ns1tonspl6)constituteanunusualIylargereplicasecoinplex,whichincIudestwomethy11FansferasesputativelyinvolvedinviralmRNAcapformation.TheS-adenosyl-1.-methionine(AdoMet)-dependent(guanine-N7)-methy11ransferase(N7-MTase)activitywasrecentlyattributedtonspl4,whereasnspl6hasbeenpredictedtobetheAdoMet-dependentreconstitutedcompleteSARS-CoVmRNAcapmethylationinvitro.(nucleoside-290)-methy1transferase.Here,WehaveWeShowthatmRNAcapinethylationrequiresathirdviralprotein,nspl,WhichactsasanessentialtriggertocompleteRNAcap-1formation.Theobiigatesequenceofmethylationeventsisinitiatedbynspl4,whichfirstmethylatescappedRNAtranscriptstogeneratecap-07MeGppp-RNs.The1atterarethenseIectively290-methyIatedbythe29O-MTasenspl6inComplexwithitsactivatornspiotogiverisetocap-I7MeGpppA290Mc-RNs.Furthermore,SensitiveinvitroinhibitionassaysOfbothactiviIiesshowthataurintricarboxylicacid,activeinSARS-CoVinfectedcells,targetsbothMTaseswithIC50valuesinthemicromolarrange,providingava1idatcdbasisforanti-coronavirusdrugdesign.CitationiBouvetM,DebarnotC,Imbert-1,SeliskoB,SnijderEJ,etal.(2010)InVitroReconstitutionofSARS-CoronavirusmRN1CapMethy1ation.P1.oSPathog6(4):el000863.doi:10.1371/journal.ppat.1000863Editor:MichaclJ.Buchmeier,UniversityofCaliforniairvine,UnitedStatesofmericaReceivedNovember26,20.