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1、Proc.Nati.Acad.Sci.USA Vol.86,pp.5626-5630,July1989 Microbiology Coronavirussubgenomicminus-strandRNAsandthepotentialfor mRNAreplicons (kneticsofRNAsynts/mRNAreplication) PHIROZEB.SETHNA,SHAN-LINGHUNG*,ANDDAVIDA.BRIANt DepartmentofMicrobiology,UniversityofTennessee,Knoxville,TN37996-0845 Communicate
2、dbyDorothyM.Horstmann,April24,1989(receivedforreviewFebruary28,1989) ABSTRACTThegenomeoftheporcinetanmisiblegas- troenteritiscoronavirusisaplus-stand,polyadenylylated, infectiousRNAmoleculeof20kilobases.Duringvirusrep- lication,sevensubgenomicmRNAsaregeneratedbywhatis thoughttobealeader-primngmensmt
3、oforma3- coterminalnestedset.Byusingradiolabeled,strand-specifc, syntheticoligodeoxynucleotideprobesinRNAblothybridiza- tionanalyses,wehavefoundaminus-strandcounterpartfor thegenomeandforeachsubgenomicmRNAspeciesinthe cytoplasmofinfectedcells.Subgenomicminusstrandswere foundtobecomponentsofdouble-st
4、randedreplicativeforms andinnumbersthatsurpassfull-lengthantigenome.Wepro- posethatsubgenomicmRNAreplication,inadditiontoleader- primedtranscription,isasignificantmechanismofmRNA synthesisandthatitfunctionstoamplifymRNAs.Itisa mechanismofamplificationthathasnotbeendescribedforany othergroupofRNAviru
5、ses.Subgenomicrepliconsmayalso functioninamannersimilartogenomesofdefectiveinterfering virusestoleadtotheestablishmentofpersistentinfections,a universalpropertyofcoronaviruses. Thepolyadenylylatedplus-strandRNAgenomeofthepor- cinetransmissiblegastroenteritiscoronavirus(TGEV)(1), likethatoftheavianin
6、fectiousbronchitiscoronavirus(2)and themousehepatitiscoronavirus(MHV)(3),isinfectious.It isthereforepresumedthatasinglemoleculeofgenomicRNA issufficientforinitiatinginfection,andmuchevidencenow supportsthehypothesisthatgenomereplicationoccurs throughafull-lengthminus-strandantigenomethatalso servesa
7、sthetemplateforleader-primedtranscriptionof subgenomicmRNAmolecules(4-6).Sinceleaderpriming initiatesatspecificinternalsitesontheminus-strandantige- nomeandproceedsthoughtothe5endofthemolecule, mRNAsaremadethatforma3-coterminalnestedset. Exceptforthe smallestspecies,coronavirusmRNAsare structurallyp
8、olycistronicbutfunctionprimarilyasmonocis- tronicmoleculeswithusuallyonlythe5-terminalopen readingframebeingtranslated(7-10).Thesubgenomic mRNAsofcoronaviruses,ifmadebytheleader-priming mechanism,wouldthereforebeexpectedtohavea5un- translatedleadersequenceof=80basesthatisidenticalto the5endofthegeno
9、meanda3noncodingterminusof300 basesthatisidenticaltothe3endofthegenome(Fig.1).This indeedseemstobethecasefromsequencedata(11-14). Sincethepromoterforsynthesisoftheminus-strand antigenomebytheTGEVRNA-dependentRNApolymerase (15)presumablyresideswithinthe276-basenoncodingregion atthe3endoftheplus-stran
10、dgenome(16),andthepromoter forgenomesynthesispresumablyresideswithinthe80-base antileadersequence(anestimatedlength)atthe3endofthe minus-strandantigenome,itisnaturaltoaskwhethersub- 208 I 4 v I.O Genome(kilobases) 432I0 -i3 7.k 9.2k H If_JECC27.7k.MN ORFS 4Probe ,_site 7.7k27.7k=mRNAs 9.2k1m_. n hp
11、FIG.1.Structuralrelationshipsamongthegenome,mRNAtran- scripts,andsyntheticoligodeoxynucleotideprobes.Thesevenopen readingframes(ORFS)deducedfromprimarysequenceofthe3end oftheTGEVgenomearedrawntoscale.TheyareidentifiedasPfor peplomerprotein,Mformatrixprotein,Nfornucleocapsidprotein, HPforhydrophobicp
12、rotein,and7.7k,27.7k,and9.2kforpotential nonstructuralproteinsof7.7,27.7,and9.2kDa,respectively.Their correspondingmRNAsareidentifiedasp,m,n,hp,7.7k,27.7k,and 9.2k.Thesitefromwhichtheoligonucleotideprobeswerederivedis indicatedbyanarrow.The5leadersequence(presumedtobe80 bases)andthe3noncodingsequenc
13、e(276bases)areindicatedby heavylinesandaredrawntoscale.The3poly(A)tailisindicated byawavyline.ThelargestmRNA,whichservesastemplatefor synthesisoftheviralpolymerase,ispresumablyidenticaltothe genome. genomicmRNAsundergoreplicationasdoesthegenome, sincetheypossess3and5endsequencesthatareidentical toth
14、oseofthegenome. Wehaveaddressedthisquestionbyseekingtheexistence ofsubgenomicminus-strandRNAmoleculesincellsinfected withTGEV.Syntheticoligodeoxynucleotideprobeswere usedthatspecificallyidentifiedbothfull-lengthandsubge- nomicplus-andminus-strandRNAspecies,andthekinetics ofsynthesisofthemostabundant
15、specieswasmeasured. EvidenceformRNArepliconsintheformofreplicative intermediateswasfound,andweproposethatthesefunction asamechanismformRNAamplification.Wefurtherpropose thatmRNArepliconscompetewithreplicatinggenomefora limitingfactorduringRNAsynthesis,perhapstheviralRNA polymerase,muchasdodefectivei
16、nterferingRNAspeciesof somedefectiveviruses,andthatthisexplainshowcorona- virusesreadilyestablishpersistentinfectionsincellculture. MATERILSANDMETHODS PreparationofRNAfromUninfectedandInfectedCells. Clone116ofthePurduestrainofTGEVwasplaque-purified andgrownonswinetesticlecellsasdescribed(1).Thevirus wasplaque-purifiedagainbyusinginfectiousgenomicRNA Abbreviations:TGEV,porcinetransmissiblegastroenteritiscorona- virus;MHV,mousehepatitiscoronavirus. *Presentaddress:DepartmentofMicrobiology,Scho
