丙型肝炎病毒核心蛋白对Dicer切割功能的影响

Hepatitis C virus core protein interacts with Dicer to inhibit processing of dsRNA

目的探讨核心蛋白与Dicer的相互作用及其对Dicer功能的影响。方法构建丙型肝炎病毒核心蛋白真核表达质粒,采用免疫荧光染色和Western blot检测核心蛋白的表达,Western blot检测核心蛋白对细胞内Dicer表达的影响,免疫共沉淀技术检测真核细胞内核心蛋白与Dicer的相互作用。体外转录合成dsRNA,检测核心蛋白对Dicer切割dsRNA作用的影响。结果在细胞内,核心蛋白能够明显恢复被shRNA抑制的虫荧光素酶基因的表达(P<0.05),而对siRNA引发的RNAi无拮抗作用。在真核细胞内,加入核心蛋白后,Dicer的表达量无明显改变,并能检测到两者相互结合。在体外抑制实验中,重组表达的Dicer能切割dsRNA产生siRNA;核心蛋白加入后,dsRNA的切割受到抑制。结论核心蛋白在真核细胞内可以结合Dicer,并通过抑制Dicer对dsRNA的切割作用,抑制RNA干扰作用。

Objective To investigate the interaction between hepatitis C virus （HCV） core protein and Dicer enzyme and determine the effect of the core protein on Dicer. Methods An eukaryotie recombinant plasmid encoding HCV core protein was constructed, and the expression of the protein in the HepG2 cells after transfection was confirmed by immunofluorescence staining and Western blot analysis. The interaction between HCV core protein and Dicer enzyme in the HepG2 cells was detected by co-immunoprecipitation. After dsRNA was synthesized in vitro, inhibitory test was used to determine the effect of core protein on the dicing function of recombinant Dicer enzyme to dsRNA. Results HCV core protein significantly rescued the expression of extrin- sic luciferase gene that was silenced by shRNA compared with control （ P 〈 0. 05 ）, but had no antagonistic effect on siRNA-induced RNAi in vitro. In HepG2 cells, core protein had no significant effect on the expression of Dicer, and a combination of them was also detected. In vitro inhibitory test demonstrated that recombinant Dicer sufficiently cut dsRNA into siRNAs. However, the addition of core protein inhibited the cutting of dsRNA and the production of siRNAs. Conclusion Core protein combines with Dicer, and inhibits the dicing function of Dicer for dsRNA to suppress RNA interference.