冠状病毒Coronavirusl论文-1988 Synthesis of virus-specific RNA in permeabilized murine coronavirus-infected cells.pdf

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VIROLOGY166,66-75(1988)SynthesisofVirus-SpecificRNAinPermeabilizedMurineCoronavirus-InfectedCellsJULIANL.LEIBOWITZANDJAMESR.DEVRIESDepartmentofPathologyandLaboratoryMedicine,P.O.Box20708,UniversityofTexasHealthScienceCenter,Houston,Texas77225ReceivedDecember29,1987;acceptedMay3,1988Wehavedevelopedapermeabilizedcellsystemforassayingmousehepatitisvirus-specificRNApolymeraseactivity.Thisactivitywascharacterizedastoitsrequirementsformono-anddivalentcations,requirementsforanexogenousenergysource,andpHoptimum.ThissystemfaithfullyreflectsMHV-specificRNAsynthesisintheintactcell,withregardtobothitstimeofappearanceduringthecourseofinfectionandtheproductssynthesized.ThesystemisefficientandtheRNAproductswereidenticaltothoseobservedinintactMHV-infectedcellsasjudgedbyagarosegelelectrophoresisandhybridization.PermeabilizedcellsappeartobeanidealsystemforstudyingcoronavirusRNAsynthesissincetheycloselymimicinvivoconditionswhileallowingmuchoftheexperimentalflexibilityoftrulycell-freesystems.01998AcademicPress.Inc.INTRODUCTIONWeissandLeibowitz,1983;Spaanetal.,1982).AThecoronavirusescompriseagroupoflargeenve-uniquefeatureofcoronavirusreplicationisthepres-lopedpositive-strandviruseswithauniquereplicationenceofacommonleadersequenceofabout70basesscheme.ThegenomicRNAisabout27kbinsize(Bour-atthe5endofeachmessagewhichispresentonlysnelletal.,1987)andsharesmanystructuralfeaturesonce(atthe5end)inthevirionRNA(Spaaneta/.,withthegenomesofotherpositive-strandviruses,i.e.,1983;Laietal.,1983,1984;Bariceta/.,1985).Theitiscappedatthe5terminus(Laietal.,1982a),ispoly-mechanismofsynthesisofallspeciesofcoronavirus-adenylatedatthe3end(Lomniczi,1977;Yogoetal.,specificRNAsislargelyunknown.1977)andcanfunctionasmessengerRNAinvitroProgressinstudyingthedetailsofthesynthesisof(Leibowitzetal,,1982;DenisonandPerlman,1986)theMHVmRNAshasbeenhamperedsomewhatbyandpresumablyinvivoaswell(DenisonandPerlman,thelackofanefficientandeasilyreproducibleinvitro1987).Thetranslationproduct(s)ofthevirionRNAistranscriptionsystemwhichfaithfullyreproducesthehypothesizedtoincludethecoronavirus-specificRNA-eventswhichoccurinintactcells.SeveralgroupsofdependentRNApolymerase,althoughthishasneverworkershavedemonstratedactinomycinD-resistantbeenrigorouslydemonstrated.RNApolymeraseactivityinextractsofcoronavirus-in-InadditiontothevirionRNA,infectedcellscontainfectedcells.DennisandBrian(1982)havereportedtheseveralotherclassesofcoronavirus-specificRNApresenceofamembrane-associatedpolymeraseactiv-(SternandKennedy,1980a;Laietal.,1981;LeibowitzityincytoplasmicextractsofTGEV-infectedcells.Aeta/.,1981;Spaaneta/.,1981).Laiandco-workerssimilaractivityhasbeendemonstratedtobepresenthavedemonstratedthatMHV-infectedcellscontainainextractsofMHV-infectedcells(Braytonetal.,1982,singleRNAspeciesofnegativepolaritywhichisge-1984;Maheyeta/.,1983).Recently,Comptonetal.nomelength(198213).Thisnegative-strandRNAis(1987)havedescribedasystembasedonanextractthoughttoserveasthetemplateforpositive-strandfromlysolecithin-treatedcells.Thesesystemshaveei-mRNAsynthesis.Formousehepatitisvirus(MHV),onetherbeendifficulttoworkwithduetotheirrelativelylowofthemostextensivelystudiedcoronaviruses,thereefficiencies,ortheyhavebeenhardtoreproduceandaresevenspeciesofMHV-specificmRNApresentinmaintainonadailybasis,ortheydonotfaithfullyreflectinfectedcells,thelargestofwhichisindistinguishablethepatternofMHVRNAsynthesisobservedininfectedfromvirionRNA(Leibowitzeta/.,1981;Laieta/.,1981;cells.Spaaneta/.,1981).StructuralanalysesoftheseRNAsTocircumventtheshort-comingsoftheexistinginhaveshownthemtomakeupanestedsetwithco-vitroMHVpolymerasesystemsandtoovercometheterminal3ends(Leibowitzetal.,1981;SternandKen-relativeexperimentallimitationsofintactcells,wehavenedy,1980a,b;Laieta/.,1981;Cheleyeta/.,1981;takenanapproachsimilartothatusedbyCondraandLazzarini(1980)forstudyingVSVreplication.Inthispa-perwereportthecharacteristicsofapermeabilizedcellTowhomrequestsforreprintsshouldbeaddressed.systemanddemonstratethatitincorporatesribonucle-0042-6822/88$3.0066Copynght01988byAcademicPress.Inc.AllrightsofreproductionIanyformreserved. MHVRNASYNTHESISINPERMEABILIZEDCELLS67otidetriphosphatesintoRNAmoleculeswhichappearGTP,200&IUTP,2.5&IofunlabeledCTP,12PMidenticaltothevirus-specificmRNAssynthesizedincreatinephosphate,200PMspermidine,10pMdlTP,MHV-infectedcells.100pg/mlcreatinephosphokinase,1mMdithiothrei-tol,150mMsucrose,10pg/mlactinomycinD,0.24TIU/mlaprotinin(Sigma),20rig/mlouabainoctahy-MATERIALSANDMETHODSdrate(Sigma).