Fig 2: RNF34 interacts with MAVS Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING, or Flag-TBK1. Luciferase assays were performed 24 h after transfection.Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53 + pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34 + pACT-2 was used to exclude the self-activation of RNF34.Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.GST-tagged RNF34 was subjected to a pull-down assay with HEK293T cell lysates. Immunoblot with an anti-MAVS antibody is shown in the top panel. Loading of the GST proteins assessed using Coomassie blue staining is shown in the bottom panel. GST was used as a negative control.Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-MAVS or Flag-vector-expressing plasmids were subjected to SDS–PAGE and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-RNF34 (upper panel) or GST (middle panel) for 2 h and then analyzed with anti-Flag antibody.Representative confocal images of immunofluorescence staining for Flag-RNF34 colocalization with endogenous MAVS in THP-1 cells infected with VSV for 12 h. Scale bar, 10 µm.Immunoblot showing the levels of the RNF34 protein in THP-1 cells infected with VSV (MOI = 1.0) for the indicated times. a-Tubulin was used as a loading control.In situ PLA assay of the RNF34-MAVS complex in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times using an anti-RNF34 or anti-MAVS antibody. RNF34-MAVS complex, red; nuclei, blue. Scale bar, 5 µm.One hundred cells in Fig 2H were counted, and the quantification of PLA signals per cell is shown.Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-mMAVS. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibody.Data information: Cell-based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *P < 0.05; **P < 0.01; and ***P < 0.001.Source data are available online for this figure.

Fig 3: RNF34 negatively regulates RLR signaling pathways AImmunoblot showing RNF34 levels in the PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2, or siRNF34-3 oligos shown in Fig 1A and B.BImmunoblot showing Flag-RNF34 and Flag-RNF34 H342A levels in the cells presented in Fig 1C and D. a-Tubulin was used as a loading control.CImmunoblot showing RNF34 levels in the control (shCtrl) and three stable RNF34-knockdown (shRNF34-1, shRNF34-2, and shRNF34-3) HEK293T cell lines shown in Fig 1E and F.DNDV-GFP replication was defined as the product of the mean fluorescence intensity of GFP-positive cells in shRNF34-1, shRNF34-2, or shRNF34-3 cells and the mean fluorescence intensity of shCtr cells.E-I(E, F, H) Luciferase activity driven by the IFN-ß promoter in HEK293T cells transfected with Flag-MAVS (E), Flag-N-RIG-I (F), or poly(I:C) (10 µg/ml; H) together with increasing amounts of the HA-RNF34 expression vector. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction. (G, I) Luciferase activity driven by the IFN-ß promoter in HEK293T cells transfected with Flag-STING and Myc-cGAS (G), or (dAT:dT) (10 µg/ml; I) together with increasing amounts of the HA-RNF34 expression vector. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction.JLuciferase activity driven by the NF-?B promoter in HEK293T cells transfected with poly(I:C) together with increasing amounts of the HA-RNF34 expression vector. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction.KImmunoblot showing the levels of the phosphorylated (-p) and total IRF3, TBK1, and RNF34 proteins in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.Data information: Cell-based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *P < 0.05; **P < 0.01; and ***P < 0.001.Source data are available online for this figure.

Fig 4: RNF34 interacts with MAVS and enhances the K27‐/K29‐linked ubiquitination of MAVS Immunoblot showing the levels of HA‐RNF34 and Flag‐N‐RIG‐I, Flag‐MAVS, Flag‐STING, and Flag‐TBK1 in the cells shown in Fig 2A.Immunoprecipitation analysis of HEK293T cells transfected with Myc‐RNF34 together with Flag‐V, Flag‐MAVS, Flag‐RIG‐I, Flag‐STING, or Flag‐TBK1. Anti‐Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti‐Myc or anti‐Flag antibody.Immunoblot showing the levels of MAVS, RIG‐I, STING, and TBK1 in HEK293T cells transfected with the Myc‐vector (Myc‐V) or Myc‐RNF34.Quantification of MAVS colocalization with RNF34 in Fig 2F.Schematic of MAVS along with predicted translation product mMAVS with the start site corresponding to Met 142.Immunoprecipitation analysis of HEK293T cells expressing Flag‐MAVS and Myc‐RNF34 together with HA‐Ub (WT; K27R; K29R; K27/29R). Anti‐Flag immunoprecipitates were analyzed using immunoblotting with an anti‐HA or anti‐Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti‐HA, anti‐Myc, or anti‐Flag antibody.Immunoblot showing levels of the RNF34 protein in cells transfected with the control siRNA and RNF34 siRNA‐1 shown in Fig 3F. α‐Tubulin was used as a loading control.Data information: Cell‐based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two‐tailed Student's t‐tests were used for statistical analyses: *P < 0.05; **P < 0.01; and ***P < 0.001.Source data are available online for this figure.

Fig 5: RNF34 negatively regulates RLR signaling pathways A, BResults of the ELISAs used to determine IFN-ß (A) and IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2, or siRNF34-3 oligos and infected with VSV for the indicated times.C, DLuciferase activity driven by the IFN-ß (C) or NF-?B (D) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction.EPlaque assay of VSV loads in supernatants from cells transfected with shCtrl, shRNF34-1, shRNF34-2, or shRNF34-3 and subsequently infected with VSV.FMicroscopy images of shCtrl, shRNF34-1, shRNF34-2, or shRNF34-3 cells infected with NDV-GFP (MOI = 1.0) for the indicated times. Gray represents cells; green represents NDV. Scale bar, 300 µm.GImmunoblot showing the levels of the phosphorylated (p) and total IRF3, TBK1, and VSV-G proteins in PMA-differentiated THP-1 cells transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.H, JRT–PCR analysis of the expression of the IFN-ß (H), IL8, ISG54, and ISG56 (J) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.IResults of the ELISA used to determine IFN-ß secretion in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.Data information: Cell-based studies were performed independently at least three times with comparable results.Cell-based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *P < 0.05; **P < 0.01; and ***P < 0.001.Source data are available online for this figure.