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

Hepatitis C virus NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell functions

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。

Objective To construct the point mutation plasmids expressing NS3/4A with different secondary structure of the amino-terminal 120 residues of NS3, and to investigate the differences in serlne protease and inhibitory effects on host cells between each subgroup. Methods The point mutation plasmids were constructed, which expressed NS3/4A with the corresponding secondary structures of subgroup, and were named as A1-2, A2-1, A2-2, BI-1, B1-2, 132-1, and 132-2, with the backbone of M-H05-5 （AI-1）. Western blot was performed to detect the expression of NS3/4A and the difference in in cis and in trans NS3 serine protease activity between each subgroup. The inhibitory effects of HCV NS3/4A with different aminoterminal secondary structures on IFN-β production and p53-dependent transcriptional activation were revealed by Luciferase reporter assay. Results Western blot revealed the successful expression of the constructs and the incomplete cleavage of NS3/4A in subgroup A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker compared with that of 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 with that of the other subgroups. Differences in inhibitory effects of HCV NS3 on IFN-β promoter activity and on p53-dependent luciferase gene transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion HCV NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell functions.