Abstract
The transcription factor interferon regulatory factor 3 (IRF3) initiates type I interferon transcription, which is required for host defense. Here, we identify RAD18 as a central E3 ubiquitin ligase that selectively targets phosphorylated IRF3 (p-IRF3) for autophagic degradation. RAD18 specifically promotes the dissociation of p-IRF3 from the IFNB promoter and in turn terminates its transcriptional activity. Mechanistically, RAD18 binds the p-IRF3 dimer located on the IFNB promoter and triggers K63 polyubiquitylation of p-IRF3 at Lys 193. The ubiquitylated p-IRF3 dimer consequently dissociates from the IFNB promoter, translocates out of the nucleus and undergoes OPTN-mediated autophagic degradation. Rad18fl/fl Lysm-cre mice resist lethal vesicular stomatitis virus infection in vivo due to IFNβ overproduction. In H1N1-infected human macrophages or monocytes from individuals with active systemic lupus erythematosus, RAD18 protein levels negatively correlate with p-IRF3 and IFNB1 mRNA levels. Thus, RAD18 functions as a break to terminate IRF3-driven IFNB1 transcription and may be a potential therapeutic target for RNA virus infection or autoimmune diseases.
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Data availability
All data generated or analyzed during this study are included in this article and the Supplementary Information. MS data are publicly available at the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD050956). RNA-sequencing data used in this study were downloaded from the NCBI Gene Expression Omnibus under repository accession numbers GSE72509 and GSE61635. The deidentified datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (32370980 and 31370879 to W.C. and 82271074 and 81670842 to W.H.) and the Natural Science Foundation of Zhejiang Province (LY17C080003 to W.C.). We thank X. Cao and N. Li (the National Key Laboratory of Medical Immunology), J. Wang, Q. Wang, H. Yao, W. Liu, D. Wang, Y. Yao, Z. Cai, L. Lu, L. Wang, W. Sheng and P. Xu (all from Zhejiang University), Y. He (Duke University), J. Ye (Huazhong Agricultural University) and L. Qi (Shanghai University) for providing experimental materials or technical assistance.
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W.C. and W.H. conceived the study, designed the experiments, interpreted the data and drafted the manuscript. Y.C. performed cellular experiments, analyzed them and generated figures. J.Z. performed flow cytometry experiments and in vitro binding and ubiquitylation assays. L. Zhao prepared RNA virus and lentivirus and assisted in cellular experiments. X.W. performed and analyzed animal experiments. L. Zhang recruited participants and collected human samples. J.W. and Y.Z. assisted in cellular experiments and interpreted the data. R.S. performed and analyzed LC–MS/MS experiments and analyzed the RNA-sequencing data. J.H. performed all quantifications of western blotting data. All the authors discussed, revised and approved the manuscript.
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Nature Immunology thanks Karen Mossman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: S. Houston, in collaboration with the Nature Immunology team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Unphosphorylated IRF3 degrades in the proteasome and phosphorylated IRF3 degrades via autophagy.
