Fig. 6

BinCARD1 activates RIG-I signaling by promoting the K63-linked ubiquitination of TRAF3. A, B An ISRE luciferase reporter assay was performed to determine the effect of BinCARD1 on the activation of RIG-I signaling. HEK293T cells were transfected with an ISRE-Luc reporter plasmid, a pRL-TK control plasmid, and a Flag-BinCARD1-expressing plasmid or an empty pCAGGS vector. At 36 h p.t., the cells were left untreated or stimulated with SeV (A) or poly (I:C) (B) for 12 h. The overexpression of BinCARD1 was confirmed by western blotting with a rabbit anti-Flag pAb. The luciferase activity of the cell lysates was analyzed with a dual-luciferase reporter assay in which firefly luciferase activity was normalized to the luciferase activity of Renilla, which had been coexpressed. The fold-change in the firefly luciferase activity of BinCARD1-overexpressing cells compared with cells transfected with empty vector is shown. ***P < 0.001. C Co-IP assays were performed to examine the interaction between BinCARD1 and RIG-I signaling pathway proteins. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and Flag-tagged RIG-I, MAVS, TRAF3, TBK1, or IRF3. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Myc mAb and subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-Myc pAb. D HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF3 and gradually increasing amounts of Myc-tagged BinCARD1. At 36 h p.t., cell lysate proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-Myc pAb. E TRAF3 impaired the binding between BinCARD1 and IAV NP. HEK293T cells were transfected with combinations of plasmids expressing BinCARD1, V5-WSNNP, and TRAF3-Flag. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-BinCARD1 mAb, and the bound proteins were eluted and subjected to western blotting with a mouse anti-BinCARD1 mAb, a rabbit anti-Flag pAb, or a rabbit anti-NP pAb. F A co-IP assay was performed to assess TRAF3 ubiquitination. HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF3 (C68A/H70A) and HA-tagged ubiquitin, with or without Myc-tagged BinCARD1. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-TRAF3 mAb and then western blotted with a rabbit anti-Flag pAb or a rabbit anti-HA mAb. G A co-IP assay was performed to determine the type of TRAF3 ubiquitination linkage mediated by BinCARD1. HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF3 (C68A/H70A) and HA-tagged ubiquitin (K63) or ubiquitin (K63R), with or without BinCARD1. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-TRAF3 mAb and subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-HA mAb. H K63-linked polyubiquitination of TRAF3 was induced by BinCARD1 in infected cells. RAW264.7 cells were transfected with an empty vector or a BinCARD1-expressing plasmid, and at 48 h p.t., the cells were infected with SeV. At 0 and 6 h p.i., cell lysate proteins were immunoprecipitated with a mouse anti-TRAF3 mAb and subjected to western blotting with a rabbit anti-ubiquitin (linkage-specific K63) mAb, a mouse anti-ubiquitin mAb, a mouse anti-TRAF3 mAb, or a mouse anti-BinCARD1 mAb. I A co-IP assay was performed to determine the effect of BinCARD1 on the interaction between MAVS and IKKε. HEK293T cells were transfected with plasmids expressing Flag-tagged IKKε and Myc-tagged MAVS, with or without BinCARD1. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Myc mAb and subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-MAVS mAb. J The effect of BinCARD1 on the phosphorylation of IRF3 was assessed. HEK293T cells were transfected with plasmids expressing HA-tagged TRAF3, Flag-tagged IRF3, and BinCARD1 individually or in various combinations. At 36 h p.t., cell lysate proteins were subjected to western blotting with a rabbit anti-HA mAb, a rabbit anti-Flag pAb, a rabbit anti-IRF3-p mAb, or a mouse anti-BinCARD1 mAb