丙型肝炎病毒核心蛋白抑制shRNA介导的RNA干扰作用

Hepatitis C virus core protein inhibits RNA interference mediated by shRNA

目的研究丙型肝炎病毒编码的蛋白对RNA干扰的抑制作用,筛选出有作用的RNA干扰抑制因子。方法构建丙型肝炎病毒不同蛋白质的真核表达质粒,加入萤火虫荧光素酶基因的RNA干扰体系,共转染HeLa细胞,检测萤火虫荧光素酶活性以观察RNA干扰效果的变化,筛选出有作用的RNA干扰抑制因子。在针对内源性GFP的RNA干扰体系中进一步确证该种抑制作用。利用共转染方法观察该蛋白抑制RNA干扰的剂量效应关系、在不同细胞系中的作用以及对siRNA介导的RNA干扰是否有相同的拮抗作用。结果加入核心蛋白后,萤火虫荧光素酶活性恢复到80%,与空白对照比较有显著性差异(P<0.05)。在绿色荧光蛋白RNA干扰体系中,核心蛋白使绿色荧光蛋白的表达强度明显增加。该种对抗RNA干扰的作用在HepG2、HEK293和HeLa细胞中均存在,且呈剂量依赖关系。在siRNA介导的RNA干扰体系中,加入核心蛋白后萤火虫荧光素酶活性无显著性变化(P>0.05)。结论核心蛋白能够对抗shRNA介导的RNA干扰作用,但不能对抗siRNA介导的RNA干扰作用,这种作用具有普遍性。核心蛋白对抗RNA干扰的作用可能有利于提高丙型肝炎病毒在宿主细胞内的生存能力并导致持续感染,亦可能在丙型肝炎病毒致病机制中发挥重要作用。

Objective To identify the potent RNA interference suppressor encoded by hepatitis C virus（HCV）.Methods The eukaryotic recombinant plasmids expressing various proteins encoded by HCV genes were constructed by inserting the corresponding DNA sequences into pcDNA3.1-3Flag,and the expression of the proteins in HepG2 cells were confirmed by immunofluorescence staining.The recombinant plasmids and the RNA interference system targeting firefly luciferase were co-transferred into HepG2 cells.The luciferase activity was assayed by dual luciferase assay system to identify the potent RNA interference suppressor,which was further confirmed using a RNA interference system targeting intrinsic GFP.The suppressing effect was observed in other two non-liver cell lines and the dose-dependent effect was studied.The effect of the potent suppressor on siRNA-mediated RNA interference was studied using a RNA interference system targeting firefly luciferase.Results The eukaryotic recombinant plasmids expressing various proteins encoded by HCV genes were constructed successfully.The core protein of HCV rescued both extrinsic luciferase activity and intrinsic GFP that were silenced by shRNA in HepG2 cells（P0.05）,in a dose-dependent manner.Similar results were observed in HEK293 and HeLa cells.However,HCV core protein did not rescue the luciferase activity silenced by siRNA（P0.05）.Conclusion HCV core protein can act as a potent suppressor of shRNA-mediated RNA interference,but can not suppress siRNA-mediated RNA interference.This ability may contribute to the persistent infection and pathogenesis of HCV.