a. Immunoblotting (left panel) and quantification (right panel) of IRF3 in mouse peritoneal macrophages (PMs) pretreated with vehicle (DMSO) or MG132 (10 μM) for 1 hr, followed by cycloheximide (CHX, 50 μg/ml) treatment for the indicated time. GAPDH was used as the loading control. b. Immunoblotting of IRF3 in HEK293T cells pretreated with DMSO or MG132 (10 μM) for 1 hr, followed by CHX (50 μg/ml) treatment for the indicated time to block protein synthesis. c. Immunoblotting of IRF3 in HEK293T cells pretreated with DMSO, 3-MA (2 mM), CQ (50 μM), BafA1 (200 nM), MG132 (10 μM) or bortezomib (PS-341, 25 nM) for 1 hr, followed by CHX treatment (50 μg/ml) for 24 hr to block protein synthesis. d. Immunoblotting of phosphorylated IRF3 (p-IRF3) and IRF3 in HEK293T cells infected with Sendai virus (SeV, MOI = 1.0) for the indicated time. e. Immunoblotting of p-IRF3 and IRF3 in HEK293T cells infected with SeV (MOI = 1.0) for 48 hr, and DMSO, 3-MA (2 mM), CQ (50 μM), BafA1 (200 nM), MG132 (10 μM) or PS-341 (25 nM) was added 24 hr before cell collection. f. Schematic structure of IRF3 protein, and its truncated mutants 5 A and 5D. g, h. Immunoblotting of Flag-fusion protein in HEK293T cells that were transfected with Flag-IRF3-5A (g), or Flag-IRF3-5D and IKKε (h), and 24 hr later, were treated as in c. i-k. Immunoblotting of p-IRF3 and IRF3 in PMs pretreated with DMSO, CQ (50 μM), or Calyculin A (100 nM) for 1 hr, followed by VSV (MOI = 0.001) (i), HSV (MOI = 10) (j), or LPS (50 ng/ml) (k) treatment for the indicated time. l. Immunoblotting of p-IRF3 and IRF3 in PMs from Atg3f/f and Atg3f/f Lysm-Cre mice. Cells were infected with VSV (MOI = 0.1) in vitro for the indicated time. m, n. Immunoblotting of p-IRF3 and IRF3 in BMDMs from Atg7f/f Lysm-Cre mice (m), Atg3f/f Lysm-Cre mice (n) and control mice. Cells were infected with VSV (MOI = 10) in vitro for the indicated time. o. Immunoblotting of IRF3, Histone, and GAPDH in the cytoplasmic (Cyto.) and nuclear (Nucl.) fractions extracted from PMs treated as in d. p. Representative immunofluorescence (IF) images of co-localization among exogenous GFP-IRF3 and mCherry-LC3B and p-IRF3 in HEK293T cells transfected with IKKε. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for the indicated time, and BafA1 (200 nM) was added 12 hr before cell collection to block autophagic degradation. Scale bar, 10 μm. q, r. Immunoblotting of p-IRF3 and IRF3 in BMDMs from Atg7f/f Lysm-Cre mice (q), Atg3f/f Lysm-Cre mice (r) and control mice. BMDMs were infected with VSV (MOI = 10) for 4 hr, followed by CHX (50 μg/ml) treatment for the indicated time before cell collection. Data in a are shown as mean ± s.e.m. and represent three independent experiments. The data were analyzed using unpaired two-sided Student’s t-tests.
Extended Data Fig. 2 RAD18 is a potential E3 ligase to promote p-IRF3 degradation in autophagosomes.
a. Immunoblotting of Flag-IRF3 ubiquitylation in HEK293T cells expressing Flag-IRF3 and HA-ubiquitin (HA-Ub). Cells were infected with SeV (MOI = 1) for 36 hr, and DMSO, CQ (50 μM), BafA1 (200 nM), or 3-MA (2 mM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. b. Immunoblotting of the Flag-IRF3 dimer (Native PAGE) and Flag-IRF3 (SDS PAGE) in HEK293T cells that were transfected with Flag-IRF3 and/or IKKε as indicated, and 24 hr later, were infected with SeV (MOI = 1) for another 24 hr. c. Quantification of the candidate E3 ligases in the Flag-IRF3 complex described as in Fig. 2c, by unlabeled quantitative mass spectrometry. d. Immunoblotting of Flag-IRF3 ubiquitylation in HEK293T cells transfected with Flag-IRF3, indicated Myc-E3 ligases, and HA-Ub. