首页|期刊导航|南京医科大学学报(英文版)|Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation
Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation
Xueping Wang Fujun Li Motoko Nagano-Fujii Lin Deng Kikumi Kitayama Hak Hotta
南京医科大学学报(英文版)2009，Vol.23Issue(4)：257-264,8.
南京医科大学学报(英文版)2009，Vol.23Issue(4)：257-264,8.
Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation
Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation
摘要
Abstract
Objective: To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus,and to analyze their serine protease activities. Methods: The point mutation plasmid constructs were generated by using the QuickChange site-directed mutagenesis kit with the backbone of M-H05-5 (A 1-1), and were named as subgroup A 1-2, A2-1, A2-2, B 1-1, B 1-2, B2-1,and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results: The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The imrnunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/SB△C as a substrate for NS3/4A serine protease, it was also found that the/n trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion: The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different secondary structure of the 120-aa sequence at N-terminus of NS3.关键词
hepatitis C virus/point mutation/activity, serine protease/secondary structureKey words
hepatitis C virus/point mutation/activity, serine protease/secondary structure分类
医药卫生引用本文复制引用
Xueping Wang,Fujun Li,Motoko Nagano-Fujii,Lin Deng,Kikumi Kitayama,Hak Hotta..Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation[J].南京医科大学学报(英文版),2009,23(4):257-264,8.基金项目
This work was supported by Japan China Sasakawa Medical Fellowship(2006-2007). （2006-2007）
