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 hamp...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4031142?pdf=render |
| _version_ | 1828337659407237120 |
|---|---|
| author | Wei-Yu Liao Ting-Yung Ke Hung-Yi Wu |
| author_facet | Wei-Yu Liao Ting-Yung Ke Hung-Yi Wu |
| author_sort | Wei-Yu Liao |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T22:18:03Z |
| format | Article |
| id | doaj.art-1fc6cfe2f000430a91455e5fe83e0dfc |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T22:18:03Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-1fc6cfe2f000430a91455e5fe83e0dfc2022-12-22T02:27:23ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0195e9842210.1371/journal.pone.0098422The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.Wei-Yu LiaoTing-Yung KeHung-Yi WuThe 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.http://europepmc.org/articles/PMC4031142?pdf=render |
| spellingShingle | Wei-Yu Liao Ting-Yung Ke Hung-Yi Wu The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis. PLoS ONE |
| title | The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis. |
| title_full | The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis. |
| title_fullStr | The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis. |
| title_full_unstemmed | The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis. |
| title_short | The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis. |
| title_sort | 3 terminal 55 nucleotides of bovine coronavirus defective interfering rna harbor cis acting elements required for both negative and positive strand rna synthesis |
| url | http://europepmc.org/articles/PMC4031142?pdf=render |
| work_keys_str_mv | AT weiyuliao the3terminal55nucleotidesofbovinecoronavirusdefectiveinterferingrnaharborcisactingelementsrequiredforbothnegativeandpositivestrandrnasynthesis AT tingyungke the3terminal55nucleotidesofbovinecoronavirusdefectiveinterferingrnaharborcisactingelementsrequiredforbothnegativeandpositivestrandrnasynthesis AT hungyiwu the3terminal55nucleotidesofbovinecoronavirusdefectiveinterferingrnaharborcisactingelementsrequiredforbothnegativeandpositivestrandrnasynthesis AT weiyuliao 3terminal55nucleotidesofbovinecoronavirusdefectiveinterferingrnaharborcisactingelementsrequiredforbothnegativeandpositivestrandrnasynthesis AT tingyungke 3terminal55nucleotidesofbovinecoronavirusdefectiveinterferingrnaharborcisactingelementsrequiredforbothnegativeandpositivestrandrnasynthesis AT hungyiwu 3terminal55nucleotidesofbovinecoronavirusdefectiveinterferingrnaharborcisactingelementsrequiredforbothnegativeandpositivestrandrnasynthesis |