| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 90 === Hepatitis C virus (HCV) is the major etiological agent of post- transfusion- associated non- A, non- B hepatitis worldwide. An estimated 3 % of the world’s population is infected with HCV, according to the World Health Organization. HCV infection most commonly results in chronic hepatitis that eventually develops into cirrhosis and hepatocellular carcinoma. The current available therapy, using interferon- α and combination with ribavirin, has worked on less than 50 % of the patients. Therefore, there is an urgent need for new therapies.
The non-structure protein 3 (NS3) of HCV ranges from a. a. 1027 to 1657 of the poly-protein and is a well- designed and multi- functional protein. The N terminal one-third (from a. a. 1027 to 1207) of NS3 protein contains a serine protease activity, which catalyzes the cleavage between NS3/ NS4A, NS4A/ NS4B, NS4B/ NS5A and NS5A/ NS5B. The NS4A protein is its functional cofactor. The C terminal two-thirds region of NS3 protein contains an ATPase activity and an RNA helicase activity. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic procession in vivo. The thesis investigates the inter-molecular effects on RNA helicase activity between NS3 and NS4A and the intra-molecular influences between the N terminal domain and the C terminal domain of the NS3 protein.
We used baculovirus expression system to produce NS3- NS4A, full-length NS3 protein, and a truncated NS3 protein that only contains the C terminal helicase domain, and assayed their helicase activity. Our data showed that NS4A up-regulated the RNA helicase activity, RNA binding ability, and the ATPase activity. Unlike the NS4A protein, the N terminal protease domain of the HCV NS3 protein down-regulated the RNA helicase activity, but enhanced the ATPase activity. Besides, the effects of the potassium ion to the helicase activities of three proteins were different. When the concentration of potassium ion was above 75 mM, the helicase activity of the NS3- NS4A protein began to decline. The helicase activity of the NS3 protein reached maximum at 75 mM KCl. But, any the helicase activity of NS3 helicase domain was greatly inhibited by K+, even at low concentration. The preliminary results should provide valuable information to further investigate the mechanisms of intermolecular and intramolecular interactions of the HCV NS3 protein. This may provide a hope to develop new therapeutic agents for hepatitis C.
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