HepG2细胞中HCV NS5A蛋白对HCV IRES启动蛋白翻译的影响

The effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells

目的探讨HepG2细胞中HCV非结构蛋白5A（NS5A）对HCV IRES启动蛋白翻译的影响，以了解HCV的复制调控机制。方法将构建的表达双荧光素酶的双顺反子载体pCMV-Rluc-IRES-Fluc和含HCV NS5A基因的表达质粒pcDNA-NS5A共转染HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，细胞免疫荧光技术检测HCV-NS5A蛋白的表达，RT-PCR检测虫荧光素酶基因mRNA水平，并与相应对照做比较，以观察HCV NS5A对HCV IRES介导虫荧光素酶翻译水平的影响。结果转染pcDNA-NS5A的HepG2细胞中虫荧光素酶活性明显高于转染pCDNA3．I-3flag的对照组，并存在剂量依赖关系；而RT-PCR虫荧光素酶基因mRNA水平在两组间差异无统计学意义。转染pcDNA-NS5A的HepG2细胞质中可见HCV NS5A蛋白的表达。结论HCV NS5A蛋白对HCV IRES介导虫荧光素酶的翻译有正调节作用，并存在剂量依赖关系．

Objective To study the effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells. Methods HepG2 cells were co-transfected with a plasmid vector containing a bicistronic transcript carrying Renilla luciferase and firefly luciferase genes separated by HCV IRES sequences, and an expressing vector producing the NSSA protein. The luciferase activity and the mRNA of the luciferase gene were then detected. The NS5A expression was confirmed by fluorescence microscopy. Results HCV NSSA protein was detected in the cytoplasm of the HepG2 cells transfected with pcDNA-NSSA, and the luciferase activity was up-regulated in the presence of the HCV NS5A protein while the expression of luciferase mRNA showed no difference. Conclusion HCV NS5A protein can upregulate the HCV IRES activity and this effect is dose-dependent with NS5A.