title: The untranslated regions of classic swine fever virus RNA trigger apoptosis.
abstract: Classical swine fever virus (CSFV) causes a broad range of disease in pigs, from acute symptoms including high fever and hemorrhages, to chronic disease or unapparent infection, depending on the virus strain. CSFV belongs to the genus Pestivirus of the family Flaviviridae. It carries a single-stranded positive-sense RNA genome. An internal ribosomal entry site (IRES) in the 5' untranslated region (UTR) drives the translation of a single open reading frame encoding a 3898 amino acid long polypeptide chain. The open reading frame is followed by a 3' UTR comprising four highly structured stem-loops. In the present study, a synthetic RNA composed of the 5' and 3' UTRs of the CSFV genome devoid of any viral coding sequence and separated by a luciferase gene cassette (designated 5'UTR-Luc-3'UTR) triggered apoptotic cell death as early as 4 h post-transfection. The apoptosis was measured by DNA laddering analysis, TUNEL assay, annexin-V binding determined by flow cytometry, and by analysis of caspase activation. Contrasting with this, only trace DNA laddering was observed in cells transfected with the individual 5' or 3' UTR RNA; even when the 5' UTR and 3' UTR were co-transfected as separate RNA molecules, DNA laddering did not reach the level induced by the chimeric 5'UTR-Luc-3'UTR RNA. Interestingly, RNA composed of the 5'UTR and of stem-loop I of the 3'UTR triggered much stronger apoptosis than the 5' or 3'UTR alone. These results indicate that the 5' and 3' UTRs act together in cis induce apoptosis. We furthered obtained evidence that the UTR-mediated apoptosis required double-stranded RNA and involved translation shutoff possibly through activation of PKR.
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan.
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Hsu
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Chung-Lun
authors.initials: CL
authors.lastname: Chen
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Shi-Wei
authors.initials: SW
authors.lastname: Huang
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan.
authors.forename: Chia-Chen
authors.initials: CC
authors.lastname: Wu
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
authors.forename: I-Hsuan
authors.initials: IH
authors.lastname: Chen
authors.affiliation: Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya University, Coimbatore, Tamil Nadu, India.
authors.forename: Muthukumar
authors.initials: M
authors.lastname: Nadar
authors.affiliation: Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan.
authors.forename: Yin-Peng
authors.initials: YP
authors.lastname: Su
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Ching-Hsiu
authors.initials: CH
authors.lastname: Tsai
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000235:genetics*; D004285:Dogs; D015967:Gene Expression Regulation, Viral; D006801:Humans; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D017304:Annexin A5; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D020169:Caspases