Replication of murine coronavirus defective interfering RNA from negative-strand transcripts.
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.
Joo
M
M
Department of Microbiology, University of Texas at Austin 78712-1095, USA.
Banerjee
S
S
Makino
S
S
D000818:Animals; D016477:Artifacts; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003673:Defective Viruses / Q000378:metabolism*; D005822:Genetic Vectors; D003698:Hepatitis Delta Virus / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; D012367:RNA, Viral