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.
【저자키워드】 coronavirus, subgenomic mRNA, negative-strand RNA synthesis, Cis-acting element, 【초록키워드】 complement, Deletion, leader sequence, reverse transcription, UTR, Quantitative, Critical, Mutagenesis, sgmRNA, genomic RNA, Bovine coronavirus, Efficiency, sequence, polymerase chain, addition, required, demonstrated, correlated, cis -acting element, positioned, 【제목키워드】 mRNA, synthesis, identification, element,