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標題: SARS冠狀病毒3'非轉譯區結構分析與細胞宿主蛋白結合之探討
Structural Analysis of the 3' UTR of SARS Coronavirus RNA and Identification of the Interacted Cellular Factors
作者: 黃士瑋
Huang, Shi-Wei
關鍵字: Severe Acute Respiratory Syndrome;急性嚴重呼吸道症候群
出版社: 生物科技學研究所
急性嚴重呼吸道症候群(Severe Acute Respiratory Syndrome,SARS)是經由一種新的冠狀病毒(coronavirus)感染所引起的,被稱為SARS冠狀病毒(SARS-associated coronavirus,SARS-CoV)。SARS冠狀病毒在病毒上的分類屬於冠狀病毒科(Coronaviridae)、冠狀病毒屬(Coronavirus),在病毒顆粒的外層具有套膜(envelope),內部的基因組則為單一正股的核糖核酸(positive-sense single strand RNA),5'端具有蓋帽結構(cap),3'端具有poly-A tail,其基因體長度約有3萬個鹼基,在所有核糖核酸病毒(RNA virus)中是基因體最大的一屬。經由前人研究顯示在老鼠肝炎病毒(mouse hepatitis virus, MHV;Hsue and Masters, 1997)跟牛冠狀病毒(bovine coronavirus, BCoV;Willams et al., 1999)中,在病毒基因組3'端非轉譯區(3' untranslated region,3' UTR)中的上游區,會形成兩個stem-loop的結構(SL-I和SL-II),但SL-I形成stem的部分可以分開而與另一個SL-II中的loop產生新的鍵結,也就是所謂的偽結(psudoknot),並且這些結構已被證實對於在病毒複製的過程當中扮演重要的角色。經由電腦程式模擬,在SARS-CoV中可能具有相似的結構並且也有機會形成偽結(Goebel et al., 2004)。進一步利用Enzymatic Structure Mapping來佐證電腦程式所預測3'端非轉譯區的結構發現,在SARS-CoV 3'端非轉譯區的上游部分,主要由兩個stem-loop所組成,但部分結構並不穩定,其中SL-I利用電腦預測可能形成雙股的部分容易打開,這段區域亦與可能形成偽結的區域相符合;以具有放射性源的SL-I跟SL-II當作探針,與非洲綠猴的腎臟細胞蛋白抽出物進行UV cross-linking,初步證實的確有細胞宿主蛋白的能和SL-I及SL-II作結合,而這些蛋白是否在病毒生活使中扮演重要的角色,則有待更進一步去探討。

Severe Acute Respiratory Syndrome (SARS) in humans is caused by a novel virus, named SARS-associated coronavirus or SARS-CoV, which is a single stranded positive sense RNA virus about 30 kb long, belonging to the family Coronaviridae. The genomic RNA consists of several ORFs which encode the respective proteins through subgenomic RNAs which are flanked by a capped 5' UTR and a polyadenylated 3' UTR on their 5' and 3' ends respectively. Secondary and tertiary structures in the 3' untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. The 3' UTR of the genomic RNA of coronaviruses as previously reported, folds into a secondary structure comprising 3 stem loops (SL-I, SL-II, and SL-III). Previous reports (Willams et al, 1999; Hsue and Masters, 1997) on other coronaviruses show that the elements present in SL-I and SL-II are essential for virus replication. We confirmed the presence of these elements contained in SL-I and SL-II in SARS-CoV using mfold computer prediction program followed by Enzymatic Structure Probing. From the results of enzymatic structure mapping we also observed a potential pseudoknot formation between the unstable bases of the stem of SL-I and the loop of SL-II as predicted by phylogenetic analysis using the structure mapping data of other coronaviruses. UV cross-linking analysis was performed using SARS-CoV 3' UTR as a radio-probe to find out binding of potential cellular factors from VERO E6 cell extract and several proteins did crosslink to the probe RNA.
Appears in Collections:生物科技學研究所

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