Fig 2: Noncanonical NF-?B signaling is necessary and sufficient for over-proliferation in TRAF3 KO cells.(A) Noncanonical NF-?B pathway schematic. Proteins shown in bold were manipulated in this study. (B) Immunoblotting of NT and TRAF3 KO1 cells transduced with pLVTHM GFP NT or p100 sgRNAs. Blot was stained for p100/p52, TRAF3, and GAPDH (loading control). (B') Quantifications of western blot (B). Histogram shows mean ± 1 s.d. (n = 3). p-values were calculated by Student’s t-test. (C) Cytometric analysis of NT and TRAF3 KO cells transduced with pLVTHM GFP NT or p100 sgRNAs. Graph shows mean ± s.d. (n = 4). p-values were calculated by one-way ANOVA followed by Tukey’s multiple comparisons test. (D) NT and TRAF3 KO1 cells transduced with pLVTHM GFP NT or p100 sgRNAs were treated with BrdU at 4 days post-confluency and immunostained for BrdU and DAPI. (E) Immunoblot of NT and TRAF3 KO1 cells transduced with NT, NIK KO1 or KO2. Blot was stained for NIK, TRAF3, and GAPDH (loading control). (E’) Quantifications of western blot (E). Histogram shows mean ± 1 s.d (n = 3). p-values were calculated by one-way ANOVA followed by Tukey’s multiple comparisons test. (F) Cytometric analysis of NT cells and TRAF3 KO1 cells transduced with control NT, NIK KO1 and KO2. Histogram shows mean ± 1 s.d. (n = 5). p-values were calculated by one-way ANOVA followed by Tukey’s multiple comparisons test. (G) NT and TRAF3 KO1 cells transduced with control NT, NIK KO1 and KO2 were treated with BrdU at 4 days post-confluency and immunostained for BrdU and DAPI. (H and I) Cytometric analysis of NT and TRAF3 KO cells treated with 7 µM BV6 at low (H) and high (I) density. Histogram shows mean ± 1 s.d. (n = 5 (H), n = 6 (I)). p-values were calculated by one-way ANOVA followed by Dunnett’s (H) or Tukey’s (I) multiple comparisons test. (J) Immunoblot of NT, TRAF3 KO1 (positive control), and NT cells expressing pWPI mScarlet NIK-?T3. Blot was stained for NIK and GAPDH (loading control). (K) Cytometric analysis of NT cells and NT pWPI mScarlet NIK-?T3 cells. Histogram shows mean ± 1 s.d. (n = 6). p-values were calculated by Student’s t-test. (L) NT and NT pWPI mScarlet NIK-?T3 cells were treated with BrdU at 4 days post-confluency and immunostained for BrdU and DAPI.Figure 5—source data 1.Source data file for Figure 5.

Fig 3: 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 IKKe. HEK293T cells were transfected with plasmids expressing Flag-tagged IKKe 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

Fig 4: Schematic model showing the role of BinCARD1 in the replication cycle of IAV. BinCARD1 interacts with IAV NP and enhances the interaction between NP and IMP a7, thereby facilitating the nuclear import of the vRNP complex and newly synthesized NP. In contrast, the role played by BinCARD1 in promoting IAV replication is counteracted by the host defense system: BinCARD1-mediated Lys63-linked polyubiquitination of TRAF3 favors the activation of the RIG-I innate immune signaling pathway, and polyubiquitinated BinCARD1 is subjected to selective autophagic degradation via the TBK1-p62 axis

Fig 5: Efb stabilizes TRAF3.a Immunoblots of WCL from MH-S infected with Newman and ?Efb (MOI = 25). b Immunoblots of WCL and IP products of WCL from HEK293T cells transfected with indicated plasmids. Ub, wild-type ubiquitin; K48, Ub with a single 48 lysine residue left; K63 Ub with a single 63 lysine residue left. c Immunoblots of IP products of WCL from MH-S infected with Newman and ?Efb (MOI = 25). d Immunoblots of WCL and IP products from HEK293T cells transfected with indicated plasmids. e Immunoblots of IP products from MH-S infected with Newman and ?Efb (MOI = 25). f Efb inhibits K48-linked ubiquitin chains on TRAF3 in vitro. g Immunoblots of WCL and IP products from HEK293T cells transfected with indicated plasmids. K/R, replace all lysine of TRAF3 with arginine; R106K, replace 106 arginine of K/R with lysine; R155K, replace 155 arginine of K/R with lysine. R106/155K, replace 106 and 155 arginine of K/R with lysine. Data are representative of three experiments with at least three independent biological replicates. Source data are provided as a Source Data file.