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

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 （AI-1）, and were named as subgroup A1-2, A2-1, A2-2, BI-1, B1-2, 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 immunofluorescence 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/SBAC as a substrate for NS3/4A serine protease, it was also found that the in 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.

氨基端不同二级结构的丙型肝炎病毒NS3／4A复合体的丝氨酸酶活性差异研究

目的探讨表达不同氨基端二级结构的丙型肝炎病毒非结构蛋白3／4A（HCVNS3／4A）各亚型丝氨酸酶活性及其对宿主细胞功能抑制作用的差异。方法以pSGS／M。HOS-5／4A为模板（A1-1），构建表达不同氨基端二级结构NS3／4A的点突变质粒，并分别命名为A1-2，A2-1，A2-2，B1-1，B1-2，B2-1，B2-2。Western印迹检测所有构建质粒的表达及各亚型顺式及反式NS3蛋白酶活性的差异。荧光素酶报告试验检测pSGS／M-H05-5／4A各个二级结构亚型对干扰素-β（IFN-β）产生及p53依赖的荧光素酶基因的转录活性的抑制作用以及各亚型之间抑制作用的差异。结果Western印迹显示所有构建质粒均成功表达，而且A2-1和B2-1亚型NS3／4A存在不完全切割现象。表明与其他亚型相比，A2—1和B2—1顺式NS3丝氨酸蛋白酶活性较弱。与其底物NS5A／SBAC共表达后，A2—1和B2-1亚型未切割NSSASB明显多于其他亚型，而切割NS5A则明显少于其他亚型。说明与其他亚型相比，A2—1和B2．1亚型的反式NS3蛋白酶活性亦较弱。荧光素酶试验结果显示，所有亚型M—H05-5／4A均显著抑制IFN-β启动子活性（P〈0．01）和p53依赖的荧光素酶基因的转录活性（P〈0．01），A2-1和B2-1亚型的抑制作用显著弱于其他亚型（P〈0．05）。结论氨基端不同二级结构的HCVNS3／4A复合体具有不同的丝氨酸酶活性和宿主细胞功能抑制作用。