- 學位論文
尋找C型肝炎病毒之非結構蛋白5A 在干擾素對抗病毒感染的機制上可能影響的細胞因子
Investigation of the Hepatitis C virus nonstructural protein 5A interacting cellular factors involved in interferon-α antiviral mechanism
摘要
C型肝炎病毒 (HCV)為導致慢性肝炎、肝硬化與肝癌的主要因子之一。依據世界衛生組織統計全世界人口約有3%的HCV帶原者，其中有高達80%的病人會在20到30年之後發生肝硬化，甚至最終死於肝癌的發生。在HCV感染的治療上，目前使用的標準治療為長效型甲型干擾素合併抗病毒藥物ribavirin，但是治療成效卻只有50-60%。其中HCV的基因型為影響慢性C型肝炎治療成效的主因之一，在不同的HCV基因型感染的病人中可發現，基因型2與3的病人對IFN-α的治療效果較基因型1為優，並且基因型1b病人常伴隨較嚴重的病程演變，因此HCV基因型1b是如何抵抗IFN-α的治療為相當重要的研究議題。 已知HCV的非結構蛋白中，E2、NS3/4A與NS5A在協助HCV抵抗干擾素的治療中，扮演著相當重要的角色，並且由實驗室先前的結果可知，不同功能性區域的NS5A的確在抑制干擾素作用上，亦分別扮演了不同程度的功能性抑制。因此為了解NS5A這些不同的功能性區域，在細胞內是否會結合著不同的細胞因子，影響干擾素的作用。首先我使用大腸桿菌之表現系統進行NS5A的表現與純化，再利用純化的NS5A作為取得細胞因子的誘餌，與干擾素處理後的細胞萃取物進行結合，再經由LC-MS/MS分析所結合的蛋白質，我們共鑑別出heat shock protein 70 (Hsp70), carbonyl reductase 1 (CBR1), Rab7 and elongation factor 1 alpha 1 (EF1A1)共4種蛋白。經由in vitro pull-down與in vivo免疫沈澱分析，最後確定可與NS5A結合的細胞因子，只有Hsp70。 已知Hsp70為細胞內重要伴護蛋白，並被認為可協助細胞因子p58IPK或FANCC進行PKR活性的抑制，因此我們分別利用in vitro pull-down與in vivo免疫沈澱，分析NS5A、PKR與Hsp70三者間的結合關係，並確定在細胞當中NS5A可作為連結PKR與Hsp70的橋樑。
並列摘要
Hepatitis C virus (HCV) establishes a chronic infection leading to fibrosis, cirrhosis and ultimately hepatocellular carcinoma. The World Health Organization estimates that more than 3% individuals are infected with this virus. The standard therapy for chronic HCV infection is a combination of PEG-IFN-α and ribavirin, which could eliminate HCV in 50-60% of the patients and is significantly altered by the virus genotypes. Comparing with HCV genotypes 2 and 3, genotyrpe 1 is accompanied with a higher rate of evolution to chronic hepatitis. HCV E2, NS3/4A and NS5A can antagonize IFN-α actions. According to the previous data from our laboratory, the different functional domains of NS5A may exert divergent inhibitory effects on IFN-α activity. In order to examine whether NS5A may affect IFN-α activity through interaction with some cellular factors, I used an E. coli expression system to express and purify the recombinant NS5A protein, which was then used as a bait to search for the interacting cellular factors. In my experiments, there were four candidate proteins identified, i.e., heat shock protein 70 (Hsp70), carbonyl reductase 1 (CBR1), Rab7 and elongation factor 1 alpha 1 (EF1A1). However, Hsp70 is the only candidate confirmed by in vitro pull-down and in vivo co-immunoprecipitation. Hsp70 is an important chaperone under cellular heat stress condition and it can assist the inhibitors of protein kinase R (PKR), namely the p58IPK and the Fanconi anemia (FA) complementation group C gene product (FANCC), to reduce the PKR activity, thereby protect cells from apoptosis. From in vitro pull-down and in vivo co-immunoprecipitation experiments, I demonstrated that NS5A might act as a bridge to bring PKR and Hsp70 together. Thus, we hypothesize that NS5A may imitate the function of p58IPK or FANCC to inhibit PKR activity, which, however, remains to elucidated.
參考文獻
延伸閱讀
- Peng, K. L. (2008). C型肝炎病毒核心蛋白調控組蛋白甲基化酵素活性之功能性研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2008.01600
- Lu, K. Y. (2012). 利用人類蛋白質體微陣列晶片探究C型肝炎病毒非轉譯區與宿主之交互作用 [master's thesis, National Central University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0031-1903201314445556
- 謝明儒（2011）。第一部分：以碳酸酐異構酶II、IX及XII RNA干擾技術探討對人類乳癌細胞侵襲及移動能力的影響第二部分：C型肝炎病毒套膜蛋白E2導致肝臟纖維化機制探討〔博士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2011.00115
- 嚴可芮（2013）。Hepatitis C Virus NS3 Protein Associates with Integrin beta1 and Influences Cell Adhesion and Migration〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2013.00853
- Kou, Y. H. (2007). Molecular mechanisms of NS3 and NS4A proteins involved in the pathogenesis of hepatitis C virus [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2007.02190