Fig 1: WDR77 negatively regulates antiviral innate immunity in mice.a–d WT, Wdr77CKO and Mavs-/- mice (n = 3 each) were infected intravenously with VSV at 2 × 107 PFU per mouse. The spleens, livers, and lungs were collected 4 h or 8 h after infection. Ifnb1 (a), Ifna4 (b) inductions and VSV RNA levels (c) were measured respectively by qPCR. The sera were collected and used for the measurement of IFN-ß by ELISA (d) (For a, Ifnb1: **p = 0.0013, ****p < 0.0001, ****p < 0.0001, nsp = 0.0815, ***p = 0.0006, nsp = 0.1397 in sequence; for b, Ifna4: ***p = 0.0004, ****p < 0.0001, ****p < 0.0001, nsp = 0.6194, ***p = 0.0008, ***p = 0.0008 in sequence; For c, VSV: nsp = 0.7616, **p = 0.0010, nsp = 0.3889, nsp = 0.1316, nsp = 0.8740, **p = 0.0023 in sequence; for d, IFN-ß: * p = 0.0216, ***p = 0.0003 in sequence). e Hematoxylin-eosin staining of lung sections from WT and Wdr77CKO mice infected intravenously with VSV at 2 × 107 PFU per mouse for 12 h. Scale bars indicate 50 µm. f WT, Wdr77CKO and Mavs-/- mice (n = 10 each) were injected intravenously with VSV at 5 × 107 PFU per mouse, and the survival rates were monitored for 15 days (*p = 0.049). g WT, Wdr77CKO and Mavs-/- mice (n = 10 each) were injected intranasally with IAV at 8 × 102 PFU per mouse, and the survival rates were monitored for 15 days (*p = 0.045). h WT, Wdr77CKO and Mavs-/- mice (n = 3 each) were infected intravenously with HSV at 1.5 × 108 PFU per mouse. The sera were collected 12 h or 24 h after infection and used for the measurement of IFN-ß by ELISA (IFN-ß: nsp = 0.5412, nsp = 0.9892 in sequence). i WT, Wdr77CKO and Mavs-/- mice (n = 10 each) were injected intravenously with HSV at 1.5 × 108 PFU per mouse, and the survival rates were monitored for 15 days (*p = 0.445). Data are representative of three independent experiments with similar results (e), or three independent experiments (a–d, and h) (mean ± SEM of three biological replicates). P-value was determined by two-tailed Student’s t-test, two-sided log-rank (Mantel-Cox) test (f, g and i). n.s. indicates no statistical significance. Source data are provided as a Source Data file.
Fig 2: Atp13a1 is essential for antiviral immune response in mouse primary cells. A) Wild-type and Atp13a1-cKO BMDMs were treated with or without SeV, VSV, or poly (I:C) as indicated for 6 h before ELISA analysis for Ifnß in the medium. B–G) Wild-type and Atp13a1-cKO BMDMs were treated with or without SeV, VSV or poly (I:C) respectively as indicated for 6 h before qPCR analysis for the induction of B) Ifnb, C) Isg54, D) Il-6, E) Ifna4, F) Cxcl10, and G) Ccl2. H) Wild-type and Atp13a1-cKO BMDMs were infected with VSV respectively for the indicated time before immunoblotting analysis. Data are representative of three independent experiments (shown as mean and SD in A–G). p value was determined by two-tailed unpaired Student's t-test, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns indicates not statistically significant.
Fig 3: WDR77 binds to MAVS and negatively regulates IFN-ß induction.a Immunoblot analysis of MAVS expression in P5 fraction containing crude mitochondria. b Purification procedure for Flag-MAVS. c Silver staining of purified Flag-MAVS. The Flag-MAVS band is indicated by an arrow and the asterisk indicated interested bands. d Peptides identified by mass spectrometry. e Plasmids as indicated were co-transfected into HEK293T cells. 36 h after transfection, cells were harvested for immunoprecipitation and immunoblotting. Asterisk indicated nonspecific bands. f–h HEK293T cells were transfected with luciferase reporters and increasing amounts of plasmids as indicated for 24 h, and then stimulated with or without SeV for 12 h. Cells were collected and IFNB1 promotor activation was detected by luciferase assay (f). IFNB1 induction was measured by quantitative PCR (qPCR) (g). Culture medium was collected and IFN-ß was detected by ELISA (h) (For f, IFNB1: ****p < 0.0001, ****p < 0.0001, nsp = 0.9540, nsp = 0.4002 in sequence; For g, IFNB1: ****p < 0.0001, ****p < 0.0001, nsp = 0.6511, nsp = 0.1233 in sequence; For h, IFNB1: ***p = 0.0002, ****p < 0.0001, nsp = 0.6413, nsp = 0.0503 in sequence). i HEK293T cells were transfected with luciferase reporters and plasmids as indicated for 24 h, and then stimulated with or without SeV for 12 h. IFNB1 promotor activation was analyzed by luciferase assay (For i, IFNB1: ****p < 0.0001, nsp = 0.0861, ****p < 0.0001, nsp = 0.1511 in sequence). j–k HEK293T cells were transfected with luciferase reporters and Flag-WDR77 plasmids for 24 h, and then stimulated with or without SeV, VSV or poly(I:C) for 12 h. IFNB1 promotor activation was analyzed by luciferase assay (j). IFN-ß was detected by ELISA in (k) (For j, IFNB1: nsp = 0.9994; ****p < 0.0001, ****p < 0.0001, ***p = 0.0001 in sequence; For k, IFN-ß: nsp > 0.9999, *p = 0.0377, **p = 0.0059, *p = 0.0312 in sequence). Data are representative of one experiment (c and d), two independent experiments with similar results (e), or three independent experiments (f–k). P-value was determined by two-tailed Student’s t-test. n.s. indicates no statistical significance. Source data are provided as a Source Data file.
