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

Abstract

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。

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.