Enhanced accumulation of coronavirus defective interfering RNA from expressed negative-strand transcripts by coexpressed positive-strand RNA transcripts.
Expression of negative-strand murine coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNA transcripts in MHV-infected cells results in the accumulation of positive-strand DI RNAs (M. Joo et al., 1996, J. Virol. 70, 5769-5776). However, the expressed negative-strand DI RNA transcripts are poor templates for positive-strand DI RNA synthesis. The present study demonstrated that DI RNA accumulation from the expressed negative-strand DI RNA transcripts in MHV-infected cells was enhanced by the coexpression of complementary RNA transcripts that correspond to the 5' region of positive-strand DI RNA. The positive-strand RNA transcripts corresponding to the 5' end-most 0.7-2.0 kb DI RNA had a similar enhancement effect. The coexpressed positive-strand RNA transcripts lacking the leader sequence or those containing only the leader sequence failed to demonstrate this enhancement effect, demonstrating that the presence of the leader sequence in the coexpressed positive-strand RNA transcripts was necessary, but not sufficient, for the enhancement of DI RNA accumulation from the coexpressed negative-strand DI RNA transcripts. Negative-strand DI RNA transcripts that were coexpressed with the partial-length positive-strand RNA transcripts were no more stable than those expressed alone, suggesting that a higher stability of the expressed negative-strand RNA transcripts was an unlikely reason for the higher DI RNA accumulation in cells coexpressing two complementary DI RNA transcripts. Sequence analyses unexpectedly demonstrated that the leader sequence of the majority of accumulated DI RNAs switched to helper virus derived leader sequence, suggesting that enhancement of DI RNA accumulation was mediated by the efficient utilization of helper virus derived leader sequence for DI RNA synthesis. Furthermore, our data suggested that this leader switching, a type of homologous RNA-RNA recombination, occurred during positive-strand DI RNA synthesis and that MHV positive-strand RNA synthesis mechanism may have a preference toward recognizing double-stranded RNA structures over single-stranded negative-strand RNA to produce positive-strand DI RNAs.
Banerjee
S
S
Department of Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712, USA.
Repass
J F
JF
Makino
S
S
D020121:5' Untranslated Regions / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D017934:Coronavirus / Q000235:genetics*; D017931:DNA Primers; D003673:Defective Viruses / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D012367:RNA, Viral / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral