Analysis of coronavirus transcription regulation

Insertion of an intergenic region from murine coronavirus mouse hepatitis virus (MHV) into an MHV defective interfering (DI) RNA led to transcription of subgenomic DI RNA in helper virus-infected cells. Using this system we studied how two intergenic regions positioned in close proximity affected subgenomic RNA synthesis. When two intergenic regions were separated by more than 100 nt, slightly less of the larger subgenomic DI RNA (synthesized from the upstream intergenic region) was made; this difference was significant when the intergenic region separation was less than about 35 nucleotides. Deletion of sequences flanking the two intergenic regions inserted in close proximity did not affect transcription. No significant change in the ratio of the two subgenomic DI RNAs was observed when the sequence between the two intergenic regions was altered. Removal of the downstream intergenic region restored transcription of the larger subgenomic DI RNA. These results demonstrated the downstream intergenic sequence was suppressing subgenomic DI RNA synthesis from the upstream intergenic region.