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

Interestingly, our study has shown that the ED of NS1 protein possesses the ability to suppress IFN response in the absence of RNA. Typically, the RBD of NS1 mediates the inhibition of IFN synthesis, and the ED of NS1 induces the inhibition of gene expression, together with its known interactors (11) . In this study, we have found that the NS1 C-terminal ED (NS1/74-225 and NS1/126-225), but not the RBD (NS1/1-73 and NS1/1-125) block IFN-β reporter activity induced by RIG-I(N). Expression of NS1/126-225 resulted in the inhibition of IRF3 activation, indicating that the NS1 protein blocks IFN-β activation through an RNA-independent manner. It was demonstrated in previous studies that influenza A viruses TX/98 and A/Viet Nam/1203/04 expressing C-terminally truncated NS1 proteins of 73, 99, or 126 amino acids were attenuated. The resultant reduced growth correlated with a high level of IFN-α/β induced by these mutant viruses (28, 38) . In addition, in both influenza B and C viruses, the C-terminal domains of the NS1 proteins were found to possess IFN antagonist activity (39, 40) . More importantly, the N terminus-truncated NS1 proteins encoded by PR8, which was To elucidate the mechanism of how the NS1 ED inhibits IFN-β activation, we speculated that the NS1 ED contacts its counterparts in RIG-I signaling leading to inhibition. For this purpose, we examined a step within the signaling pathway that NS1/126-225 targets and found that NS1/126-225 acted downstream of MAVS and upstream of TBK1. The Co-IP assays showed unexpectedly that NS1/126-225 binds to TRAF3, which interacts with MAVS forming a platform for RNA virus signaling. We also tested the binding of TRAF3 to the full-length NS1 protein and found that this interaction exists, and co-localized in the cytoplasm. The NS1/126-225 protein mainly localized in the cytoplasm. TRAF3 expression led to a marked increase in the NS1/126-225 cytoplasmic localization, suggesting that NS1/126-225 inhibits the activation of the IFN-β pathway. Although all types of influenza virus NS1 proteins interact with TRIM25, only part of NS1 prevents IRF3 activation, indicating that TRIM25 is not required for the inhibition of IRF3 activation. In this study, we did not observe the interaction between NS1/126-225 and RIG-I, consistent with the results described in a previous publication (41) . Consequently, RIG-I seems to be non-essential for the optimal inhibition of IFN production in IAV-infected cells. However, our study demonstrated that the influenza A virus NS1 ED targets TRAF3, subsequently inhibits IFN production, implying that TRAF3 is a key factor involved for IAV to escape host innate immune responses.