Effects of Hepatitis C Virus Core Protein on DExH/D Box RNA Helicase: NS3 and CAP-Rf

• Main Authors: Ming-Feng Lin, 林明鋒
• Other Authors: Yan-Hwa Wu Lee
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/73517204120342995109

碩士 === 國立陽明大學 === 生物化學研究所 === 89 === Over 1% of the people worldwide have been infected hepatitis C virus (HCV). Its persistent infection may result in chronic active hepatitis, cirrhosis and hepatocellular carcinoma. Several studies have implicated that HCV core protein, in addition to being a component of the viral capsid, performs functions to disrupt host’s defense and may regulate viral replication. Our previous study demonstrated that HCV core protein could interact with two DExH/D box related family proteins, viral NS3 and cellular CAP-Rf. Analyzing the effect of HCV core protein on enzymatic activities of NS3 and CAP-Rf may help us to elucidate possible roles of these interactions in the HCV life cycle and their biological significances. In this study, GST-fusion proteins of NS3 and CAP-Rf as well as their respective mutants with mutation at their DECH and DEAD box were purified and used for biochemical analysis. Results revealed that mutant type of GST-NS3 or GST-CAP-Rf fusion proteins exhibited no ATPase and helicase activities. Our results also indicated that both ATPase and helicase activities of GST-NS3 are much more robust than those of GST-CAP-Rf. Additionally, the ATPase and helicase activities of GST-CAP-Rf, but not those activities of GST-NS3, were enhanced about 2-3 fold by GST-core protein. Results also indicated that although the HCV core protein interacted with the NS3, no effect on its protease activity was found. To map the interacting region between the HCV core protein and NS3 protein, various GST-NS3 truncated proteins were purified and analyzed by in vitro GST pull down assay. Results reveal that the interaction region was located at N terminal 50 residues of HCV core protein and 38-248 amino acid region of NS3, suggesting that the interaction is within the protease domain of NS3 but not overlapped with the NS4A-interacting region. All together, our results suggest that although the HCV core protein can associate with two DExH/D box related family proteins, it imparts differential effects on their enzymatic activities. In viewing the biological role of RNA-helicase in cellular gene expression, this feature of core protein may at least partially account for its role in regulating cellular gene expression. On the other hand, although interaction between viral proteins is necessary for viral replication and viral particle assembly, the role of this NS3-core associated complex in viral life cycle remains to be established.