The synthesis of the negative-strand [(−)-strand] complement of the ∼30 kilobase, positive-strand [(+)-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis -acting elements required for (−)-strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (−)-strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantitate the synthesis of bovine coronavirus (BCoV) defective interfering (DI) RNA (−) strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis -acting RNA elements within the 3′-terminal 55 nucleotides (nts) which function in the synthesis of (−)- or (+)-strand BCoV DI RNA. The major findings are as follows: (i) nts from -5 to -39 within the 3′-terminal 55 nts are the cis -acting elements responsible for (−)-strand BCoV DI RNA synthesis, (ii) nts from −3 to −34 within the 3′-terminal 55 nts are cis -acting elements required for (+)-strand BCoV DI RNA synthesis, and (iii) the nucleotide species at the 3′-most position (−1) is important, but not critical, for both (−)- and (+)-strand BCoV DI RNA synthesis. These results demonstrate that the 3′-terminal 55 nts in BCoV DI RNA harbor cis -acting RNA elements required for both (−)- and (+)-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis -acting elements required for (−)-strand RNA synthesis in coronaviruses.
【초록키워드】 Coronaviruses, knowledge, qRT-PCR, Genome, complement, RNA, reverse transcription, Quantitative, Critical, mechanism, nucleotide, Coronavirus replication, Bovine coronavirus, RNA synthesis, Coronaviral, genome replication, element, polymerase chain, defined, responsible, identify, detect, required, cis -acting element, Northern blot, 【제목키워드】 RNA, synthesis, element, negative, Harbor,