丙型肝炎病毒核心蛋白对双链RNA依赖蛋白激酶活性的影响

Effects of hepatitis C virus core protein on activity of double-stranded RNA-dependent protein kinase

目的探讨丙型肝炎病毒（HCV）核心蛋白对双链RNA依赖的蛋白激酶（PKR）活性的影响。方法将表达HCV核心蛋白的真核表达质粒pCMH6K—Core转染人肝癌细胞系BEL-7402，应用干扰素（IFN）。α-2b诱导内源性PKR的表达及活化，利用Western blot检测核心蛋白对PKR磷酸化的影响；将荧光素酶质粒pGL3-Promoter与不同剂量的pCMH6K—Core共转染BEL-7402细胞，进行荧光素酶活性检测，以反映核心蛋白对细胞蛋白合成的影响。计量资料以均数±标准差（x±s）表示，组间均数比较用t检验或单因素方差分析。结果在IFNα-2b刺激下，表达HCV核心蛋白的BEL-7402细胞中，PKR的磷酸化水平明显低于空白质粒转染组和未转染组，而3组总PKR的表达水平无明显差别。在共转染荧光素酶质粒与核心蛋白表达质粒的BEL-7402细胞中，荧光素酶活性较共转染空白质粒组增强，且荧光素酶潘性的增高与核心蛋白的表达量呈剂量依赖效应，0．5μg、1．0μg和1．5μg的pCMH6K-Core转染组荧光素酶活性分别为空白质粒组的（1．941士0．199）倍、（2．868±0．275）倍和（3．839±0．338）倍，各组比较，P值均＜0．05。结论在人肝癌细胞系BEL-7402中，HCV核心蛋白能够抑制内源性PKR的活性，从而促进细胞蛋白合成。

Objective To explore the effects of hepatitis C virus （HCV） core protein on the activity of double-stranded RNA-dependent protein kinase （PKR）. Methods The human hepatoma cell line BEL- 7402 was transfected with the HCV core gene-containing eukaryotic expression vector pCMH6K-Core （at various concentrations）, or empty vector, or no vector; a group of cells was cotransfected with the luciferase reporter plasmid pGL3-promoter. The cells were treated with interferon （IFN） α-2b to induce the expression and activation of endogenous PKR, or left untreated to serve as controls. The effect of core protein on PKR phosphorylation was detected by western blotting. Lucfferase activity was detected to reflect effects of the core protein on the synthesis of cellular proteins. The t-test and F test were used for statistical analyses. Results In the case of 1FNα stimulation, PKR phosphorylation levels were significantly lower in the HCV core protein expressing cells than in the cells transfected with empty plasmid or with no vector, but the total PKR expression level was not significantly different among these three groups of cells. Cells co-transfected with lueiferase plasmid and the core protein expressing vector showed significantly higher levels of luciferase expression than the cells co-transfeeted with the empty vector. Moreover, the luciferase activity and core protein expression levels increased in a dose-dependent manner, with the luciferase activity of the cells treated with 0.5μg, 1.0 μg and 1.5μg pCMH6K-Core being 1.941±0.199 times, 2.868±0.275 times and 3.839±0.338 times higher than that of the empty vector group （all P 〈0.05）. Conclusion In the human hepatoma cell line BEL-7402, the HCV core Drotein can hthibit the activity of endogenous PKR, thereby promoting cell protein synthesis.