Skip to main content
Article thumbnail
Location of Repository

The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis

By Wei-Yu Liao, Ting-Yung Ke and Hung-Yi Wu

Abstract

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

Topics: Base Sequence, Coronavirus, Bovine, DNA Replication, DNA, Viral, Molecular Sequence Data, RNA, Viral, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Nucleic Acid
Year: 2015
DOI identifier: 10.1371/journal.pone.0098422
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/86244
Journal:
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://hdl.handle.net/11455/86... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.