Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/49202
標題: 冠狀病毒負股RNA生合成所須之cis-acting RNA elements以及蛋白質
Identification of Cis-acting Rna Elements and Proteins Required for Minus-Strand RNA Synthesis in Coronavirus
作者: 吳弘毅
關鍵字: 生物技術, 畜牧獸醫類
基礎研究
摘要: 病毒的複製與致病機轉息息相關,對於病毒複製的研究亦是奠定抗病毒藥物及相關疫苗發展的重要基礎。對於大部分在細胞質進行複製的正股RNA 病毒而言,其cis-acting RNAelements 會和病毒以及宿主蛋白質做特異性的結合,進而形成replication complex,而此replication complex 形成的過程是啟動病毒負股RNA 生合成的首要步驟。冠狀病毒也是正股的RNA 病毒之一,因此我們假設冠狀病毒也是利用類似的機轉以進行負股基因體的形成。為了確認參與冠狀病毒負股基因體形成過程中所須之cis-acting RNA elements,以及與cis-acting RNA elementsts 產生特異性結合的宿主與病毒蛋白質,本研究計畫擬採取的實驗步驟為(i) 利用mutation analyses 以及 real-time RT-PCR 確認正股基因體的cis-actingelements 與負股基因體的生合成有關。(ii) 利用electrophoretic mobility shift assay (簡稱EMSA)及UV crosslinking assay 分析可能與cis-acting RNA elements 形成 replication complex之蛋白質。我們目前的結果顯示(i)冠狀病毒正股基因體3’UTR 的cis-acting RNA elementpseudoknot 會影響負股基因體的形成。(ii) 冠狀病毒正股基因體5’UTR 的cis-acting RNAelement stem-loop III 能與宿主以及病毒的蛋白質結合。我們希望藉由本計畫之執行,能夠系統性的釐清個別cis-acting RNA element 在負股基因體形成的重要性以及確認參與形成replication complex 的蛋白質,以了解冠狀病毒的複製機轉。
To understand the mechanism of virus replication is not only important to viral pathogenesis,but also the critical basis of the development of vaccines and the design of antiviral drugs. For mostof plus-strand RNA viruses, their replication processes occur entirely in cytoplasm. The first step ofthe processes is to initiate synthesis of minus-strand RNA genome through replication complexformed by specific binding between viral cis-acting RNA elements and cellular and viral proteins.Coronavirus is one of plus-strand RNA viruses and we, therefore, hypothesize that coronavirusutilizes the similar mechanism to synthesize minus-strand RNA genome. The proposal focuses ontwo specific aims: (1) to determine the coronavirus cis-acting RNA elements required for synthesisof minus-strand RNA genome, and (2) to identify cellular and viral proteins involved in the bindingof the cis-acting RNA elements. The experimental approaches will primarily involve (1) mutationanalyses, (2) real-time RT-PCR, (3) electrophoretic mobility shift assay (EMSA), and (4) UVcrosslinking assay. Our preliminary data suggest that (1) the cis-acting RNA element pseudoknot inthe 3'-untranslated region is required for synthesis of minus-strand RNA and (2) cellular and viralproteins are able to bind cis-acting RNA element stem-loop III in the 5'-untranslated region.Accordingly, we expect to systematically identify the individual cis-acting RNA element importantfor synthesis of minus-strand RNA and proteins bound to cis-acting RNA elements.
URI: http://hdl.handle.net/11455/49202
其他識別: NSC99-2313-B005-024
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=2158367&plan_no=NSC99-2313-B005-024&plan_year=99&projkey=PD9909-0042&target=plan&highStr=*&check=0&pnchDesc=%E5%86%A0%E7%8B%80%E7%97%85%E6%AF%92%E8%B2%A0%E8%82%A1RNA%E7%94%9F%E5%90%88%E6%88%90%E6%89%80%E9%A0%88%E4%B9%8Bcis-acting+RNA+elements%E4%BB%A5%E5%8F%8A%E8%9B%8B%E7%99%BD%E8%B3%AA
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