The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-ß Production by Targeting TNF Receptor-Associated Factor 3

Qian, Wei; Wei, Xiaoqin; Guo, Kelei; Li, Yongtao; Lin, Xian; Zou, Zhong; Zhou, Hongbo; Jin, Meilin

With the aim of elucidating the mechanism by which IAV NS1 protein counteracts the host innate immune responses, we generated four truncated H5N1 NS1 proteins, NS1/1-73, NS1/74-225, NS1/1-125, and NS1/126-225 ( Figure 1A) . In order to investigate the function of wtNS1, and its truncated peptides, we assessed its effect on IFN-β promoter activity using a luciferase reporter assay in 293T cells. Our results showed that wtNS1, NS1/74-225, and NS1/126-225 significantly decreased the IFN-β reporter activities driven by RIG-I or RIG-I(N). Conversely, NS1/1-73 did not change the activity of IFN-β reporter, and NS1/1-125 only slightly increased the activity compared to an empty vector control ( Figure 1B) . In addition, we found that wtNS1 and all truncated peptides had inhibitory effects on IFN-β reporter activities induced by Sev or rNS1-SD30 virus infection or transfection of poly(I:C) ( Figure 1B) . This suggests that NS1 N-terminal RBD alone is sufficient to inhibit the activation of IFN-β only in the presence of dsRNA; the C-terminal ED of NS1 could inhibit the activity of IFN-β reporter in all tested conditions. Driven by RIG-I(N), NS1/126-225 caused a dose-dependent inhibition of IFN-β promoter activity and IFN-β transcription ( Figure 1C) . Previous studies indicated that IAV NS1 sequesters dsRNA and binds RIG-I at its RBD, subsequently inhibiting the activation of IRF3 and preventing the induction of IFN-β (11, 16) . Our findings reveal that C-terminal ED of NS1 (NS1/126-225) blocked RIG-I(N)-mediated IFN-β induction in an RNA binding-independent manner.