| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 96 === 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.
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