Fig 4: WDR77 inhibits MAVS aggregation and activation.a HEK293T cells were co-transfected with Flag-WDR77 and HA-MAVS for 24 h followed by SeV stimulation. Cells were harvested and subjected to immunoprecipitation assay and immunoblotting. b WT or WDR77-/- #1 HEK293T cells were stimulated with or without SeV or VSV for 12 h. Cells were collected and subjected to immunoprecipitation assay and immunoblotting. c HEK293T cells were stimulated with SeV and harvested at various time points as indicated for immunoprecipitation assay and immunoblotting. The sample shown in lane 7 was 4% of input (whole cell lysate (WCL) harvested 32 h post infection). d HEK293T cells were transfected with increasing amounts of Flag-WDR77-expressing plasmids for 24 h, and then transfected with Flag-MAVS plasmids for 12 h. Cells were harvested for subcellular fractionation. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation. e WDR77-/- #1 cells were transfected with increasing amounts of Flag-WDR77-expressing plasmids for 24 h, and then stimulated with or without SeV for 8 h. Cells were harvested for subcellular fractionation. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation. f WT and WDR77-/- HEK293T cells transfected with or without Flag-WDR77 were stimulated with or without VSV for 8 h. Cells were harvested for subcellular fractionation. S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions were subjected to SDD-AGE to examine MAVS aggregation. WCLs were subjected to SDS-PAGE to examine IRF3 phosphorylation. g WT or Wdr77-/- MEF cells were stimulated with SeV for indicated times. Cells were harvested for subcellular fractionation. S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions were subjected to SDD-AGE to examine MAVS aggregation. h WT or WDR77-/- #1 HEK293T cells were stimulated with SeV. Cells were harvested at specified time points post infection and subjected to immunoblotting. i WT or WDR77-/- #1 HEK293T cells were stimulated with SeV. Cells were harvested at specified time points post infection and IFNB1 induction was measured by qPCR (IFNB1: all nsp > 0.9999, ***p < 0.0001, **p = 0.0018). Data are representative of two independent experiments with similar results (a–h), or three independent experiments (i) (mean ± SD of three biological replicates with 2 technical replicates). P-value was determined by two-tailed Student’s t-test. n.s. indicates no statistical significance. Source data are provided as a Source Data file.
Fig 5: ATP13A1 is required for maintenance of proper MAVS protein level. A) Various plasmids were transfected into wild-type or ATP13A1 -/- HEK293T cells. Twenty-four hours after transfection, the cells were infected with SeV for 12 h before qPCR analysis for the induction of IFNB. B) Wild-type and ATP13A1 -/- HEK293T cells were infected with SeV for 12 h. Whole cell lysates were then harvested for immunoblotting analysis. C) Whole cell lysates and P5 fractions from wild-type or ATP13A1 -/- HEK293T cells were analyzed by immunoblotting. D) ATP13A1 -/- HEK293T cells were transfected with FLAG-ATP13A1-expressing vectors for 24 h. The cells were then infected with VSV for 12 h before immunoblotting analysis. Asterisk indicated nonspecific bands. E) MAVS -/- and MAVS-/- & ATP13A1-/- double knockout HEK293T cells were transfected with UTR-MAVS for 24 h, and then treated with CHX for the indicated time before immunoblotting analysis (left). Protein level of MAVS normalized to tubulin was quantified (right). F) Wild-type and Atp13a1 -/- MEF cells were stained for immunofluorescent microscopic imaging. Nuclei were stained with DAPI. Mitochondria were stained with MitoTracker Red. Scale bar represents 10 micrometers. G) Flow cytometry analyses of mitochondrial membrane potential in wild-type and Atp13a1 -/- MEF cells with JC-1 MitoMP Detection Kit. Data are representative of three independent experiments (shown as mean and SD in (A), mean and SEM in (E)). p value was determined by two-tailed unpaired Student's t-test, ***p< 0.001. ns indicates not statistically significant.
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