Two murine coronavirus genes suffice for viral RNA synthesis

We identified two mouse hepatitis virus (MHV) genes that suffice for MHV RNA synthesis by using an MHV-JHM-derived defective interfering (DI) RNA, DIssA. DIssA is a naturally occurring self-replicating DI RNA with nearly intact genes 1 and 7. DIssA interferes with most MHV-JHM-specific RNA synthesis, except for synthesis of mRNA 7, which encodes N protein; mRNA 7 synthesis is not inhibited by DIssA. Coinfection of MHV-JHM containing DIssA DI particles and an MHV-A59 RNA temperature-sensitive mutant followed by subsequent passage of virus at the permissive temperature resulted in elimination of most of the MHV-JHM helper virus. Analysis of intracellular RNAs at the nonpermissive temperature demonstrated efficient synthesis of DIssA and mRNA 7 but not of the helper virus mRNAs. Oligonucleotide fingerprinting analysis demonstrated that the structure of mRNA 7 was MHV- JHM specific and therefore must have been synthesized from the DIssA template RNA. Sequence analysis revealed that DIssA lacks a slightly heterogeneous sequence, which is found in wild-type MHV from the 3' one-third of gene 2-1 to the 3' end of gene 6. Northern (RNA) blot analysis of intracellular RNA species and virus-specific protein analysis confirmed the sequence data. Replication and transcription of another MHV DI RNA were supported in DIssA- replicating cells. Because the products of genes 2 and 2-1 are not essential for MHV replication, we concluded that expression of gene I proteins and N protein was sufficient for MHV RNA replication and transcription.