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1、VIROLOGY217,495–507(1996)ARTICLENO.0144MolecularCharacterizationofTransmissibleGastroenteritisCoronavirusDefectiveInterferingGenomes:PackagingandHeterogeneity1ANAME´NDEZ,CRISTIANSMERDOU,ANDERIZETA,FA´TIMAGEBAUER,andLUISENJUANESCentroNacionaldeBiotecnologõBa,CSIC,Depa
2、rtmentofMolecularandCellBiology,CampusUniversidadAuto´noma,Cantoblanco,28049Madrid,SpainReceivedSeptember12,1995;acceptedJanuary9,1996Threetransmissiblegastroenteritisvirus(TGEV)defectiveRNAswereselectedbyserialundilutedpassageofthePUR46straininSTcells.TheseRNAsof22,
3、10.6,and9.7kb(DI-A,DI-B,andDI-C,respectively)weredetectedatpassage30,remainedstableuponfurtherpassageincellculture,andsignificantlyinterferedwithhelpermRNAsynthesis.RNAanalysisfrompurifiedvirionsshowedthatthethreedefectiveRNAswereefficientlypackaged.Virionsofdifferen
4、tdensitiescontainingeitherfull-lengthordefectiveRNAsweresortedinsucrosegradients,indicatingthatdefectiveandfull-lengthgenomeswereindependentlyencapsidated.DI-BandDI-CRNAswereamplifiedbythereversetranscription-polymerasechainreaction,cloned,andsequenced.DI-BandDI-Cgen
5、omesareformedbythreeandfourdiscontinuousregionsofthewild-typegenome,respectively.DI-Ccontains2144nucleotides(nt)fromthe5*-endofthegenome,twofragmentsof4540and2531ntmostlyfromgene1b,and493ntfromthe3*endofthegenome.DI-BandDI-CRNAsincludesequenceswiththepseudoknotmotifa
6、ndencodingthepolymerase,metalionbinding,andhelicasemotifs.DI-BRNAhasastructurecloselyrelatedtoDI-CRNAwithtwomaindifferences:itmaintainstheentireORF1bandshowsheterogeneityinthesizeofthe3*enddeletion.ThisheterogeneitymapsatthebeginningoftheSgene,whereothernaturalTGEVre
7、combinationeventshavebeenobserved,suggestingthateitheraprocessoftemplateswitchingoccurswithhighfrequencyatthispointorthatthederivedgenomeshaveaselectiveadvantage.q1996AcademicPress,Inc.INTRODUCTIONquencesbydiscontinuoustranscription(JeongandMa-kino,1994;Laietal.,1983
8、;Spaanetal.,1983;ZhangandTransmissiblegastroenteritisvirus(TGEV)isamem-Lai,1994).ThefirstmechanismproposedtoexplaintheberoftheCoron
