J. Virol., Sep 1996, 5769-5776, Vol 70, No. 9
Copyright © 1996, American Society for Microbiology

Replication of murine coronavirus defective interfering RNA from negative-strand transcripts

M Joo, S Banerjee and S Makino
Department of Microbiology, University of Texas at Austin 78712-1095, USA.

The positive-strand defective interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV), when introduced into MHV- infected cells, results in DI RNA replication and accumulation. We studied whether the introduction of negative-strand transcripts of MHV DI RNA would also result in replication. At a location downstream of the T7 promoter and upstream of the human hepatitis delta virus ribozyme domain, we inserted a complete cDNA clone of MHV DI RNA in reverse orientation; in vitro-synthesized RNA from this plasmid yielded a negative-strand RNA copy of the MHV DI RNA. When the negative-strand transcripts of the DI RNA were expressed in MHV-infected cells by a vaccinia virus T7 expression system, positive-strand DI RNAs accumulated in the plasmid-transfected cells. DI RNA replication depended on the expression of T7 polymerase and on the presence of the T7 promoter. Transfection of in vitro-synthesized negative-strand transcripts into MHV-infected cells and serial passage of virus samples from RNA-transfected cells also resulted in accumulation of the DI RNA. Positive-strand DI RNA transcripts were undetectable in sample preparations of the in vitro-synthesized negative-strand DI RNA transcripts, and DI RNA did not accumulate after cotransfection of a small amount of positive-strand DI RNA and truncated-replication- disabled negative-strand transcripts; clearly, the DI RNA replicated from the transfected negative-strand transcripts and not from minute amounts of positive-strand DI RNAs that might be envisioned as artifacts of T7 transcription. Sequence analysis of positive-strand DI RNAs in the cells transfected with negative-strand transcripts showed that DI RNAs maintained the DI-specific unique sequences introduced within the leader sequence. These data indicated that positive-strand DI RNA synthesis occurred from introduced negative-strand transcripts in the MHV-infected cells; this demonstration, using MHV, of DI RNA replication from transfected negative-strand DI RNA transcripts is the first such demonstration among all positive-stranded RNA viruses.

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