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

In order to test the effect of NS1/126-225 on various components of the RLR pathway, NS1/126-225 and expression (Figure 3A) . By contrast, wtNS1 significantly decreased the RLR adaptor-mediated IFN-β promoter activity. As expected, NS1/126-225 exhibited a decrease in the IFN-β mRNA level induced by RIG-I(N) or MAVS ( Figure 3A ). In addition, the secretion of IFN-β and the transcription level of ISGs triggered by TBK1 or IRF3(5D) in the presence of NS1/126-225 were tested. NS1/126-225 induced nearly a complete loss of the inhibition of IFN-β and ISGs, including OASL, PKR, and Mx1, whereas wtNS1 strongly blocked the production of IFN-β and ISGs mRNA expression (Figures 3B,C) . Together, these data indicate that NS1/126-225 significantly inhibits the cellular antiviral response at the level between MAVS and TBK1. In the RLR-mediated signaling pathway, TRAF3 serves as a critical link between the adaptor MAVS and downstream regulatory kinases that are essential for IRF3 activation (29, 30) . Thus, we hypothesized that TRAF3 is the target of NS1/126-225. Co-IP experiments revealed that NS1/126-225 interacted selectively with TRAF3 but not other components (Figures 4A,B) . In another experiment, wtNS1 and NS1/74-225 also interacted most potently with TRAF3 ( Figure 4C ). This association was confirmed under physiological conditions in an experiment that detected this interaction by overexpression of Flag-TRAF3 in infected A549 cells, where RIG-I served as a positive control ( Figure 4D) . Furthermore, PR8 NS1 also interacted with TRAF3 in infected A549 cells (Figure 4E ). To address whether the wtNS1 protein physically interacts with endogenous TRAF3, we performed endogenous IP assays on H5N1/HM or PR8-infected cell lysates. Our results showed that TRAF3 could be co-precipitated by the NS1 antibody ( Figure 4F ). In addition, the NS1 proteins of the strain A/Shanghai/02/2013(H7N9) and other avian The promoter activities were detected by the dual-luciferase assay system. All luciferase assays were repeated at least three times, and the data shown are mean ± SD from one representative experiment. Significance was analyzed with a two-tailed Student's t-test (*P < 0.05 or **P < 0.01, ***P < 0.001). H9N2 strain did not bind TRAF3 ( Figure S1 in Supplementary Material). These results demonstrate that NS1/126-225 or wtNS1 interacts with TRAF3 in a strain-specific manner. Based on the findings that NS1/126-225 or wtNS1 interacted with TRAF3, we next asked whether the two molecules co-localize in cells. Confocal microscopy revealed the co-staining of NS1/ 126-225 or wtNS1 and TRAF3 in cells, suggesting the co-localization of the two proteins ( Figure 4G) . Normally, expression of wtNS1 in HeLa cells resulted in a nuclear localization with minor cytoplasmic staining. TRAF3 overexpression led to a marked increase in the cytoplasmic localization of wtNS1 or NS1/126-225. These results indicate that NS1/126-225 or wtNS1 colocalize with TRAF3 in cells and TRAF3 overexpression led to a marked increase of NS1/126-225 or wtNS1 cytoplasmic localization.