[3H]CTP(ICN)wasaddedat62.5&i/mlCellsandvirusyieldingafinalCTPconcentrationof4.5FM.Followingincubationat37fortheindicatedtimes(40minforMonolayerculturesof17CL-1cellsweregrownasmostexperiments),thereactionwasstoppedbythedescribedpreviously(SturmanandTakemoto,1972;additionofanequalvolumeof2%sodiumdodecylsul-Leibowitzeta/.,1981).Theoriginandgrowthofthefate(Sigma)tothecultures.A59(MHV-A59)andJHM(MHV-JHM)strainsofmousePolymeraseactivitywasassayedbyprecipitatingla-hepatitisvirushavebeendescribed(RobbandBond,beledRNAfromreplicateculturesbytheadditionof1979).trichloroaceticacid(TCA)containing1Yosodiumpyro-Permeabilizationofcellsandradioactivelabelingphosphate(Sigma)toafinalconcentrationof5%.TCA-insolubleprecipitateswerecollectedonglassfiberfil-Monolayerculturesof17CI-1cellsweretrypsinizedtersandextensivelywashedwith5%TCA,andtheandinfectedinsuspensionasdescribedpreviouslyTCA-precipitableradioactivitywasquantitatedbyliquid(RobbandBond,1979)atamultiplicityofinfectionscintillationcounting.equalto3PFUpercell.Infectedormock-infectedcellswereplatedin35-mm6-wellclusterdishes(Costar)atExtractionandelectrophoresisofRNA2X1O6cells/wellinDulbeccosmodifiedEaglesme-dium(DME)containing2%fetalbovineserumandin-RNAwasextractedfromcellculturesandperme-cubatedat37.At3hrpostinfectionactinomycinDabilizedcellsasdescribed(Witteketal.,1984;Cabirac(Sigma)wasaddedtotheculturesat10pg/mltoinhibiteta/.,1986).Thecellsweredissolvedin8MguanidiumhostDNA-dependentRNAsynthesis.Atthetimesindi-hydrochloride,0.1M2-mercaptoethanol,and0.2Mcatedforeachindividualexperiment,thedishesweresodiumacetate,pH5.0,andtheDNAwasshearedbyplacedoniceandwashedtwicewithserum-freeDME.passagethroughahypodermicneedle.TheRNAwasBufferA[50mMTris,pH8.0,4.5mMMgAc,,20mMselectivelyprecipitatedovernightbytheadditionofeth-KCI,5mMNaCI,150mll/lRNase-freesucrose(Swartz/anoltoaconcentrationof33%.TheprecipitatedRNAMannBiotech)]wasthenaddedtothecultures.Forourwascollectedbycentrifugationanddissolvedin50standardpermeabilizationconditions,syntheticlyso-mMsodiumacetate,pH5.2,10mMEDTA,1YoSDS,lecithin(L-d-lysophosphatidylcholine,palmitoyl,Sigma)1mg/mlproteinaseKanddigestedat37for1hr.Afterwasaddedtoaconcentrationof150pg/mlandthephenolextractiontheRNAwasethanolprecipitatedcellswereheldonicefor90sec.Lysolecithinwasre-andcollectedbycentrifugationpriortofurtheranalysis.movedfromtheculturesbyaspiratingthebufferfol-RNAtobeanalyzedbygelelectrophoresiswasdis-lowedbyonewashwithbufferAwithoutlysolecithin.solvedinabuffercontaining50%formamide,4%form-BufferBwasthenaddedtotheculturesandthecellsaldehyde,20mMMops[3-(IV-morpholino)propanesul-wereincubatedat37.ThemakeupofbufferBvaried,fonicacid),5mMsodiumacetate,1mMEDTA,pH7.0,asdescribedunderResults,overthecourseoftheandelectrophoresedin0.8%agarosegelscontainingworkreportedhere.InourinitialexperimentsbufferBformaldehyde(Leracheta/.,1977).contained30mMTris,pH8.0,33mMNH&I,5mMPlasmidsNaCI,20mMKCI,4.5mMMgAc,,200PMeachGTP,ATP,andCTP(Pharmacia),12PMcreatinephosphateTheplasmidsusedinthisworkinclude9344(kindly(Sigma),200@Ispermidinetrihydrochloride(Sigma),providedbyDr.SusanWeiss,UniversityofPennsylva-10&dTTP(Pharmacia),100pg/mlcreatinephospho-nia),acDNAcloneofMHV-A59whichencompassesakinase(75units/mg,bovineheart,SigmaTypeIll),portionofgene7allofgenes5and6,andthe3pottion1mMdithiothreitol(ResearchOrganics),150mMofgene4(Budzilowiczetal.,1985).Previouslyunde-RNase-freesucrose,10pg/mlactinomycinD.[3H]UTPscribedmolecularclonesofMHV-JHMusedinthis(ICN,30Ci/mmol)wasaddedat25&i/mltoaconcen-workare118-8a,acDNAclonewhichextendsfromtrationof1.8PM.Experimentstooptimizepolymerasethe3poly(A)ofthegenometoposition119ingene7,activityledtothefollowingstandardconcentrationsforadistanceof1648bases(Spaanetal.,1983);370-1,bufferB(finalpHof8.0):30mMTris,pH8.0,44mMacDNAcloneextendingfromthePvullsiteatpositionNaCI,20mMKCI,6mMMgAc,,1m/l/lATP,200PM1044ofgene7(Spaanetal.1983)tothePstlsiteat 68LEIBOWITZANDDEVRIESposition2589ofgene3(Schmidteta/.,1987),adis-tablished,weinvestigatedtheabilityofpermeabilizedtanceofalmost4.05kbp;414-8a,whichextendsfromcells,inthepresenceofactinomycinD,toincorporatenucleotide2350tonucleotide199ingene3(SchmidtlabeledprecursorsintoTCA-precipitablematerial.eta/.,1987);and478-38a,aclonewhichextendsfromTheseinitialexperimentswerepet-formedatapHoftheDdelsiteatposition350ingene3intoMHVgene8.0,4.5mh/lMg2+,5mMNa+,20mMK+,and33mll/l2foranadditional1.8kbp.Amolecularclonerepre-NH,+.TheseconditionswerebaseduponthoseusedsentingtheBarnHIfragmentK(5.9kbp)ofthelepori-byBraytoneta/.(1982)inacell-freeMHVpolymerasepoxvirusmalignantrabbitfibromavirus(Strayereta/.,system.Permeabilizedcellswereincubatedwith1983a,b)wasusedasacontrolforsomeexperiments.