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1、VIROLOGY216,174–183(1996)ARTICLENO.0044ReplicationofSyntheticDefectiveInterferingRNAsDerivedfromCoronavirusMouseHepatitisVirus-A591WILLEMLUYTJES,HELEENGERRITSMA,andWILLYJ.M.SPAANDepartmentofVirology,InstituteofMedicalMicrobiology,LeidenUniversity,2300AHLeiden,TheNetherlan
2、dsReceivedSeptember27,1995;acceptedNovember17,1995Wehaveanalyzedthereplicationofdeletionmutantsofdefectiveinterfering(DI)RNAsderivedfromthecoronavirusmousehepatitisvirus(MHV)-A59inthepresenceofMHV-A59.UsingtwoparentalDIRNAs,MIDIandMIDIDH,atwinsetofdeletionmutantswasgenera
3、tedwithprogressivelyshorterstretchesof5*sequencecolinearwiththegenomicRNA.Alldeletionmutantscontainedin-frameORFs.WeshowthatintransfectedcellsandafteronepassagetheDIRNAsweredetectableandthattheiraccumulationwaspositivelycorrelatedwiththelengthof5*sequencetheycontained.How
4、ever,accumulationoftwotwinmutants,D2,inwhichsequencesfromnucleotideposition467werefusedtothosefromposition801,wasundetectable.Inpassage4cells,butnotintransfectedorinpassage1cells,recombinationwithgenomicRNAledtotheappearanceoftheparentalDIRNAs.Theaccumulationoftheseparent
5、alRNAswasinverselycorrelatedwiththelengthof5*sequenceonthedeletionmutantsandwashighestintheD2samples.InsharpcontrasttothedatareportedforMHV-JHM-derivedDIRNAs,weshowthatMHV-A59-derivedmutantRNAsdonotrequireaninternalsequencedomainforreplication.Thedatasuggestthatcoronaviru
6、sreplicationinvolvesanRNAsuperstructureatthe5*endofthegenomeoronecomprisingbothendsofthegenomicRNA.Wealsoconcludefromtherecombinationdatathatin-framemutantswithimpairedreplicationsignalsaremorefitthanout-framemutantswithintactreplicationsignals.q1996AcademicPress,Inc.INTR
7、ODUCTIONrentlyunderdebate(forarecentreview,seeVanderMostandSpaan,1995).CoronavirusesareenvelopedanimalvirusesthatAllviralRNAsproducedininfectedcellscontainthecausediseasesinlivestockandpetsandareamongthesame5*leaderandthesame3*enddefinedbytheagentsofhumancommoncold.Theypo
8、ssessapositive-bodysequenceofthesmallestmRNA.ProgenyvirionsstrandedRNAgenomeof27–32kbinahelicaln
