The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.
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.
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan ROC.
Wei-Yu
WY
Liao
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan ROC.
Ting-Yung
TY
Ke
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan ROC.
Hung-Yi
HY
Wu
D001483:Base Sequence; D017938:Coronavirus, Bovine / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral; D012367:RNA, Viral