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 24 hr, and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. e. Immunoblotting of p-IRF3, IRF3, and Flag-tagged protein in HEK293T cells that were transfected with Flag-RAD18 or empty vectors, and 24 hr later, were infected with SeV (MOI = 1) for the indicated time. f. Immunoblotting of p-IRF3, IRF3, and RAD18 in BMDMs that were infected with lentivirus encoding RAD18 or control lentivirus, and 24 hr later, were infected with VSV (MOI = 10) for the indicated time. g. The activity of IFNβ-luciferase (IFNβ-Luc) and ISRE-luciferase (ISRE-Luc) reporters in HEK293T cells expressing IRF3-5D and the candidate E3 ligases, detected by dual-luciferase reporter assay. h. Gene typing of Rad18−/− mice using PCR with specific primers. i. Immunoblotting of IRF3 dimer (Native PAGE), p-IRF3, IRF3, Flag-fused protein, and ATG7 (SDS PAGE) in wild-type (WT) and ATG7 KO HEK293T cells that were transfected with Flag-RAD18 or empty vector, and 24 hr later, were infected with SeV (MOI = 1) for the indicated time. j. Immunoblotting of p-IRF3 and IRF3 in HEK293T cells transfected Flag-RAD18 or empty vector. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 36 hr, and DMSO, 3-MA (2 mM), CQ (50 μM), BafA1 (200 nM), MG132 (10 μM) or PS-341 (25 nM) was added 24 hr before cell collection. k. Immunoblotting of p-IRF3, IRF3, and RAD18 in WT and RAD18 KO HEK293T cells that were infected with SeV (MOI = 1) for the indicated time, and DMSO or CQ (50 μM) was added 24 hr before cell collection. l. Immunoblotting of IRF3 dimer (Native PAGE), p-IRF3 and IRF3 (SDS PAGE) in WT, ATG7 KO or RAD18 KO HEK293T cells that were infected with SeV (MOI = 1) for the indicated time. Data in c and g are shown as mean ± s.e.m. and represent three independent samples per group. Data in g were analyzed using unpaired two-sided Student’s t-tests.
Extended Data Fig. 3 RAD18 binds p-IRF3 and triggers its K63-polyubiquitylation at Lys193 residue.
a. Reciprocal co-immunoprecipitations (Co-IPs) of endogenous p-IRF3 and RAD18 in HEK293T cells that were infected with SeV (MOI = 1) for the indicated time, and BafA1 (200 nM) was added 16 hr before collection. Cell lysates were immunoprecipitated with anti-IRF3 and anti-RAD18 antibodies. b. Representative IF images of co-localization between endogenous IRF3 and RAD18 in BMDMs that were pretreated with BafA1 (200 nM) for 2 hr and then infected with VSV (MOI = 10) for 4 hr. Scale bar, 10 μm. c. Schematic of an in vitro binding assay using separately purified IRF3 and RAD18 protein. d. Immunoblotting of Flag-IRF3 ubiquitylation in HEK293T cells transfected with Flag-IRF3, V5-RAD18 and HA-Ub (WT, KO, K6R, K11R, K27R, K29R, K33R, K48R or K63R). Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 36 hr, and BafA1 (200 nM) was added 12 hr before cell collection. e. Immunoblotting of Flag-IRF3 ubiquitylation in HEK293T cells transfected with Flag-IRF3, V5-RAD18 and HA-Ub (WT, KO, K6-only, K11-only, K27-only, K29-only, K33-only, K48-only or K63-only). Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 36 hr, and BafA1 (200 nM) was added 12 hr before cell collection. f. A structure-guided cross-species sequence alignment of full-length IRF3 showing the consensus Lysine (K) positions. The alignment was performed in UniProt (https://www.uniprot.org/) using the Clustal Omega algorithm. Lysine residue (K) in blue, Serine residue (S), and Threonine residue (T) in red. g. Immunoblotting of Flag-IRF3 ubiquitylation in IRF3−/− HEK293T cells transfected with V5-mRAD18, HA-K63-Ub and Flag-mIRF3 WT or Flag-mIRF3-K188R. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 36 hr, and BafA1 (200 nM) was added 12 hr before cell collection. h. The activity of the IFNβ-luciferase reporter in IRF3−/− HEK293T cells transfected with IKKε, RAD18, and IRF3 WT or IRF3 K → R mutants, detected by dual-luciferase reporter assay. Data in h are shown as mean ± s.e.m. and represent three biological replicates. The data were analyzed using unpaired two-sided Student’s t-tests.