[3H]UTPor[3H]CTPinthepresenceofthethreeotherunlabeledribonucleotidetriphosphafes,actinomycinSouthernblothybridizationD,andanenergyregeneratingsystem,inbufferB.ThePlasmidsweredigestedwiththeappropriaterestric-amountofTCA-precipitableradioactivitywasseveral-tionenzymeaccordingtothemanufacturerssug-foldgreaterinMHV-infectedcellsthaninmock-in-gestedconditions.Theresultingdigestswereelectro-fectedcontrols(datanotshown).Thesynthesisofra-phoresedina1o/oagarosegelandtransferredtonitro-dioactivitylabeledmaterialfromlabeledribonucleotidecelluloseasdescribedpreviously(Southern,1975).triphosphatesrequiredpermeabilization;cellsinwhichNitrocellulosefilterswereprobedeitherwithrandom-thelysolecithintreatmentwasomitteddidnotincorpo-primedcDNApreparedwith[32P]dCTPusingpurifiedrateanyradioactivity(Table1).TheTCA-precipitableMHV-A59virionRNAastemplate(WeissandLeibo-materialsynthesizedinpermeabilizedcellswasdem-witz,1983)orwiththepermeabilizedcellreactionprod-onstratedtobeRNAinsubsequentexperimentsontheucts.Hybridizationwasperformedat42in509/ofor-basisofitbeingcompletelysensitivetoRNasediges-mamide,3XSSPE,5XDenhardts.ThefiltersweretionandcompletelyresistanttodigestionwithDNasewashedtwicein0.1XSSPE,1%SDSat20andthen(Table2).washedfouradditionaltimesinthesamebufferat50.Theabilityofpermeabilized,MHV-infectedcellstoincorporate[c~-~~P]CTPintoacid-precipitablematerialRESULTSStandardizationofpermeabilizationandpolymeraseTABLE1reactionconditionsCHARACTERIZATIONOFMHVRNAPOLYMERASEOurinitialexperimentsweregearedtowarddeter-ACTIVITVINPERMEABILIZEDCELLSminingtheoptimalconditionsforpermeabilizingMHV-Percentageactivityinfected17CL-1cells.CellswereinfectedwithMHV-presentundercontrolA59ormock-infectedandincubateduntilapproxi-Reactionconditionsreactioncondition@mately60%ofthecellswereinvolvedinsyncytia.Fortheexperimentsreportedherethiswasusuallybe--Permeabilization0tween8.5and9.5hrpostinfection,atimewhenMHV--Spermidine66-CreatinephosphokinaseandspecificRNAsynthesiswasmaximal.Atthistimethecreatinephosphate51cellswerewashedasdescribedunderMaterialsand-Mg+19MethodsandpermeabilizedinbufferAcontaininglyso--Mg*+,+Mn*+(6mM)11lecithinwhichwasvariedinconcentrationfrom20to-Mg+,+Ca*+(5,10,or20mM)0250pg/ml.Afterpermeabilizationthecellswere-GTP17-UTP2stainedwithtrypanbluetodeterminethepercentage-UTP,-GTP6ofcellswhichhadbeenmadepermeabletothedye+50rMPMSFb78ateachlysolecithinconcentration.Thesepreliminary+Aprotininb121experimentsdemonstratedthatalysolecithinconcen-+40units/mlRNasin*106trationof150pg/mlpermeabilizedvirtuallyallofthe+Ouabain,1rig/ml125+Ouabain,20ng/mP131MHV-infectedcellsandgreaterthan95%oftheunin-fectedcellswithoutmakingthecellstoofragiletowith-Theresultsofseveraldifferentexperimentsaresummarized.Allstandthesubsequentincubations.Higherconcentra-dataarepresentedasapercentageoftheactivityobtainedinappro-tionsoflysolecithinimpairedourabilitytosubsequentlypriatecontrolpolymerasereactionsinpermeabilizedcells.Precisemaintainthecellsforthepolymerasereaction(datanotreactionconditionsfortheexperimentsvaryingdivalentcationsaregiveninthelegendofFig.2.Allotherreactionswereperformedun-shown).Ourstandardpermeabilizationconditionsderthestandardcationconditionsasdiscussedinthetext.werethereforesetat150pg/mllysolecithin.PerformedintheabsenceofinhibitorsotherthanactinomycinD.Oncetheconditionsforpermeabilizationwerees-cPerformedinthepresenceofaprotinin. MHVRNASYNTHESISINPERMEABILIZEDCELLS69TABLE2to8.4.ThereforeweadoptedpH8.0forourstandardreactionconditions.EFFECTOFRNaseANDDNaseDIGESTIONONTCA-PRECIPITABLEMATERIALSYNTHESIZEDINPERMEABILIZEDCELLSThemagnesiumrequirementofMHVRNAsynthesisinpermeabilizedcellswasthendetermined.InfectedTreatmentandmock-infectedcellswerepermeabilizedatpH8.0Sourceofmaterialandthemagnesiumconcentrationwasvariedfrom0digestedNoneRNaseDNasebto9mMinreplicatecultures.AscanbeseeninFig.2PermeabilizedcellscandinTable1,therewasastrictrequirementforMg*+Mock-infected1,198201,220inthissystem.IntheabsenceofMg*+thepolymeraseMHV-infected7,881298,110activitywasreducedto19%oftheamountobservedlnvivalabeledcellsat6.0mMMg*+.AlthoughMg*+wasneededformea-Mock-infected59633609suringtheMHVpolymeraseactivityinpermeabilizedMHV-infected109,00090109,500cellstheoptimumwasratherbroad.TherequirementaSamplescorrespondingtotheamountofRNApresentina35.formagnesiumcannotbereplacedbyeitherMn*+ormmwell(forpermeabilizedcells)oraloo-mmtissueculturedish(inCa*+(Tablel),bothofwhichresultedinlessactivityvivalabeledcells)weredigestedwith100pgRNaseAand10pgthanthatobtainedwhenmagnesiumwassimplyomit-RNaseTlfor60minat37andTCAprecipitatedinthepresenceoftedfromthereactionmix.AmagnesiumconcentrationcarriertRNA.bSamplesidenticaltothosedigestedwithRNaseweredigestedof6.0mMwaschosenforourstandardreactioncondi-withRNase-freeDNase1(Promega)for60minat37.tions.Infectedanduninfectedcellswerepermeabilizedat8hrpostin-Tofurtheroptimizethesystemwenextinvestigatedfection,labeledwith[32P]CTPandthecellularRNAwasextractedasthemonovalentcationrequirementsoftheMHVpoly-describedunderMaterialsandMethods.merase/permeabilizedcellsystem.Infectedandmock-dInfectedanduninfectedcellswerelabeledwith100&i/ml[3H]uridineinthepresenceofactinomycinDandtheRNAwasex-infectedcellswerepermeabilizedasdescribedabove,tracted.exceptthatthepHandmagnesiumconcentrationwereheldconstantat8.0and6.0mM,respectively,andtheNaf,K+,andNH,+concentrationswerevariedasde-wasdependentuponaddinganexcessofATPascom-scribedbelow.InitialexperimentsdeterminedthatK+paredtothethreeotherribonucleotidetriphosphates.andNH4+seemedtobeinterchangeableinthissys-AnATPconcentrationof1.OmMachievedthebestre-sults.HigherlevelsofATPmadethepermeabilizedcellsextremelyfragileandinhibitedincorporation(datanotshown).Previousinvestigatorsusingcell-freesystemshaddemonstratedpHoptimaforMHV-specificRNA-de-pendentRNApolymeraseactivityat8.4,8.0,or7.4,dependinguponthesystemused.TodeterminetheoptimumpHformeasuringMHV-specificpolymeraseactivityinpermeabilizedcells,infectedandmock-in-fectedcellswerepermeabilizedandassayedforpoly-meraseactivityasdescribedabove,withtheexceptionthatthepHofbuffersAandBwasvariedbetween7.0FIG.1.DeterminationofpHoptimumforMHVRNApolymeraseand8.4amongreplicatecultures.Inthisassay,incor-activityinpermeabilizedcells.CellswereinfectedwithMHVA59atporationoflabeledsubstrateinthepresenceofactino-am.o.i.of3,ormock-infected,andincubateduntil8.5hrpostinfec-mycinDintoTCA-precipitablematerialincreasedastion.ThecellswerepermeabilizedasdescribedunderMaterialsandthepHwasraisedfrom7.0to8.4(Fig.1).TheincreaseMethods.ReplicatecultureswereassayedforMHVRNApolymer-ofpolymeraseactivityasthepHwasraisedproceededaseactivityusingtheoriginalformulationofbufferB(33mMNH&I,5mMNaCI,20mMKCI,4.5mMMgCIP,asdescribedunderMateri-inastep-wisefashion,withthegreatestincrementinalsandMethodswiththeexceptionthatthepHwasvariedbetweenactivityoccurringasthepHwasincreasedfrom7.2to7.0and8.4amongthereplicatecultures.After40minofincubation7.4.FurtherincreasesinthepHfrom7.4to8.4hadatheassaywasterminatedandtheamountofradioactivityincorpo-relativelysmalleffectupontheactivity,withthegreat-ratedintoTCA-precipitablematerialwasdetermined.AlldatapointsestportionofthatincreaseoccurringasthepHwasrepresentthemeanofduplicatesamples.Theresultswerecalcu-latedbysubtractingtheamountofradioactivityinmock-infectedchangedfrom7.6to8.0.TheabilityofpermeabilizedsamplesfromthatincorporatedintoMHV-infectedsamplesundercellstosynthesizeactinomycinD-resistantRNAre-identicalconditions.TheresultsareexpressedinarbitraryunitswithmainedalmostconstantasthepHwasvariedfrom8.0themaximumactivitybeingsetat100. 70LEIBOWITZANDDEVRIESpermeabilizedcellstoincorporateradiolabeledCTPaswellasUTPintoTCA-precipitablematerialsuggeststhatthepolymeraseactivitywearedetectingisnotduetothepolyuridylatepolymerasepresentinthecyto-plasmofmammaliancells(HayashiandMcFarlane,1979).ProteaseinhibitorsandRNaseinhibitorshaveboth0-beenreportedtoincreasetheRNA-dependentRNA03.0,Tr--9.0polymeraseactivitypresentinextractsofWestNilevi-MAGNSIUMCONCENTRAT!ONrus-infectedcells(GrunandBrinton,1986).Wethere-FIG.2.DeterminationofthemagnesiumoptimumforMHVRNAforedeterminedtheeffectofaddingPMSForaprotinin,polymeraseactivityinpermeabilizedcells.Cellswereinfectedwithtwoproteaseinhibitors,ontheabilityofpermeabilizedMHV-A59ormock-infectedandincubateduntil9hrpostinfection.MHV-infectedcellstodirectthesynthesisofactinomy-ThecellswerepermeabilizedandincubatedinaformulationofbufferBwhichcontained30rnMTris,33mMNH&I,5mMNaCI,20mMtinD-resistantRNA.AsshowninTable1,50&IPMSFKCI,pH8.0,asdescribedunderMaterialsandMethodswiththeex-decreasedincorporationofCTPintoTCA-precipitableceptionthatthemagnesiumconcentrationwasvariedbetween0RNAbyabout20%.However,aprotininincreasedac-and9mMamongreplicatecultures.At40minincubationtheassaytivitybyabout20%.WeattributethedifferenteffectswasterminatedandtheamountofradioactivityincorporatedintoTCA-precipitablematerialwasdetermined.Alldatapointsrepresentofthesecompoundstothemuchbroaderspectrumofthemeanofduplicatesamples.Theresultswerecalculatedbysub-activityofPMSF,adrugwhichinhibitsmostserinees-tractingtheamountofradioactivityinmock-infectedsamplesfromterases(FahrneyandGold,1963;LaskowskiandSea-thatincorporatedintoMHV-infectedsamplesunderidenticalcondi-lock,1972).Surprisingly,theadditionofplacentaltions.RNaseinhibitorhadlittleeffectonRNAsynthesisbypermeabilizedcells.TheeffectofouabainontheMHVpolymerase/per-tern,Na+wasrequiredforactivity,andatotalmonova-meabilizedcellsystemwasinvestigatedbecauseoflentcationconcentrationgreaterthan80rnMresultedthedependenceofthesystemonanexogenousen-inadecreaseinpolymeraseactivity(datanotshown).ergysource.OuabainisaninhibitoroftheNa+/K+-de-TheMHVpolymeraseactivitypresentinpermeabilizedpendentATPasepresentintheplasmamembrane(Ru-cellswasrelativelyinsensitivetomonovalentcationohoandKyte,1974).Wereasonedthatafterpermeabil-concentrations,aslongasthetotalmonovalentcationizationthisenzymemightbecompetingwiththeMHVremainedbelow80mn/l.K+wasnotrequired;theomis-polymerasecomplexforATP.IfthishypothesisistruesionofK+frombufferBdecreasedactivityby5-l09/o.wefeltthattheadditiononaninhibitoroftheATPaseAtconcentrationsabove80mMNa++K+thepolymer-tothesystemmightstimulatetheMHVpolymeraseac-aseactivitydecreasedsomewhat.Weadoptedfinaltivity.Thisdidappeartobethecase.Ouabainatcon-concentrationsof44mMNa+and20mMK+inbuffercentrationsof1and20rig/mlincreasedthepolymer-Bforourstandardreactionconditions.Theseconcen-aseactivityto125and131%ofthatobservedincon-trationswereconvenienttouseandapproximatelyintrols.WecouldnotincreasetheconcentrationofATPthecenterofthebroadoptimumconcentrationsofabove1mMtodirectlytesttheideathatouabainex-monovalentcations.erteditsstimulator-yeffectonMHVpolymeraseactivityTherequirementsoftheMHVpolymerase/perme-byincreasingthebiologicallyeffectiveATPconcentra-abilizedcellsystemforvariouscofactorsweredeter-tioninourreactionsinceconcentrationsofATPgreatermined.AsshowninTable1,theomissionofspermi-than1mn/lcausedthepermeabilizedcellstodetachdinedecreasedtheactivityto669/oofthatobservedfromthesubstrateandsubsequentlydisintegrate.Wewiththecompletesystem.Therewasarequirementforthereforeincludedouabainat1rig/mlandaprotininatanenergyregeneratingsystem;theomissionofCPK0.24TIU/mlinallsubsequentexperiments.andcreatinephosphatedecreasedactivityto51%ofcontrolvalues.Thesystemalsorequiredallfourribonu-Characterizationoftheproductssynthesizedincleotidetriphosphates.TheomissionofeitherGTPorpermeabilizedcellsUTPdecreasedincorporationoflabeledCTPby83and98%,respectively.TheomissionofbothUTPandGTPTheTCA-precipitablematerialsynthesizedinMHV-decreasedsynthesisby94%ofthatobservedintheinfectedpermeabilizedcellswasidentifiedasRNAbycompletesystem.Theseresultssuggestedthattheac-itssensitivitytoRNase.ItwasnotsensitivetoDNasetivityweweredetectingwasnotapolynucleotideter-(Table2).TofurthercharacterizetheRNAsynthesizedminaltransferase-likeactivity.Similarly,theabilityofinoursystem,weextractedRNAfrompermeabilized MHVRNASYNTHESISINPERMEABILIZEDCELLS71cellslabeledwith[a-32P]CTPandfromparallelculturesRNAspeciessynthesizedinthepermeabilizedcellsisofintactMHV-infectedcellslabeledwith[3P]ortho-verysimilartotheMHV-specificRNAsobservedinin-phosphateinthepresenceofactinomycinD.Thesetactcells.sampleswerethenanalyzedbyelectrophoresisonaFurtherevidenceofthevirus-specificnatureoftheseformaldehydegel.TheautoradiographshowninFig.3RNAswasobtainedbySouthernblothybridization.illustratesthattherelativeamountsandsizesoftheMHV-specificplasmidclonesandaplasmidclonede-rivedfromtheunrelatedmalignantrabbitfibromavirusweredigestedwiththeappropriaterestrictionenzymeabtoexcisetheclonedinsertandresolvedbyagarosegelelectrophoresis.Thebandatapproximately3.0kbp(Fig.4A,lanea)representsthecloningvectorpGEM-1.Thebandatapproximately4.3kbp(Fig.4A,lanesb-e)representspBR322.Thebandatapproximately2.8kbp(Fig.4A,lanef)representspUC19.Replicatefiltersofmolecularclonesrepresentingthemost310kboftheMHVgenomewerehybridizedwitheitherrandom-primedcDNAsynthesizedfromapurifiedvirionRNAtemplate(Fig.4B),RNAextractedfromMHV-infectedpermeabilizedcellslabeledwith[a-32P]UTPafterper-meabilization(Fig.4C),orRNApreparedfrommock-infectedpermeabilizedcells.Asexpected,therandom-primedcDNAprobehybridizedtoalloftheMHV-spe-cificclones(lanesa-e)anddidnotrecognizetheplasmidcontainingthemalignantrabbitfibromavirusBarnHIfragmentK(lanef).ThelabeledRNAsynthe-sizedafterpermeabilizationofinfectedcellsalsohy-bridizedspecificallywiththeMHVinserts,althoughitdidnotgiveasstrongasignalasrandom-primedcDNAprobe(Fig.4C).Theapparentbandatabout3.7kbpinPanelC,lanee,asartifactualsincenoDNAispresentatthatpositionintheethidiumbromidestainedgel.Thesignalwithclones118-8aand414-8awasconsid-erablyweakerthanthesignalobtainedwiththeotherMHV-specificinserts.Weattributethesedifferencesinsignal,atleastinpart,totheloweramountofthesetwoinsertspresentinthegel(Fig.4A).Thisisalsoreflectedintherelativesignalsobtainedwiththerandom-primedprobe.ThespecificityofthehybridizationreactionwasconfirmedbythelackofhybridizationofMHV-infectedpermeabilizedreactionproductwithanirrelevantplas-midinsert(Fig.4C,laneg)andthefailureofperme-abilizedcellreactionproductsfrommock-infectedcellstohybridizewiththeseMHVclones(datanotshown).Additionally,theextentofhybridizationofthereactionproductsfrompermeabilizedMHV-infectedcellstoMHVcDNAclonesboundtonitrocellulosecircleswassimilartothatofRNApreparedbylabelingintactMHV-FIG.3.GelanalysisofRNAproductssynthesizedinpermeabilizedcells.CellswereinfectedwithMHVA59,incubateduntil8.5hrpost-infectedcellswith[32P]orthophosphateinthepres-infection,andpermeabilized.PermeabilizedcellswerelabeledwithenceofactinomycinD(datanotshown).