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

Transcription factor IRF3 is a key innate immune system component that mediates IFN-β induction. Once IRF3 is phosphorylated, it forms a dimer, translocates into the nucleus from the cytoplasm, and induces the expression of IFN-β and ISGs through specifically binding to their promoter regions (27) . In order to further investigate how NS1/126-225 inhibits the signaling that mediates type I IFN production, we used an IRF3-luciferase reporter plasmid, allowing for the measurement of IRF3 activation. As shown in Figure 2A , IRF3-luciferase reporter activation by RIG-I(N) was blocked in 293T cells overexpressing NS1/126-225 or wtNS1. We next addressed whether NS1/126-225 affected the dimerization and nuclear localization of endogenous IRF3 mediated by RIG-I(N). Non-reduced SDS-PAGE and immunoblot analysis of cell lysates after RIG-I(N) transfection showed that NS1/126-225 or wtNS1 expression produced a considerable reduction in the activated dimer form of IRF3 (Figure 2B) . Similarly, RIG-I(N)induced phosphorylation of IRF3 was strongly repressed by NS1/126-225 or wtNS1 and that phosphorylated IRF3 was largely distributed in nuclear fractions ( Figure 2C) . ELISA assays of IFN-β in the medium of cells transfected with plasmids encoding NS1/126-225 or wtNS1 along with RIG-I(N) showed that both NS1/126-225 and wtNS1 inhibited the production of IFN-β ( Figure 2D) . Together, these data indicate that NS1/126-225 inhibits the expression of type I IFN induced by RIG-I(N) through blocking the phosphorylation of IRF3. Previous studies have shown that a recombinant VSV-GFP system can be used as a strategy to screen proteins possessing IFN-antagonizing activity (28) . In the present study, we employed recombinant VSV-GFP to investigate if NS1/126-225 serves as an antagonist of IFN production. When 293T cells expressed NS1/126-225, a high level of VSV-GFP replication was present, consistent with wtNS1 protein (Figure 2E) , suggesting that the inhibitory effect of NS1/126-225 on IFN production is also present during actual viral infection.