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J. Virol., 04 1995, 2313-2321, Vol 69, No. 4
Copyright © 1995, American Society for Microbiology

Two murine coronavirus genes suffice for viral RNA synthesis

KH Kim and S Makino
Department of Microbiology, University of Texas at Austin 78712-1095.

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 1 proteins and N protein was sufficient for MHV RNA replication and transcription.

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