Research Article: The 3′-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

Date Published: May 22, 2014

Publisher: Public Library of Science

Author(s): Wei-Yu Liao, Ting-Yung Ke, Hung-Yi Wu, Volker Thiel.

http://doi.org/10.1371/journal.pone.0098422

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.

Partial Text

After entry into a cell, the positive-strand [(+)-strand] RNA virus genome first serves as a template for synthesis of viral proteins required for subsequent virus replication. The synthesis of the negative-strand [(−)-strand] complement from the (+)-strand genome is the first step of genome replication and is assumed to be initiated from the 3′ end of the (+)-strand RNA genome. The 3′ end of the synthesized (−)-strand RNA genome is then employed as the site for the initiation of the (+)-strand RNA genome. Therefore, the RNA elements on the 3′ end of (+)- and (−)-strand RNA genome may play critical roles in targeting the viral replication complex to initiate the synthesis of their counterparts.

Many cis-acting elements required for coronavirus replication have been identified in the past two decades [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. The identification of the cis-acting elements specific for (−)-strand RNA synthesis in coronavirus, however, has been hampered due to insufficiencies in the techniques used to detect and quantitate RNA (−) strand species. In this study, we employed the method of head-to-tail ligation and qRT-PCR to examine the cis-acting elements required for (−)-strand BCoV DI RNA synthesis [26], [37]. This method has the advantages of detecting and quantitating low copy number of (−)-strand BCoV DI RNA as well as distinguishing between T7 RNA polymerase-generated (−)-strand DI RNA transcripts and coronaviral polymerase-generated (−)-strand DI RNA, and thereby allows us to systematically examine the cis-acting RNA elements required for (−)-strand DI RNA synthesis and to gain insight into the mechanism of coronavirus replication.

 

Source:

http://doi.org/10.1371/journal.pone.0098422