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1、VIROLOGY166,66-75(1988)SynthesisofVirus-SpecificRNAinPermeabilizedMurineCoronavirus-InfectedCellsJULIANL.LEIBOWITZANDJAMESR.DEVRIESDepartmentofPathologyandLaboratoryMedicine,P.O.Box20708,UniversityofTexasHealthScienceCenter,Houston,Texas77225ReceivedDecember29,1987;acceptedMay3,1988
2、Wehavedevelopedapermeabilizedcellsystemforassayingmousehepatitisvirus-specificRNApolymeraseactivity.Thisactivitywascharacterizedastoitsrequirementsformono-anddivalentcations,requirementsforanexogenousenergysource,andpHoptimum.ThissystemfaithfullyreflectsMHV-specificRNAsynthesisinthe
3、intactcell,withregardtobothitstimeofappearanceduringthecourseofinfectionandtheproductssynthesized.ThesystemisefficientandtheRNAproductswereidenticaltothoseobservedinintactMHV-infectedcellsasjudgedbyagarosegelelectrophoresisandhybridization.Permeabilizedcellsappeartobeanidealsystemfo
4、rstudyingcoronavirusRNAsynthesissincetheycloselymimicinvivoconditionswhileallowingmuchoftheexperimentalflexibilityoftrulycell-freesystems.01998AcademicPress.Inc.INTRODUCTIONWeissandLeibowitz,1983;Spaanetal.,1982).AThecoronavirusescompriseagroupoflargeenve-uniquefeatureofcoronavirusr
5、eplicationisthepres-lopedpositive-strandviruseswithauniquereplicationenceofacommonleadersequenceofabout70basesscheme.ThegenomicRNAisabout27kbinsize(Bour-atthe5endofeachmessagewhichispresentonlysnelletal.,1987)andsharesmanystructuralfeaturesonce(atthe5end)inthevirionRNA(Spaaneta/.,wi
6、ththegenomesofotherpositive-strandviruses,i.e.,1983;Laietal.,1983,1984;Bariceta/.,1985).Theitiscappedatthe5terminus(Laietal.,1982a),ispoly-mechanismofsynthesisofallspeciesofcoronavirus-adenylatedatthe3end(Lomniczi,1977;Yogoetal.,specificRNAsislargelyunknown.1977)andcanfunctionasmess
7、engerRNAinvitroProgressinstudyingthedetailsofthesynthesisof(Leibowitzetal,,1982;DenisonandPerlman,1986)theMHVmRNAshasbeenhamperedsomewhatbyandpresumablyinvivoaswell(DenisonandPerlman,thelackofanefficientandeasilyreproducibleinvitro1987).Thetranslationproduct(s)ofthevirionRNAistransc
8、riptionsystemwhichf
