Hsu Wei-Li, Chen Chung-Lun, Huang Shi-Wei, Wu Chia-Chen, Chen I-Hsuan, Nadar Muthukumar, Su Yin-Peng, Tsai Ching-Hsiu
Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan.
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
PLoS One. 2014 Feb 12;9(2):e88863. doi: 10.1371/journal.pone.0088863. eCollection 2014.
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.
经典猪瘟病毒（CSFV）在猪身上可引发多种疾病，从包括高烧和出血在内的急性症状，到慢性病或隐性感染，这取决于病毒株。CSFV属于黄病毒科瘟病毒属。它携带单链正链RNA基因组。5'非翻译区（UTR）中的内部核糖体进入位点（IRES）驱动一个单一开放阅读框的翻译，该阅读框编码一条3898个氨基酸长的多肽链。开放阅读框之后是一个3'UTR，其包含四个高度结构化的茎环。在本研究中，一种由CSFV基因组的5'和3'UTR组成的合成RNA，不含任何病毒编码序列，中间由一个荧光素酶基因盒隔开（命名为5'UTR-Luc-3'UTR），在转染后4小时就引发了凋亡性细胞死亡。通过DNA梯状分析、TUNEL检测、流式细胞术测定膜联蛋白-V结合以及分析半胱天冬酶激活来检测凋亡。与此形成对比的是，在用单独的5'或3'UTR RNA转染的细胞中仅观察到微量的DNA梯状条带；即使5'UTR和3'UTR作为单独的RNA分子共转染，DNA梯状条带也未达到嵌合的5'UTR-Luc-3'UTR RNA诱导的水平。有趣的是，由5'UTR和3'UTR的茎环I组成的RNA引发的凋亡比单独的5'或3'UTR要强得多。这些结果表明5'和3'UTR顺式共同作用诱导凋亡。我们进一步获得证据表明，UTR介导的凋亡需要双链RNA，并且可能通过PKR的激活涉及翻译阻断。
