丙型肝炎病毒非结构蛋白5A对干扰素α-2b诱导的信号传导和转录激活因子1磷酸化及核转移的影响

Effect of HCV NSSA on STAT1 phosphorylation and nuclear translocation induced by IFNα-2b

目的探讨丙型肝炎病毒(HCV)非结构蛋白5A(NSSA)对干扰素α-2b诱导的Janus激酶-信号传导和转录激活子(JAK-STAT)信号传导途径中STAT1磷酸化及核转移的影响。方法用表达HCV NS5A的质粒(pCNS5A)转染Huh7细胞,应用免疫细胞化学技术检测HCV NS5A的表达,用免疫荧光和Western blot方法检测HCV NS5A对干扰素α-2b诱导的STAT1磷酸化和核转移的影响。结果转染了pCNS5A的Huh7细胞质可见HCV NS5A蛋白的表达；以干扰索α-2b诱导30 min后,STAT1磷酸化及核转移在转染了表达HCV NS5A的质粒组比转染空白载体pRC/CMV组及未转染组减少,而未转染组及转染空白载体pRC/CMV组间无明显差别。结论表达HCV NS5A的质粒pCNSSA成功转染至Huh7细胞,HCV NS5A减弱干扰素α-2b诱导的STAT1的磷酸化及核转移,提示NS5A影响干扰素α-2b的JAK-STAT信号传导途径可能是HCV干扰素抵抗的机制之一。

Objective To study the effect of HCV NS5A on the Janus kinase （JAK）/signal transducer and activation of transcription （STAT 1） phosphorylation and translocation induced by IFN α -2b and to understand the possible molecular mechanism of HCV interferon resistance. Methods Hepatocellular carcinoma cell line Huh7 was transiently transfected with HCV NS5A protein expression plasmid pCNS5A and blank plasmid pRC/CMV respectively; the cells of the same cell line, without transfection, served as controls. Immunocytochemistry was used to prove the successful transfection. Immunofluorescence and Western blot were performed to observe the difference in STAT1 phosphorylation and nuclear translocation between HCV NS5A- expressed and non-HCV NSSA-expressed cells after 30 minutes of IFN α -2b induction. Results HCV NS5A protein was detected in the cytoplasm of Huh7 cells transfected with pCNSSA, indicating the successful transfection. In comparison to the blank plasmid-transfected and non-transfected group, STAT1 phosphorylation and sequential nuclear import were reduced in the presence of HCV NS5A protein. Conclusion HCV NS5A can, to some extent, inhibit phosphorylation and nuclear translocation of STAT1 in IFN α -2b-induced JAK/STAT pathway, which may be a possible mechanism of HCV interferon resistance.