[LY-~P]UTP,250&i/ml,underourstandardconditionsfor40minandtheRNAwasextracted(lanea).Areplicateculturewasnotper-Kineticsofsynthesismeabilizedbutrathertheintactcellswerelabeledwith500&ilml[3zP]orthophosphateinthepresenceofactinomycinDfor40min(laneb).TheRNAsampleswereelectrophoresedonan0.8%aga-Thetimecourseofincorporationoflabelinperme-rosegelcontainingformaldehydeandautoradiographed.abilizedcellswasdeterminedbypreparingreplicate LEIBOWITZANDDEVRIESabcdefabcdefabcdef2.1120..1.3-1,,I2,E2,4,5,Ei,NI//,g344y+Y370-1iIFIG.4.HybridizationofRNAproductsofpermeabilizedMHV-infectedcellstoMHV-specificplasmidclones.PurifiedplasmidDNAwasdigestedwithPstl,plasmids478-38a(lanea),414.8a(laneb),370-l(lanec),118-8a(laned),and9344(lanee),orBarnHIinthecaseoftheclonedmalignantrabbitfibromavirusBarnHIfragmentK(lanef).Replicatesamplesoftherestrictiondigestswereresolvedbyelectrophoresisina1%agarosegel.(A)Thepatternobtainedbyethidiumbromidestaining.Thepositionsofmolecularweightmarkers,aHindIlldigestofhDNA,areindicatedtotherightofthephotograph.(B)AfterSoutherntransfertoanitrocellulosefiltertheDNAwashybridizedtoarandom-primedMHV-A59cDNAprobe.Theautoradiographwasexposedfor7hr.(C)Areplicatefiltertothatshownin(B)washybridizedtoRNAsynthesizedinpermeabilizedMHV-A59-infectedcellsincubatedwith[a-32P]CTPafterpermeabilization.Theautoradiographwasexposedfor72hr.(D)AschematicshowingtheapproximatemappositionsoftheMHVclonesusedinthisexperiment.ThefilledrectanglerepresentstheMHVleadersequenceatthe5endofthegenome.culturesofMHV-infectedandmock-infectedcells,in-permeabilizedcellsoccurredat11hrpostinfectioncubatingthemfor8.5hr,permeabilizingthemusingthe(datanotshown).SimilarexperimentswithMHV-JHMstandardconditionswehaddeveloped,andlabelingyieldedsimilarresults,althoughpolymeraseactivitythemforthetimesindicatedinFig.5.Theaccumula-appeared1hrlaterthanduringMHV-A59infection.IttionofradioactivityinTCA-precipitableproductsin-shouldbenotedthattheinfectionproceededsome-creases,althoughnotinalinearfashion,overthefirstwhatslowerintheexperimentsreportedherethanin40minoflabeling.AfterthattimetheamountofTCA-ourpreviouslyreportedwork(Leibowitzeta/.,1981).precipitableradioactiveproductinthecellsdecreasesThereasonsforthisdiscrepancyarenotknownatthisdramatically.time.ThekineticsofthedevelopmentoftheMHV-specificDISCUSSIONRNApolymeraseactivityoverthecourseofinfectionwasdeterminedinpermeabilizedcells.AsshowninInthisworkwereportthedevelopmentandcharac-Fig.6,theaccumulationofMHVRNApolymeraseac-terizationofapermeabilizedcellsystemforassayingtivityininfectedcells(PanelA),asdetectedbyouras-MHV-specificRNApolymeraseactivity.Thisactivitysay,faithfullymirroredthekineticsofactinomycinD-wascharacterizedastoitsrequirementsformono-andresistant,[3H]uridineincorporationintoTCA-precipita-divalentcations,requirementsforanexogenousen-blematerial(PanelB)duringaseriesof1-hrpulses.ergysource,pHoptimum,anditstimeofappearancePolymeraseactivityisfirstdetectableat5hrpostinfec-duringthecourseofinfection.TheRNAproductssyn-tionintheseexperiments.SubsequentexperimentsthesizedinpermeabilizedcellsweredemonstratedtoshowedthatthepeaklevelofpolymeraseactivityinbeMHV-specificbyagarosegelelectrophoresis. MHVRNASYNTHESISINPERMEABILIZEDCELLS73sizedaccuratelyreflecttheRNAspeciessynthesizedin101intactMHV-infectedcells.AllsevenoftheMHVmRNAspeciesaremadeinapproximatelythesameratiosastheyareinvivo.Thiscontrastswithtrulycell-freesys-temsinwhichtheRNAproductssynthesizedwerenotcharacterizedastotheprecisemolecularspeciesofo~!LY-Y-YRNAsynthesized(Braytonetal.,1982,1984;Maheyeta/.,1983)orthoseinwhichthemajorproductwas0102030406060genomelength(Comptonetal.,1987).AlthoughthereasonsforthisdifferenceintheRNAproductssynthe-FIG.5.Thekineticsofincorporationof[3H]CTPintoMHV-specificsizedareunknown,apossibleexplanationforthisob-RNAinpermeabilizedcells.CellswereinfectedwithMHV-A59(0)orservationisthelossofasolublefactorresponsibleformock-infected(A),incubateduntil8.5hrpostinfection,andperme-abilized.ReplicatecultureswereincubatedinbufferBcontainingregulatingMHVtranscriptionduringpreparationofcell-62.5&i/mlof[3H]CTPunderstandardreactionconditions(Materialsfreeextracts.Otherexplanationsarepossibleaswell,andMethods)for0,10,20,40,60,and80min.Atthesetimestheandadditionalworkisneededtoidentifyputativefac-cellsweresolublizedandtheamountofradioactiveprecursorincor-torsneededfortheappropriateregulationofMHVRNAporatedintoTCA-precipitablematerialwasdetermined.synthesis.Thekineticsoftheaccumulationofpolymeraseac-ThepurposeofthepresentworkwastodevelopandtivityweobservedparallelstheincreaseofactinomycincharacterizeasystemforstudyingtheMHV-specificD-resistanturidineincorporationwhichoccursduringRNApolymerasethatwasmoreamenabletoexperi-MHVinfection.Noearlypeakofpolymeraseactivityormentalmanipulationsthanintactcells.Toavoiddiffi-uridineincorporationinintactcellswasdetected.Incultiesinreproducinginvitrotheintracellularenviron-thisregardourresultsaresimilartothoseofComptonmentwhichevolvesduringMHVinfectionweelectedetal.