The Mechanisms of Hepatitis C Virus NS4A Protein on the Inhibition of Protein Synthesis and theInternal Cleavage of NS3 Protein

• Main Authors: Yi-Ming Wang, 王怡敏
• Other Authors: Shin Chang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/23492695413222383664

碩士 === 國立臺灣大學 === 微生物學研究所 === 93 === Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. Previous studies have demonstrated that the viral nonstructural protein NS4A interacts with NS3 and is a cofactor of NS3 serine protease essential for the proteolytic processing of the viral polyprotein. NS4A protein was also demonstrated to inhibit cellular and viral protein synthesis. By performing GST pull down assay, our laboratory has previously identified eEF1A that specifically interacted with NS4A. The purpose of this study was to examine whether NS4A inhibits protein translation via interacting with eEF1A. By performing luciferase assay, the inhibition of translation by NS4A protein was confirmed in this study. The eEF1A C-terminal domain, responsible for binding with eEF1B and tRNA was identified to be involved in the interaction. Furthermore, mutations at Val-23 [NS4A(V23A)] and at Ile-25 and Val-26 [NS4A(I25AV26A)] disrupted the interaction between NS4A and eEF1A. The effect of NS4A on translation inhibition was also decreased with V23 and I25AV26A mutations. In contrast, a mutation at Val-24 [NS4A(V24A)] remained the ability of NS4A to interact with eEF1A and the inhibition effect on luciferase activity. These results suggest NS4A may interfere with protein synthesis by forming complex with eEF1A. This may be involved in HCV infection and helps its survival in host cells. It was reported the NS3 protein is internally cleaved in the presence of NS4A when expressed in HepG2, COS-7, and NIH3T3 cells. The internal cleavage of NS3 protein required not only NS4A as a cofactor but also the activity of NS3 serine protease. However, in this study, we found that NS4A(I25AV26A) that retains the function to act as a cofactor of NS3 was unable to induce the internal cleavage of NS3. It suggests that the serine protease activity of NS3 is not sufficient for its internal cleavage. In addition, the internal cleavage products of NS3 appeared to have higher oncogenic potential than the intact NS3. In this study, soft agar analysis was performed to compare the transformation activity of NS3 in the presence of NS4A or NS4A(I25AV26A). Preliminary data did indicate a higher transformation activity of NS3 in the presence of wild type NS4A. Nevertheless, the numbers of transforming colonies were low and may need to be further confirmed. The possible roles of the interactions among the viral nonstructural proteins, on the oncogenesis of HCV infection remain to be elucidated.