(a) Survival rate (%) of mice. March5^+/+ and March5^+/− mice (6–7 weeks of age) were infected with VSV (2 × 10⁸ p.f.u. per mouse) via tail vein injection. Mortality was monitored for 9 days (n=15 mice per group). (b) VSV replication analysis using qPCR in March5^+/+ and March5^+/− mouse tissues. March5^+/+ and March5^+/− mice were infected with VSV; 4 days later, tissues from the spleen, lung, liver and brain were collected and analysed for VSV RNA replicates using qPCR. Error bars, mean±s.e.m. (n=9 mice per group). (c) Bioassay of IFN-β or IL-6 production in serum of March5^+/+ and March5^+/− mice 12 h after infection with VSV (left, 2 × 10⁸ p.f.u. per mouse) or 200 μg of poly(I:C) injection (right) via the tail vein. Error bars, mean±s.e.m. (n=9 mice per group). (d) Quantitative PCR analysis of Ifnb1, Oas1, Ifit1, Mx1, Gbp2b and Isg15 mRNA expression in the spleen, lung and liver of March5^+/+ and March5^+/− mice 4 days after VSV infection. Graphs represent fold-induction of indicated genes relative to mRNA levels in March5^+/+. Error bars, mean±s.e.m. (n=3 mice per group). *P<0.05, **P<0.01, ***P<0.001 versus March5^+/+ by Student's t-test. All data are representative of at least three independent experiments.

(a) Immunoblot analysis of MARCH5 expression levels in MARCH5-depleted Raw264.7 cells. See full blots in . (b) Twenty-four hours after infection, PR8-GFP or VSV-GFP virus replication assay by fluorescence microscopy (fluorescence, upper; phase-contrast microscopy, bottom) and virus titration by fluorescence analysis or plaque assay in siControl or siMARCH5 expressing Raw264.7 cells. Error bars, mean±s.e.m. (n=3). Scale bars, 100 μm. (c) Determination of the virus titration using fluorescence analysis or plaque assay in March5^+/+ and March5^+/− BMDM cells infected with PR8-GFP or VSV-GFP virus. Error bars, mean±s.e.m. (n=3). (d,e) Bioassay of IFN-β or IL-6 (ELISA) in supernatants of MARCH5-depleted Raw264.7 (d) or March5^+/+ and March5^+/− BMDM cells (e) infected or transfected with PR8-GFP, VSV-GFP, poly(I:C) or 5′ppp-dsRNA. After cells were transfected with siControl or siMARCH5 for 24 h, followed by infection with virus or transfection with poly(I:C) for indicated times. Error bars, mean±s.e.m. (n=3). All data are representative of at least three independent experiments.

(a) After Myc-MARCH5^WT or Myc-MARCH5^H43W expression vectors were introduced into MARCH5^−/− HEK293T cells, cells were infected with PR8-GFP or transfected with poly(I:C) for 24 h. Promoter activity of IFN-β, ISRE, IFN-α or IRF3. Graphs represent fold-induction relative to the luciferase activity in control cells. Error bars, mean±s.e.m. (n=3). (b) Immunoblot analysis of endogenous or ectopic expression levels of MARCH5 in MARCH5^+/+ or MARCH5^−/− HEK293T cells. See full blots in . (c,d) Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis (c). Bioassay of IFN-β or IL-6 production (ELISA) (d). Error bars, mean±s.e.m. (n=3). Scale bars, 100 μm. (e,f) IRES, MARCH5^WT-Flag or MARCH5^H43W-Flag stably expressing Raw264.7 cells were stimulated by infection with VSV-GFP for 24 h. Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis (left). Quantification of GFP intensity or virus titration by plaque assay (right; e). Bioassay of IFN-β or IL-6 production (ELISA) in supernatants of cultured Raw264.7 stable cells (f). Error bars, mean±s.e.m. (n=3). All data are representative of at least three independent experiments. Scale bars, 100 μm.

(a) IFN-β promoter activity in N-RIG-I, MDA5, MAVS, TRIF, TRAF-6, TRAF-3, IKK-α or IRF3 with Myc-MARCH5^WT (0, 50, 100, 200, 400 and 800 ng)-transfected HEK293T cells for 36 h. Graphs represent fold-induction of IFN-β relative to the luciferase activity in control cells. Error bars, mean±s.e.m. (n=3). (b) Bioassay of IFN-β (ELISA) in supernatants of HEK293T cells transfected with MAVS or TRIF with Myc-MARCH5^WT (0, 100, 200 and 400 ng). Error bars, mean±s.e.m. (n=3). (c) Immunoblotting of p-IRF3, p-p65, total IRF and total p65 in lysates of HEK293T cells. After transfection with control or Myc-MARCH5^WT for 24 h, cells were stimulated by transfection with poly(I:C) for the indicated time. All data are representative of at least three independent experiments. See full blots in .