Extended Data Fig. 4 The sequence of phosphorylation and ubiquitylation of IRF3 after RNA virus infection.
a. Co-IPs of Flag-RAD18 and HA-IRF3-WT, HA-IRF3-5A or HA-IRF3-5D mutant in IRF3−/− HEK293T cells transfected with or without IKKε. BafA1 (200 nM) was added at 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. b. The in vitro binding between RAD18 and activated Flag-IRF3-WT, Flag-IRF3-5A, or Flag-IRF3-5D mutant in the presence of double-strain ISRE DNA. The samples were immunoprecipitated with anti-IRF3 antibodies. c. Immunoblotting of Flag-IRF3 ubiquitylation in IRF3−/− HEK293T cells transfected with RAD18, HA-Ub-K63 and Flag-IRF3-WT, Flag-IRF3-5A or Flag-IRF3-5D mutant. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for the indicated time, and 3-MA (2 mM) was added 16 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. d. Immunoblotting of Flag-IRF3 ubiquitylation in IRF3−/− HEK293T cells transfected with RAD18, HA-Ub-K63 and Flag-IRF3 WT, Flag-IRF3-K77L or Flag-IRF3-K193R mutant. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for the indicated time, and 3-MA (2 mM) was added 16 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. e. The in vitro ubiquitylation of activated Flag-IRF3-WT, Flag-IRF3-K77L or Flag-IRF3-K193R mutant that incubated with purified Flag-RAD18 protein, recombinant E1, E2 and ubiquitin in the presence of double-strain ISRE DNA. f. The in vitro binding between RAD18 and activated Flag-IRF3-WT, Flag-IRF3-K77L or Flag-IRF3-K193R mutant in the presence of double-strain ISRE DNA. The samples were immunoprecipitated with anti-IRF3 antibodies.
Extended Data Fig. 5 The effect of E3 ligases and Pin1 on p-IRF3 degradation and the type I IFN response.
a. The relative mRNA levels of Isg15, Rantes, Cxcl10, and Ifit1 in BMDMs treated as in Fig. 3a, were determined by qPCR assay. b. Co-IPs of Flag-IRF3 and indicated V5-E3 ligase in HEK293T cells analyzed by immunoblotting. Twenty-four hours after transfection with the indicated plasmids, HEK293T cells were infected with SeV (MOI = 1) for 24 hr, and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. SE, short exposure; LE, long exposure. The red asterisks indicate the bands of indicated E3 ligases. c. Co-IPs of Flag-IRF3 and V5-Pin1 in HEK293T cells transfected with or without IKKε. BafA1 (200 nM) was added at 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. d. Immunoblotting of p-IRF3, IRF3, indicated E3 ligases and Pin1 in the indicated gene KO HEK293T cells that were infected with SeV (MOI = 1) for 36 hr. HEK293T cells were transfected with a control sgRNA or the sgRNA targeting indicated E3 ligase or Pin1, and then selected with puromycin (1 μg/ml) to get single clones of positive cells. e. The activity of IFNβ-luciferase and ISRE-luciferase reporters in HEK293T cells that were transfected with IRF3-5D plasmids and a scramble control siRNA (siCtrl) or siRNAs targeting indicated E3 ligase and Pin1, detected by dual-luciferase reporter assay. f, g. The relative mRNA levels of Isg15, Rantes, Cxcl10, and Ifit1 (f) as well as VSV gRNA levels (g) in BMDMs that were transfected with a scrambled control siRNA (siCtrl) or siRNAs targeting indicated E3 ligase or Pin1, and 36 hr later, were infected with VSV (MOI = 10) for another 8 hr. Data in a and e-g are shown as mean ± s.e.m. and represent three biological replicates. These data were analyzed using unpaired two-sided Student’s t-tests.
