急性呼吸道症候群冠狀病毒核殼蛋白質 與細胞因子Daxx之專一性結合作用

Specific interaction between SARS-CoV nucleocapsid protein and cellular factor Daxx

鄭又華(Yiu-Hua Cheng)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02243

摘要

在2002年中國南方廣東省，爆發了一種非典型肺炎疾病Severe Acute Respiratory Syndrome (SARS)，並且在2003年迅速擴散到世界各地。受到SARS感染的病人會出現以下症狀，包括: 發燒、呼吸困難、淋巴球數目減少、下呼吸道感染、腹瀉等，並且造成約10%的致死率。經由科學研究者的努力在SARS病患及和臨床檢體一起培養的非洲綠猴腎臟細胞Vero E6 中，成功分離出一株新型冠狀病毒，命名為SARS-CoV，同時利用猴子模式證實此為引起SARS的元兇。SARS-CoV為一含有套膜之病毒，基因體為一單股正向的RNA，長約30,000個核苷酸。病毒顆粒由四種結構性蛋白質: spike(S)、membrane(M)、envelope(E)，及nucleocapsid(N) 所組成。
SARS-CoV N蛋白質會與病毒RNA纏繞，負責病毒顆粒的包裹及釋放，並且也會自己形成雙體結構。除此之外在血清缺乏的哺乳類動物細胞中也發現N 蛋白質會誘發細胞凋亡並促使肌動蛋白重新組裝。在本篇論文中首先利用高通量酵母菌功能性基因模組由200個跟細胞凋亡相關之蛋白質中篩選出一個細胞因子Daxx (Death-associated protein)會與N蛋白質產生交互作用。後續更利用酵母菌二次雜交系統分析發現N蛋白質是與Daxx區域第570-740個胺基酸處結合。Daxx的羧端區域會與許多蛋白質產生交互作用包括: SUMO、Fas、PML及ETS-1等。推測N蛋白質是否會受到SUMO的調控修飾。為了證實這個假設，將可以表現N蛋白質之質體及三種可以表現不同SUMO蛋白質之質體: SUMO-1、SUMO-2及SUMO-3共轉染入293T細胞。由西方墨點法實驗發現N蛋白質可能主要受到SUMO-1及SUMO-2蛋白質的轉譯後修飾。除此之外，也由活體外實驗推測在N蛋白質N-terminal的第1-235個胺基酸處存在兩個SUMO-1的 sumoylation sites。未來可以進一步去研究受到SUMO修飾的N蛋白質在病毒生活史及致病過程中可能扮演的角色。本論文研究找出一個新的宿主蛋白質Daxx會與病毒結構蛋白質N產生交互作用，對於後續研究病毒蛋白質的功能具有貢獻。

關鍵字

急性呼吸道症候群 ； 核殼蛋白質

並列摘要

In November 2002, an atypical pneumonia, Severe Acute Respiratory Syndrome (SARS), emerged in Guangdong Province, Southern China, and spread worldwide in 2003. SARS infection exhibits a wide clinical course, mainly characterized by fever, dyspnea, lymphopenia, lower tract respiratory infection, and diarrhea, and causes death in approximately 10% of cases. A novel coronavirus termed SARS-CoV was isolated from SARS patients and Vero E6 cells inoculated with clinical specimens, and identified to be the causative agent of SARS in monkey model experiment. SARS-CoV is an enveloped, positive-sense single-stranded RNA virus with about 30,000 nucleotides in length. The virus particle consists of four structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N).
The SARS-CoV N protein possesses RNA-binding activity and is capable of undergoing self-dimerization. The N protein was also demonstrated to induce apoptosis and actin reorganization in mammalian cells under stress conditions. In this study, Daxx (Death-associated protein) was identified to be an N-interacting protein by performing functional yeast array analysis. Yeast two hybrid system further demonstrated that the N protein specifically bound to the C-terminal domain of Daxx protein from 570 to 740 amino acid residues. The C-terminal region is also involved in binding of Daxx to many proteins such as, SUMO, Fas, PML, and Ets-1. It is possible that the SARS-CoV N protein can be modified by SUMO proteins. To test this hypothesis, 293T cells were cotransfected with plasmids encoding the N protein and various SUMO proteins including SUMO-1, SUMO-2, and SUMO-3. Western blot analysis indicated that the N protein may be preferentially modified by SUMO-1 and SUMO-2 proteins. In addition, in vitro sumoylation assay identified two sumoylation sites in the N-terminal 235 amino acid residues of the N protein. Functional significances of the SUMO-modified N protein involved in the life cycle and pathogenesis of SARS-CoV need to be further elucidated. This study suggests a new link between host cell machinery and a SARS-CoV structural component, and will help us to understand the role of N protein .

並列關鍵字

SARS ； nucleocapsid ； Daxx

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