(a) Immunoprecipitation between Myc-MARCH5 and Flag-MAVS in HEK293T cells. HEK293T cells were co-transfected with indicated plasmids for 36 h and interaction was analysed by immunoblotting with indicated antibody. (b) Confocal microscopy analysis of Flag-MAVS^WT and Myc-MARCH5^WT localization in HEK293T cells. Scale bars, 10 μm. (c,d) Immunoprecipitation analysis of Flag-MAVS or Myc-MARCH5-truncated mutants. HEK293T cells were co-transfected with indicated plasmids and interaction was analysed by immunoblotting with indicated antibodies. Flag-MAVS fragments (WT: full length, ΔCARD; Δ10–77, ΔPR; Δ103–173, ΔTM; Δ514–535) (c). Myc-MARCH5 fragments (WT; full length, ΔR; 76–278, ΔT234; 1–139; d). (e,f) GST pull-down assays using GST-tagged wild-type and mutants of MARCH5. The GST-conjugated MARCH5 wild-type protein was purified from insect and incubated with cell lysates treated with/without poly(I:C) (e). The GST-MARCH5 mutants were purified from bacteria and their interactions to MAVS were analysed immunoblotting with anti-Flag antibody. The bottom panel shows the Coomassie Blue-stained gel (f). (g) IFN-β and ISRE promoter activity in Flag-MAVS expressing HEK293T cells transfected with Myc-MARCH5 fragments. Error bars, mean±s.e.m. (n=3). (h) Promoter activity of IFN-β and ISRE in HEK293T cells transfected with Myc-MARCH5^WT and Flag-MAVS fragment constructs. Graphs represent fold-induction relative to the luciferase activity in control cells. Error bars, mean±s.e.m. (n=3). All data are representative of at least three independent experiments. See full blots in .

(a,b) Interaction between MARCH5 and MAVS in HEK293T cells. Cells were transfected with Flag-N-RIG-I (a) or poly(I:C) (b); interaction was determined by immunoblotting with anti-MAVS antibody after lysates were immunoprecipitated with anti-MARCH5 or anti-Myc antibody. (c) After cells were infected with PR8 virus for 24 h, cell lysates were immunoprecipitated with anti-MARCH5 antibody or control IgG. Interaction was evaluated using anti-MAVS antibody. (d) Immunoblot analysis of endogenous MAVS expression levels in March5^+/+ and March5^+/− BMDM cells infected with PR8-GFP. (e) Immunoblot analysis of Flag-MAVS expression levels in HEK293T cells. Cells were transfected with Flag-MAVS^WT and Myc-MARCH5^WT, with or without treatment of MG132 (10 μM). (f) Immunoblot analysis of Flag-MAVS^WT expression levels in HEK293T cells. Cells were co-transfected with Flag-MAVS and Myc-MARCH5^WT or Myc-MARCH5^H43W. (g,h) Cells were transfected with Flag-N-RIG-I (g) or poly(I:C) (h) for 36 h. Lysates were mixed with or without 6 × native sample buffer, followed by SDS–PAGE. (i) After Myc-MARCH5^WT or Myc-MARCH5^H43W expression vector was reintroduced into MARCH5^−/− HEK293T cells, cells were infected with PR8-GFP. The lysates were mixed with or without 6 × native sample buffer, followed by SDS–PAGE. (j) CHX chase analysis of endogenous MAVS in MARCH5^+/+ or MARCH5^−/− HEK293T cells. After Myc-MARCH5^WT-expressing vector was reintroduced to MARCH5^−/− HEK293T cells, cells were exposed to CHX for indicated times before harvest, with or without transfection with poly(I:C). Numbers below lanes indicate MAVS expression levels relative to GAPDH. All data are representative of at least three independent experiments. See full blots in .

(a) Ubiquitination assay of endogenous MAVS by using anti-MAVS antibody in poly(I:C)-transfected HEK293T cells. Levels of ubiquitination were determined by anti-Ub antibody. (b,c) Ubiquitination assays in Flag-MAVS^WT and Myc-MARCH5^WT-expressing HEK293T cells. After cells were transfected with Flag-MAVS^WT and control or Myc-MARCH5^WT, ubiquitination levels of Flag-MAVS were determined by immunoblotting with anti-Ub, K48-Ub or K63-Ub-specific antibodies (b). Cells were co-transfected with Flag-N-RIG-I and Myc-MARCH5WT or H43W. Endogenous ubiquitination levels of MAVS were determined by anti-Ub antibody after IP with endogenous anti-MAVS antibody (c). (d) Ubiquitination of endogenous MAVS in MARCH5^+/+ or MARCH5^−/− HEK293T cells transfected with Flag-N-RIG-I. (e) In vitro ubiquitination assay of Flag-MAVS. Myc-MARCH5^WT and Flag-MAVS^WT were incubated with a reaction mixture with ubiquitin, E1, UbcH5b as E2, and ubiquitination levels were determined by immunoblotting with anti-Ub antibody. See full blots in .

(a) Expression of Flag-MAVS lysine mutants by Myc-MARCH5^WT in HEK293T cells. (b) Ubiquitination assay of Flag-MAVS lysine mutants by Myc-MARCH5^WT. Ubiquitination of MAVS lysine mutants was assessed by immunoblotting with anti-Ub antibody. (c) Promoter assay of IFN-β, ISRE or NF-κB in HEK293T cells transfected with Myc-MARCH5^WT and Flag-MAVS^WT or lysine mutants for 36 h. Graphs represent fold-induction relative to the luciferase activity in control cells. Error bars, mean±s.e.m. (n=3). All data are representative of at least three independent experiments. See full blots in .