Study The Role Of NS2 On TLR7 Activation In A549 Cells Infected By Respiratory Syncytial Virus

Objective: To investigate the changes of transcription level of nonstructural protein NS2 in respiratory syncytial virus(RSV)infection of human type II alveolar epithelial cells(A549),study on the role of NS2 in TLR7 activation of A549 cells infected by RSV,in order to provide new ideas of the prevention of RSV infection.Method: After A549 cells infected by RSV in vitro,this study divided into normal control group,RSV infection group,RSV NS2 small interfering RNA silencing(NS2si RNA)group and TLR7 agonists(Resiquimod,R848)group.A549 cells in each group were infected 4h,12 h,24h,48 h and culture supernatant were collected.(1)RSV NS2,TLR7 m RNA expression in each group were detected by real-time quantitative PCR(RT-PCR)at different time points;(2)The expression of TRIF,TRAF6 and p-Ikappa B-alpha protein in each group were detected by Western-blot at different time points;(3)INF-alpha,INF-beta in cell culture supernatant of each group was detected by enzyme-linked immunosorbent method(ELISA).Result:(1)Real-time PCR: The expression of TLR7 m RNA increased after infection and reached 8 times at 48 h compared with the normal control group,the difference was statistically significant(P<0.01);In TLR7 agonists group,the expression of TLR7 m RNA increased significantly and reached 17.3 times at 48 h compared with the normal control group,the expression of TLR7 m RNA increased and reached 1.2 times at 24 h compared with RSV infection group,the difference was statistically significant(P<0.01);In NS2 si RNA group,the expression of TLR7 m RNA increased significantly compared with the normal control group but decreased compared with RSV infection group,the expression of 48 h infection was 70% in the RSV infected group and the difference was statistically significant from 12h(P<0.05).The expression of NS2 m RNA increased after infection;In TLR7 agonists group,the expression of NS2 m RNA increased and reached1.1 times at 24 h compared with RSV infection group,the difference was not statistically significant(P>0.01);In NS2 si RNA group,the expression of NS2 m RNA increased significantly and decreased compared with RSV infection group,the expression of 24 h infection was 38%,48 h infection was 39% in the RSV infected group and the difference was statistically significant(12h P<0.05;24h,48 h P<0.01).The results showed that RSV infection could increased the expression of TLR7 m RNA,and NS2 played a certain role in promoting the activation of TLR7 in RSV infected A549 cells.(2)Western-Blot: In the normal control group,the expression of TRIF,TRAF6 and p-Ikappa B-alpha was low,with the infection time increased,the expression increased,the difference was statistically significant(P<0.01);In TLR7 agonists group,the expression of TRIF,TRAF6 and p-Ikappa B-alpha was significantly increased compared with RSV infection group,the difference was statistically significant(P<0.05);In NS2 si RNA group,the expression of TRIF,TRAF6 and p-Ikappa B-alpha increased compared with the normal control group,but decreased compared with RSV infection group,the difference was statistically significant(P<0.05).The results showed that RSV NS2 could activate the expression of TRIF,TRAF6 and p-Ikappa B-alpha protein.(3)ELISA: In RSV infection group,the IFN-alpha,IFN-beta level increased compared with normal control group and was time-dependence,the difference was statistically significant(P<0.01);In TLR7 agonists group,the IFN-alpha,IFN-beta level increased gradually with the infection time increased compared with RSV infection group,the difference was statistically significant(P<0.05);In NS2 si RNA group,the IFN-alpha,IFN-beta level increased gradually with the infection time increased compared with RSV infection group,the difference was statistically significant(P<0.01).The results indicated that NS2 inhibited the expression of IFN-alpha and IFN-beta in the process of RSV infection.Conclusions: NS2 plays an important role in TLR7 activated A549 cells infected by RSV,TRAF6,TRIF,p-Ikappa B-alpha activated,leading to the downstream I interferon IFN-alpha,IFN-beta expression decreased.