(1987)andSawickiandSawicki(1986).Thesere-topursueapathwhichwouldleaveasmuchofthecellsultsdifferfromthoseofearlierworkers(Braytoneta/.,machineryinplaceaspossible.Thesystemwehave1982,1984)whodetectedapeakofpolymeraseactiv-developedhasseveraladvantageswhencomparedtoityat2hrpostinfectionfollowedbyafallinactivitypriorthecell-freesystemsdevelopedbyotherworkers.Al-toasubsequentincreasetomaximallevels.Therea-thoughitisdifficulttocomparetherelativeefficienciessonsforthesedifferencesisnotknown.Itcouldrelateofdifferentsystemsduetothedifferentwaysinwhichtothedifferentcelllinesusedbydifferentlaboratoriestheexperimentalresultshavebeenpresented,wecancalculatetheamountofRNAsynthesizedinoursys-tem.Usingtheoptimizedreactionconditions,one35-mmwellofMHV-infectedpermeabilizedcellsincorpo-ratedabout900fmolofUMPintoMHV-specificRNA.ThisisestimatedtobeaboutfivefoldmoreRNAsynthe-sisonapercellbasisthanthatobtainedfromacell-freeextractpreparedfrompermeabilizedcells(Comptoneta/.,1987).Otherworkersusingcell-freesystemshavereportedyieldsonthebasisoffemtomolesofUMP/h/mgprotein(DennisandBrian,1982;Maheyeta/.,1983;Braytoneta/.,1982,1984).Thesehavebeenintherangeof150-400fmol/hr/mgprotein.One35-mmwellofMHV-infected17CL-1cellscontainsabout4500246610pgofprotein,providingayieldonapermilligrambasisHamPosthktblHamPosthfectiiwhichisapproximately1800fmolofUMP/mgprotein/FIG.6.TheaccumulationofMHV-specificRNApolymeraseactivity40min,afigurewhichmakesitatleastfourtimesmoreduringinfection.Replicateculturesof17CI-1cellswereinfectedwithefficientthanthepreviouslydescribedcell-freesys-MHVA59(m)ormock-infected(A)andincubatedfor2hr.Atthattems.Therearenodataatthistimetosuggestthattime,andathourlyintervalsthereafter,duplicatesetsofculturesoursystem,oranyotherMHVpolymeraseassay,wereeitherpermeabilizedandassayedfortheincorporationofradio-iscapableofinitiatingthesynthesisofnewstrandsactiveCTPintoTCA-precipitablematerialunderstandardreactionconditions(A)orexposedtoactinomycinD(5pg/ml)for15minandofRNA.labeledwith100&iof[3H]uridinefor1hrandthensolublizedwithAsecondadvantageofpermeabilizedcellsforstudy-SDSandassayedforincorporationoflabelintoTCA-precipitablema-ingMHVRNAsynthesisisthattheproductssynthe-terial(B). 74LEIBOWITZANDDEVRIESorberelatedtothevastlydifferentreactionconditionsmerasegeneproductincellsinfectedwithmurinecoronavirusthatareemployedbythedifferentmethodsofassayingA59.Virology157,565-568.DENNIS,D.E.,andBRIAN,D.A.(1982).RNA-dependentRNApolymer-MHVpolymeraseactivity.aseactivityincoronavirus-infectedcells.J.Viral.42,153-l64.ThesystemwehavedescribedforassayingtheFAHRNEY,D.E.,andGOLD,A.M.(1963).Sulfonylfluorides.I.RatesofMHV-inducedRNA-dependentRNApolymeraseactiv-reactionwithacetylcholinesterases,a-chymotrypsinandtrypsin.ityshouldproveusefulforotherworkers.Itissimpleto1.Amer.Chem.Sot.85,997-l003.setup,providesasystemwhichshouldbeamenableGRUN,1.B.,andBRINTON,M.A.(1986).CharacterizationofWestNilevirusRNA-dependentRNApolymeraseandcellularterminaladen-topulse-chase-typeexperiments,andfurnishesasys-ylylanduridylyltransferasesincell-freeextracts.1.Viral.60,1113-ternwheremacromoleculessuchasRNAtemplatesor1124.purifiedproteinscanbeaddedandtheireffectonMHVHAYASHI,T.T.,andMCFARLANE,K.(1979).Comparisonofendoge-synthesisobserved.Itmayalsoserveasapointofde-nousandexogenousRNAprimersofpoly(U)polymeraseinrathe-parturefordevelopingacompletelycell-freesystempaticribosomes.Biochem.1.177,895-902.LAI,M.M.C.,BARIC,R.S.BRAYTON,P.R.,andSTOHLMAN,S.A.whichbetterreflectstheintracellularsynthesisofMHV(1984).CharacterizationofleaderRNAsequencesonthevirionRNAsthanthecurrentlyavailablesystems.andmRNAsofmousehepatitisvirus,acytoplasmicRNAvirus.Proc.Nat/.Acad.Sci.USA81,3626-3630.ACKNOWLEDGMENTS&I,M.M.C.,BRAMON,P.R.,ARMEN,R.C.,PAUON,C.D.,PUGH,C.,andSTOHLMAN,S.A.(1981).MousehepatitisvirusA59:mRNAThisworkwassupportedbyPublicHealthServiceGrantNS20834structureandgeneticlocalizationofthesequencedivergencefromtheNationalInstituteofNeurologicandCommunicativeDis-fromhepatotropicstrainMHV-3.J.Viral.39,823-834.easesandStroke.TheauthorsthankDr.MarkSchoenbergforper-&I,M.M.C.,PATON,C.D.,BARIC,R.S.,andSTOHLMAN,S.A.formingsomeofthepreliminaryworkwhichinspiredustocontinue(1983).PresenceofleadersequencesinthemRNAofmousehep-theseexperimentsandDr.EmeliaOleszakforathoughtfulreadingatitisvirus.J.Viral.46,1027-l033.ofthemanuscript.L~I,M.M.C.,PATTON,C.D..andSTOHLMAN,S.A.(1982a).FurthercharacterizationofmRNAsofmousehepatitisvirus:PresenceofREFERENCEScommon5-endnucleotides.1.Viral.41,557-565.LAI,M.M.C.,PATTON,C.D.,andSTOHLMAN,S.A.(1982b).Replica-BARIC,R.S.,STOHLMAN,S.A.,RAZAVI,M.K.,andL~I,M.M.C.(1985).tionofmousehepatitisvirus:NegativestrandedRNAandreplica-Characterizationofleader-relatedsmallRNAsincoronavirus-in-tiveformRNAareofgenomelength.1.Viral.44,487-492.fectedcells:Furtherevidenceforleader-primedmechanismofLASKOWSKI,M.,andSEALOCK,R.W.(1972).Proteinproteaseinhibi-transcription.virusRes.3,19-33.tors-Molecularaspects.InTheEnzymes,3rded.