Abstract

While the 5 * cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA tran-scription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize the cis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of the cis-acting signal for replication in the 5 * untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5*-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA tran-scription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5 * untranslated region, suggesting that the secondary structure formed by this 35-nt