- 學位論文
中東型呼吸道症候群冠狀病毒RNA包裹訊號
RNA Packaging Signal of Middle East Respiratory Syndrome Coronavirus
摘要
中東型呼吸道症候群 (Middle East Respiratory Syndrome)首發病例於2012年9月發現，之後經分析確定為新種冠狀病毒感染所引起之致命呼吸道疾病，並命名為中東型呼吸道症候群冠狀病毒 (Middle East Respiratory Syndrome Coronavirus，MERS-CoV)。冠狀病毒為帶有正股RNA基因體及套膜的病毒，其基因體約26-32 kb，可表現病毒複製時所需的非結構性蛋白質及結構性蛋白質，其中結構蛋白質N被認為是與其基因體結合的主要蛋白質。實驗室先前於SARS-CoV的研究中，發現SARS-CoV的RNA包裹訊號位於其基因體第19888-19950個核苷酸之間，且SARS-CoV可透過N蛋白質與此長63個核苷酸的RNA包裹訊號結合，進而被包裹至類病毒顆粒內。本篇論文的目的為探討MERS-CoV的RNA包裹訊號及MERS-CoV N蛋白質對於包裹RNA genome的重要性。將表現MERS-CoV四種結構蛋白質的質體轉染入Huh7細胞株中，轉染三天後收集其細胞及細胞培養液，以超高速離心的方式沉澱，建立類病毒顆粒的產出和純化系統。同時，透過基因體之序列及二級結構分析，推測MERS-CoV RNA包裹訊號位於基因體19712-19969的258個核苷酸片段。進一步收集帶有此258個核苷酸片段及EGFP序列RNA (EGFP-PS)的類病毒顆粒，感染帶有MERS-CoV receptor DPPIV的Huh7細胞株，證實此258個核苷酸片段確實帶有MERS-CoV RNA包裹訊號。當將表現EGFP-PS RNA 及MERS-CoV M、E及S蛋白質之質體同時轉染入Huh7細胞中時，發現不帶有N蛋白質的類病毒顆粒，在感染Huh7細胞株時無法偵測到EGFP蛋白質的表現，顯示MERS-CoV的RNA包裹需有N蛋白質的參與。未來可進一步探討具有RNA包裹功能的最小基因體RNA片段及重要的結構與序列，以及N蛋白質與RNA包裹訊號結合的區域。
並列摘要
The first case of Middle East respiratory syndrome was found in September 2012, Saudi Arabia. Laboratory testing identified this case as a novel coronavirus infection. The coronavirus is now later named Middle East respiratory syndrome coronavirus (MERS-CoV). Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with genomic RNA about 26-32 kb in length. The viral particle consists of four structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N). N protein is considered to be the most important protein that interacts with the viral genome. Our laboratory has previously demonstrated that SARS-CoV RNA packaging signal is located in the RNA fragment of SARS-CoV genome from nt 19888-19950. The RNA fragment can be assembled into virus-like particles (VLPs) in a N protein-dependent manner. The purpose of this study is to determine the RNA packaging signal of MERS-CoV and the involvement of N protein in the package of viral genome. To establish a system for producing MERS-VLPs, plasmids encoding the four structural proteins of MERS-CoV were co-transfected into Huh7 cells. Three days post-transfection, VLPs were collected and purified from the transfected cells and culture medium following ultracentrifugation. Meanwhile, the sequence and secondary structure of MERS-CoV genome were analyzed for prediction of MERS-CoV RNA packaging signal. VLPs carrying RNA sequence of EGFP fused to the putative MERS-CoV RNA packaging signal spanning nt 19712-19969 of the viral genome were used to incubate with MERS-CoV-permissive Huh7 cells. The results demonstrated that the 258-bp RNA sequence is sufficient to function as a packaging signal and be packaged into the MERS-VLPs. In addition, MERS-CoV RNA was packaged into the VLPs in a N protein-dependent manner. The RNA-interacting domain of the viral N protein and the minimal RNA sequences and structures critical for the viral genome package need to be future determined.
參考文獻
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