Objective To investigate the role of nonstructural protein NS2 in respiratory syncytial virus (RSV) infected human type Ⅱ alveolar epithelial cells (A549) activated TLR7 during the signal transduction.Methods This study divided into normal control group,RSV infection group,RSV NS2 small interfering RNA silencing (NS2 siRNA) group and TLR7 agonists (resiquimod,R848) group.A549 cells in each group were infected with different time points and culture supernatant were collected.The expression of TRIF,TRAF6 and p-IkappaB-alpha protein in each group was detected by Western blot at different time points.Type I interferon (IFN)-α,IFN-β in cell culture supernatant of each group detected by enzyme-linked immunosorbent method (ELISA).Results In the normal control group,the expression of TRIF,TRAF6 and p-IκB-α was low,with the infection time increased,the expression of them increased.In NS2 siRNA group,the expression of the three proteins increased compared with the normal control group,but decreased compared with RSV infection group.In RSV infection group and NS2 siRNA group,the IFN-α,IFN-β level increased compared with normal control group and was time-dependent,the difference was statistically significant(P ＜0.01);In TLR7 agonists group,the IFN-α,IFN-β level increased gradually with the infection time increased compared with RSV infection group,the difference was statistically significant (P ＜ 0.05).Conclusion NS2 activates TLR7 in the process of A549 infection,and inhibits the expression of IFN-α and IFN-β.%目的 探讨非结构蛋白NS2在呼吸道合胞病毒(RSV)感染人Ⅱ型肺泡上皮细胞(A549)活化TLR7信号转导过程中的可能作用.方法 以RSV感染体外培养的A549细胞,设立正常对照组、RSV感染组、RSV NS2小干扰RNA沉默组(NS2 siRNA+ RSV组)及TLR7激动剂组(R848+RSV组).各组分别于病毒感染后的4、12、24、48 h收集细胞和培养上清液.Western blot法检测各组不同时间点TRIF、TRAF6及p-IκB-α蛋白表达;ELISA法检测各组细胞培养上清液中Ⅰ型干扰素α(IFN-α)、IFN-β含量的变化.结果 正常对照组中TRIF、TRAF6、p-IκB-α蛋白的表达量较低,经RSV感染之后,随感染时间增加,表达量升高;在NS2siRNA+ RSV组三者表达量较正常对照组上调,但相对于RSV感染组,表达下降,表明RSV NS2可以活化TRIF、TRAF6及p-IκB-α蛋白的表达.RSV感染组及NS2 siRNA+ RSV组、IFN-α、IFN-β含量随感染时间增加逐渐升高,4h开始升高,差异有统计学意义(P＜0.01);R848+RSV组IFN-α和IFN-β呈时间依赖性增加,感染4h升高,差异有统计学意义(P＜0.05).结论 NS2在病毒感染A549细胞过程中激活TLR7,抑制了IFN-α和IFN-β的表达.
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