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標題: 豬瘟病毒核糖核酸3''端非轉譯區參與轉譯機制之研究
The study of the 3'' untranslated region of Classical swine fever virus RNA is involved in translational regulation
作者: 黃士瑋
Huang, Shih-Wei
關鍵字: Classical swine fever virus;豬瘟病毒;translational regulation;轉譯
出版社: 生物科技學研究所
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豬瘟(Classical swine fever)或稱豬霍亂(Hog chorea),是由豬瘟病毒(Classical swine fever virus, CSFV)所引起一種豬隻急性或超急性發熱性的傳染性疾病,全身性嚴重地出血為主要病徵,而感染率與死亡率均高達95%,對於養豬業造成嚴重經濟損失。豬瘟病毒屬於黃病毒科(Flaviviridae)之正股RNA病毒,基因體5''端不含cap結構而3''端亦不含poly-A tail,而兩端各有一段非轉譯區(untranslated region, UTR),5'' UTR內含有一段內部核醣體進入區(internal ribosomal entry sites, IRES)的區域,作為病毒轉譯出蛋白的重要起始位置,此區域與C型肝炎病毒(Hepatitis C virus, HCV)、牛病毒性下痢病毒(Bovine viral diarrhea virus, BVDV)皆具有相似結構與功能。豬瘟病毒之3''端非轉譯區(3'' UTR)由四個莖環(stem-loops),分別命名為SLI、SLII、SLIII和SLIV。對於正股RNA病毒而言,其3''端非轉譯區通常參與病毒轉譯、複製以及兩者間之調控。然而3'' UTR是否參與並如何調節病毒蛋白表現仍所知有限,我們利用一段RNA,其兩端分別具有豬瘟病毒的5'' UTR與3'' UTR,中間區段以螢火蟲冷光基因作為報導基因,轉染至豬腎臟細胞中來探討3'' UTR是否能夠調節病毒轉譯,我們發現豬瘟病毒3''端非轉譯區對於轉譯具有正向與負向調控之能力。其中SLI對於轉譯調控扮演了抑制之角色。SLI中負向調控轉譯之區域位於最末端CGGCCC序列。接著我們藉由豬瘟病毒3''端非轉譯區置換成C型肝炎病毒3''端非轉譯區或poly(A)皆提高了轉譯活性,而加入CGGCCCC序列於C型肝炎病毒3''端非轉譯區或poly(A)之3''端,同樣抑制蛋白表現。經由序列比對分析結果,我們發現此六個核苷酸序列可能與IRES中IIId1配對鍵結,而IIId1為起始轉譯中40S核醣體次單元結合之區域。因此,我們認為位於3''末端之序列確實參與豬瘟病毒轉譯調控。此外,單獨的SLIV雖然對於轉譯作用不大,但可能與其他莖環或宿主蛋白結合而促進轉譯,單獨的SLII和SLIII對於轉譯具有正向調控之能力,但一同存在而無SLII和SLIII之情況下,對於轉譯並無加成之效果。正股RNA病毒之基因體可作為轉譯與複製負股的模板,但其方向相反,無法同時發生於同一條正股RNA,而與真核細胞內的mRNA相較之下,病毒基因體可表現之蛋白有限,病毒如何精準地調控蛋白質合成,以正股作為模板進行複製,宿主細胞蛋白亦可能參與其中。我們希望藉由研究中初步之結果,可更清楚了解病毒轉譯調控之機制。

The 3'' untranslated region (UTR) usually harbors signals for translation, replication and the switch of the translation and replication for a positive-sense RNA virus. In particular, viruses are entirely reliant on the translation apparatus of their host cells to synthesize the polypeptides that are necessary in the viral life cycle. Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, is the causative agent of classical swine fever (CSF). Viral protein synthesis of CSFV is initiated in a cap-independent manner by a highly conserved structure known as internal ribosome entry site (IRES), located in the 5'' UTR. The structure and function of CSFV IRES are similar to that of the well-characterized Hepatitis C virus (HCV), comprising two main structural domains marked as II and III. The 3'' UTR of CSFV is comprised of four stem-loops designated as SLI, SLII, SLIII, and SLIV. To understand whether the 3'' UTR is involved in internal ribosome entry site (IRES)-mediated translation in CSFV, we performed a reporter assay in which the luciferase expression is under the control of CSFV 5'' and 3'' UTRs. We found that the 3'' UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3'' -end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3'' -ends such as the poly(A) or the 3'' UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3'' UTR. After sequence comparison and alignment, we propose that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40S ribosomal subunit binding site for the translational initiation, located at the 5'' UTR. In conclusion, our data support that the 3'' -end terminal sequence can play a role in regulating the translation of CSFV. Furthermore, we have found SLs could regulate translation cooperately. Since the template for translation and replication of a positive-sense RNA virus is the same, the switch between these two processes should be in a tight regulation. In this research, we contribute to a small piece in the big jigsaw puzzle, to extend understanding of how virus precisely regulates translation.
其他識別: U0005-3105201216034300
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