Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(−)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (−)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (−)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (−)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (−)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (−)-strand sgmRNA. (3) Nucleotides positioned from −15 to −34 of the sgmRNA 7 3'-terminal region are required for efficient (−)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (−1) of sgmRNA 7 is correlated to the efficiency of (−)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (−)-strand sgmRNA synthesis in BCoV.