(P,D.Boyer,BOURSNELL,M.E.G.,BROWN,T.D.,FOULDS,I.J.,GREEN,P.F.,TOM-Ed.),pp.375-473.AcademicPress,NewYork.LEY,F.M.,andBINNS,M.M.(1987).CompletionofthesequenceLEIBOWIT~,J.L.,WEISS,S.R.,PAAVOLA,E.,andBOND,C.W.(1982).ofthegenomeofthecoronavirusavianinfectiousbronchitisvirus.Cell-freetranslationofmurinecoronavirusRNA.J.Viral.43,905-J.Gen.Viral.68,57-77.913.BRAMON,P.R.,LAI,M.M.C.,PATTON,C.D.,andSTOHLMAN,S.A.LEIBOWITZ,J.L.,WILHELMSEN,K.C.,andBOND,C.W.(1981).Thevi-(1982).CharacterizationoftwoRNApolymeraseactivitiesinducedrus-specificintracellularRNAspeciesoftwomurinecoronavi-bymousehepatitisvirus.J.Viral.42,847-853.ruses:MHV-A59andMHV-JHM.Virology114,39-51.BRAMON,P.R.,STOHLMAN,S.A.,andLA&M.M.C.(1984).FurthercharacterizationofmousehepatitisvirusRNA-dependentRNALERACH,H.,DIAMOND,D.,WOZNEY,J.M.,andBOEDTKER,H.(1977).polymerases.Virology133,197-201.RNAmolecularweightdeterminationsofgelelectrophoresisunderBu~z~~ow~cz,C.J.,WILCZYNSKI,S.P.,andWEISS,S.R.(1985).Threedenaturingconditions,acriticalreexamination.BiochemisUy96,intragenicregionsofcoronavirusmousehepatitisvirusstrainA594743-4751.genomeRNAcontainacommonnucleotidesequencethatisho-LOMNICZI,B.(1977).BiologicalpropertiesofaviancoronavirusRNA.mologoustotheBendoftheviralmRNAleadersequence.J.Viral.J.Gen.Viral.36,531-533.53,834-840.CABIRAC,G.F.,MULLOY,J.J.,STRAYER,D.S.,SELL,S.,andLEIEJOWIT~,MAHEY,B.W.J.,SIDDELL,S.,WEGE,H.,andTERMEULEN,V.(1983).1.L.(1986).TranscriptionalmappingofearlyRNAfromregionsofRNA-dependentRNApolymeraseactivityinmurinecoronavirus-thegenomeoftheShopefibromaandmalignantrabbitfibromainfectedcells.1.Gen.Viral.64,103-l11.virusgenomes.Virology153,53-69.ROBE,1.A.,andBOND,C.W.(1979).Pathogenicmurinecoronavi-CHELEY,S.,ANDERSON,R.,CUPLES,M.J.,LEECHAN,E.C.M.,andruses.I.Characterizationofbiologicbehaviorinvitroandvirus-MORRIS,V.L.(1981).Intracellularmurinehepatitisvirusvirus-spe-specificintracellularRNAofstronglyneurotropicJHMVandweaklycificRNAscontaincommonsequences.Virology112,596-604.neurotropicA59Vviruses.Virology94,352-370.COMPTON,S.R.,ROGERS,D.B..HOLMES,K.V.,FERTSCH,D..REMEN-RUOHO,A.,andKYTE,J.(1974).Photoaffinitylabelingoftheouabain-ICK,J.,andMCGOWAN,J.1.(1987).Invitroreplicationofmousebindingsiteon(Na++K+)adenosinetriphosphatase.Proc.Nat/.hepatitisvirusstrainA59.J.Viral.61,1814-l820.CONDRA,J.H.,andLAZZARINI,R.A.(1980).ReplicativeRNAsynthesisAcad.Sci.USA71,2352-2356.andnucleocapsidassemblyinvesicularstomatitisvirus-infectedSAWICKI,S.G.,andSAWICKI,D.L.(1986).Coronavirusminus-strandpermeablecells.1.L&o/.36,796-804.RNAsynthesisandeffectofcycloheximideoncoronavirusRNADENISON,M.R.,andPERLMAN,S.(1986).Translationandprocessingsynthesis.J.Viral.57,328-334.ofmousehepatitisvirusvirionRNAinacell-freesystem.1.Viral.SCHMIDT,I.,SKINNER,M.,andSIDDELL,S.(1987).Nucleotidese-60,12-18.quenceofthegeneencodingthesurfaceprojectionglycoproteinDENISON,M.,andPERLMANS.(1987).Identificationofputativepoly-ofcoronavirusMHV-JHM.1.Gen.Viral.68,47-56. MHVRNASYNTHESISINPERMEABILIZEDCELLS75SOUTHERN,E.M.(1975).DetectionofspecificsequencesamongSTRAYER,D.S.,CABIRAC,G.F.,SELL,S.,andLEIBOWITZ,1.L.(198313).DNAfragmentsseparatedbygelelectrophoresis./.Mol.Viol.98,Malignantrabbitfibromavirus:Observationsonthecultureand503-518.histopathologiccharacteristicsofanewvirus-inducedrabbittu-SPAAN,W.J.,DELIUS,H.,SKINNER,M.,ARMSTRONG,J.,ROTTIER,P.,mor.J.Natl.Cancerlnst.71,91-104.SMEEKENS,S.,VANDERZEIJST.B.A.M.,andSIDDELL,S.G.(1983).STRAYER,D.S..SKALETSKY,E.,CABIRAC,G.F.,SHARP,P.A.,CORBEIL,CoronavirusmRNAsynthesisinvolvesfusionofnon-contiguousL.B.,SELL,S.,andLEIBOWIT~,J.L.(1983a).Malignantrabbitfi-sequences.EMBOI.2,1839-l844.bromaviruscausessecondaryimmunosupressioninrabbits.].Im-SPAAN,W.J.,ROTTIER,P.J.,HORZINEK,M.C..andVANDERZEIJST,munol.130,399-404.B.A.M.(1981).Isolationandidentificationofvirus-specificSTURMAN,L.S.,andTAKEMOTO,K.K.(1972).EnhancedgrowthofamRNAsincellsinfectedwithmousehepatitisvirus(MHV-A59).murinecoronavirusintransformedmousecells.infect.lmmun.6,Virology108,424-434.501-507.SPAAN,W.J.,ROTUER,P.J.,HORZINEK,M.C..andVANDERZEIJST,B.A.(1982).SequencerelationshipsbetweenthegenomeandtheWEISS,S.R.,andLEIBOWIT~,1.L.(1983).CharacterizationofmurineintracellularRNAspecies1,3,6,and7ofmousehepatitisviruscoronavirusRNAbyhybridizationwithvirus-specificcDNAprobes.strainA59.J.Viral.42,432-439.J.Gen.Viral.64,127-l33.STERN.D.F.,andKENNEDY,S.I.T.(1980a).CoronavirusmultiplicationWITTEK,R.,HANGGI,M.,andHILLER,G.(1984).Mappingofagenestrategy.I.Identificationandcharacterizationofvirus-specifiedcodingforamajorlatestructuralpolypeptideonthevacciniavirusRNA.J.Viral.34,665-674.genome.J.Viral.49,371-378.STERN,D.F.,andKENNEDY,S.I.T.(1980b).CoronavirusmultiplicationYOGO,Y.,HIRANO.N.,HINO,S.,SHIBUTA.H.,andMATUMOTO,M.strategy.II.Mappingtheavianinfectiousbronchitisvirusintracel-(1977).PolyadenylateinthevirionRNAofmousehepatitisvirus./.lularRNAspeciestothegenome./.Viral.38,440-449.Biochem.82,1103-l108.