Mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic RNA transcription on RNA replication.

Previously, we established asystem whereby an intergenic region frommouse hepatitis virus (MHV)inserted into an MHV defective interfering (DI)RNA ledtotranscription ofa subgenomic DIRNA inhelper virus-infected cells. Byusing this system, theduration ofa primary transcription initiation activity which transcribes subgenomic-size RNAsfromthegenomic-size RNA template inMHV-infected cells was examined. Efficient DIgenomic andsubgenomic RNA synthesis was observed whentheDIRNA was transfected at1,3, 3.5,5,and6hpostinfection, indicating thatallactivities whicharenecessaryforMHV RNA synthesis are present continuously during thefirst 6h ofinfection. Theeffect ofsubgenomic DIRNAsynthesis onDIgenomic RNA replication was thenexamined. Replication efficiency oftheDIgenomic RNA whichsynthesized the subgenomic RNA was approximately 70%olowerthanthatofDIgenomic RNA whichdidnotsynthesize the subgenomic DIRNA inMHV-infected cells. Cotransfection oftwodifferent-size DIRNAsdemonstrated that replication ofthelarger DIRNA was strongly inhibited byreplication ofthesmaller genomic DIRNA. Cotransfection oftwoDIRNA species ofthesame length intoMHV-infected cells demonstrated thatreduced replication ofthegenomic DIRNA whichsynthesizes thesubgenomic RNA didnotaffect thereplication of cotransfected DIRNA,demonstrating thatthereduction inDIgenomic RNA replication worksonlyincis, not intrans.Therefore, thepreviously proposed hypothesis thatcoronavirus subgenomic RNA synthesis may inhibit thereplication ofgenomic RNA bycompeting fora limited amountofvirus-derived factors seems unlikely. Possible mechanisms ofcoronavirus transcription arediscussed.