Fig 1: 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.
Fig 2: ATP13A1 positively regulates RIG‐I‐MAVS signaling pathway. A) qPCR and ELISA analysis for the induction and expression of IFNB in wild‐type or ATP13A1 −/− HEK293T cells with various stimuli as indicated. vRNA refers to genomic RNA isolated from VSV. B) Induction of CCL5, CXCL10 and ISG54 in wild‐type and ATP13A1 −/− HEK293T cells with or without SeV infection. C) Immunoblotting analysis of cell lysates from wild‐type or ATP13A1 −/− HEK293T cells with VSV infection for the indicated time. D) Wild‐type or ATP13A1 −/− HEK293T cells were infected with VSV‐GFP (MOI = 0.1) for 12 h before microscopic imaging (left) and plaque assay (right). E) qPCR analysis for the transcription of IFNB in wild‐type and ATP13A1‐knockdown HEK293T cells with various stimuli as indicated. F) qPCR and ELISA analysis for the transcription and expression of Ifnb in wild‐type and Atp13a1 −/− MEF cells with various stimuli as indicated. G) Wild‐type or Atp13a1 −/− MEF cells were infected with VSV‐GFP (MOI = 0.1) for 12 h before microscopic imaging (left) and plaque assay (right). H) Immunoblotting analysis for FLAG‐ATP13A1 expression in ATP13A1 −/‐ HEK293T cells. Asterisk indicated nonspecific bands. I) qPCR analysis for the induction of IFNB in HEK293T cells following transient expression of the indicated plasmids. Data are representative of three independent experiments (shown as mean and SD in (A,B,D–G,I)). 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: Identification of ATP13A1 involved in antiviral signaling. A) A diagram of the recombination construct used for generation of I‐5 cells. B) IFNB, CXCL10 and ISG54 induction in HEK293T cells and I‐5 cells upon SeV infection for 12 h. C) Transcription of GSDMD‐N in HEK293T cells and I‐5 cells upon SeV infection. D) HEK293T cells and I‐5 cells were infected with SeV for 12 h before microscopic imaging. E) Knockdown efficiency of MAVS by sgRNA in I‐5 cells stabling expressing Cas9 (I‐5(Cas9)). F) Microscopic imaging of I‐5 (Cas9) cells with or without SeV infection for 12 h. G) Outline of the genome‐scale CRISPR‐Cas9 screening. H) P‐value of the hits identified from the screening. I) shRNA as indicated were transfected into HEK293T cells, which were infected with or without SeV for 12 h before ELISA and qPCR analysis. Data are representative of three independent experiments (shown as mean and SD in B,I). p value was determined by two‐tailed unpaired Student's t‐test. ****p < 0.0001. ns indicates not statistically significant.
Fig 4: WDR77 interacts with MAVS and inhibits its activity.a–c HEK293T cells were transfected with luciferase reporters and plasmids as indicated for 24 h. Cells were collected and IFNB1 (a), ISRE (b) and NFKB1 (c) promotor activation were detected by luciferase assay (For a, IFNB1: ****p < 0.0001; ****p < 0.0001; nsp = 0.9496 in sequence; For b, ISRE: ****p < 0.0001; ****p < 0.0001; nsp = 0.8583, nsp = 0.4715 in sequence; For c, NF-KB1: nsp = 0.2570 in sequence). d HEK293T cells were transfected with HA-WDR77, Flag-PRMT5, RIG-I, MAVS, TRAF2, TRAF3, TRAF5, TRAF6, TBK1, IRF3 or IKKβ-expressing plasmids as indicated for 36 h. Cells were collected and subjected to immunoprecipitation assay and immunoblotting. Asterisk indicated nonspecific bands. e Immunofluorescence assay of co-localization of HA-WDR77 (green) with Flag-MAVS (red) in HEK293T cells following SeV infection for 8 h. Nuclei were stained with DAPI (blue). Scale bars indicate 10 μm (Colocalization radio: ****p < 0.0001). f Diagram of MAVS truncations used in Co-IP experiments (top panel). Various MAVS truncations were co-expressed with Flag-WDR77 in HEK293T cells for 36 h before immunoprecipitation and immunoblotting (bottom panel) were performed. Asterisk indicates nonspecific bands. g Diagram of WDR77 truncations used in Co-IP experiments (top panel). Various WDR77 truncations were co-expressed with HA-MAVS in HEK293T cells for 36 h before immunoprecipitation and immunoblotting (bottom panel) were performed. h WT or WDR77-/- #1 HEK293T were transfected with various WDR77 truncations for 24 h, and then stimulated with or without SeV for 12 h. IFNB1 induction was measured by qPCR (ISRE: all ****p < 0.0001; nsp = 0.9998). Data are representative of three independent experiments with similar results (d, e (left), f and g), or three independent experiments (a–c, e (right) and h) (mean ± SD of three biological 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: WDR77 dampens antiviral immune response in mouse primary cells.a–f PEMs from WT, Wdr77CKO and Mavs-/- mice respectively were collected. PEMs were then stimulated for 6 h with or without VSV, SeV, poly(I:C), HSV or HT-DNA as indicated. Culture mediums were then collected and IFN-β was measured by ELISA (a). Ifnb1 (b), Ifna4 (c), Isg56 (d), Cxcl10 (e), Il-6 (f) induction were measured by qPCR (For a, IFN-β: **p = 0.0021 ****p < 0.0001, ****p < 0.0001, nsp = 0.0790, nsp = 0.9993 in sequence; for b, Ifnb1: all ****p < 0.0001, nsp = 0.1813, nsp = 0.8208 in sequence; for c, Ifna4: all ****p < 0.0001, nsp = 0.9997, nsp = 0.9998 in sequence; for d, Isg56: all ****p < 0.0001, nsp = 0.1378; For e, Cxcl10: all ****p < 0.0001, nsp = 0.5755, nsp = 0.7986 in sequence; for f, Il-6: nsp = 0.9872, ****p < 0.0001, nsp = 0.9932, nsp = 0.7531, nsp = 0.9849 in sequence). g BMDMs from WT, Wdr77CKO and Mavs-/- mice respectively were collected. BMDMs were then stimulated for 6 h with or without VSV, SeV, poly(I:C), HSV or HT-DNA as indicated. Ifnb1 induction was measured by qPCR. (All ****p < 0.0001, nsp = 0.1224, nsp = 0.9878 in sequence). h PEMs from WT, Wdr77CKO and Mavs-/- mice were collected respectively. PEMs were infected with or without SeV for 6 h. Cells were collected for subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation and WCL were subjected to SDS-PAGE to examine IRF3 phosphorylation. Data are representative of two independent experiments with similar results (h), or three independent experiments (a–g) (mean ± SEM of three biological 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.
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