Extended Data Fig. 6 The interaction between IRF3 and RAD18 and the degradation of RAD18.
a. Representative IF images of the subcellular localization of endogenous IRF3 in HEK293T cells infected with SeV (MOI = 1) for the indicated time. The nucleus was stained with DAPI. Scale bar, 10 μm. b. Representative IF images of co-localization among endogenous IRF3, LC3B, and p-IRF3 in HEK293T cells that were infected with SeV (MOI = 1) for 48 hr. BafA1 (200 nM) was added 24 hr before cell collection. Scale bar, 10 μm. c. Schematic structure of IRF3 protein, its truncated mutant IRF3Δ1-57aa and point mutant IRF3-R81L. d. Co-IPs of exogenous V5-RAD18 and Flag-IRF3 WT or its indicated mutants in HEK293T cells expressing IKKε. BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. e. Representative IF images of co-localization between GFP-IRF3 mutants and mCherry-RAD18 in IRF3−/− HEK293T cells expressing IKKε. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 24 hr, and BafA1 (200 nM) was added 12 hr before cell collection. Scale bar, 10 μm. f. Schematic structure of RAD18 protein and its truncated mutants with a single domain deletion. g. Co-IPs of GFP-IRF3 and Flag-RAD18 WT or Flag-RAD18 truncated mutants in HEK293T cells that were transfected with or without IKKε, and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. h. Immunoblotting of the ubiquitylation of RAD18 in BMDMs infected with VSV (MOI = 10) for 12 hr. DMSO or MG132 (10 μM) was added 12 hr before cell collection. i, j. Immunoblotting of RAD18, p-JAK1, and JAK1 in BMDMs that were treated with different doses of mouse IFNβ(mIFNβ) for 2 hr (i) or treated with 50 ng/ml mIFNβ for the indicated time (j).
Extended Data Fig. 7 RAD18 regulates the transcriptional activity and ubiquitylation of IRF3.
a. The relative IFNB1 mRNA levels in HEK293T cells that were transfected with V5-RAD18 plasmids (RAD18 overexpression) or empty vector (Ctrl), and 24 hr after infection, were infected with SeV (MOI = 1) for the indicated time. b. The relative IFNB1 mRNA levels in WT HEK293T cells, RAD18−/− HEK293T cells, or RAD18−/− HEK293T cells reconstituted with RAD18 WT or RAD18-C28F. These cells were infected with SeV (MOI = 1) for the indicated time. c. ChIP-PCR analysis of IRF3 binding to the IFNB1 promoter in HEK293T cells shown as in a. d. ChIP-PCR analysis of IRF3 binding to the IFNB1 promoter in HEK293T cells shown as in b. e, f. Co-IPs of V5-Pin1 (e) or V5-TRIM21 (f) with Flag-IRF3 WT or Flag-IRF3-R81L mutant in HEK293T cells that were transfected with IKKε. BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. g. The relative IFNB1 mRNA levels in HEK293T cells shown as in Fig. 5f. h. Representative IF images of the subcellular localization of IRF3 in WT or Rad18−/− BMDMs before or 16 hr after VSV (MOI = 10) infection. The Rad18−/− BMDMs were also reconstituted with RAD18 WT or RAD18-C28F using a lentivirus system. Scale bar, 10 μm. i. Representative IF images of co-localization between HA-Ub-K63 and GFP-IRF3 or indicated GFP-IRF3 mutants in HEK293T cells. To activate exogenous GFP-IRF3, HEK293T cells were transfected with IKKε and infected with SeV for the indicated time. BafA1 (200 nM) was added 16 hr before cell collection. Scale bar, 10 μm. Data in a-d and g are shown as mean ± s.e.m. and represent three biological replicates. These data were analyzed using one-way ANOVA followed by a Tukey test for multiple-group comparisons.
Extended Data Fig. 8 RAD18 regulated the IRF3-p300 interaction and the transcriptional activity of IRF3/IRF7/IRF5.
a. Reciprocal Co-IPs of endogenous IRF3 and p300 in WT or RAD18−/− HEK293T cells expressing IKKε. Cells were infected with SeV (MOI = 1) for the indicated time, and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-IRF3 antibodies or anti-p300 antibodies. b. Reciprocal Co-IPs of endogenous IRF3 and p300 in WT or Rad18−/− BMDMs that were infected with VSV (MOI = 10) for the indicated time, and BafA1 (200 nM) was added 12 hr before cell collection. The Rad18−/− BMDMs were also reconstituted with RAD18 WT or RAD18-C28F using a lentivirus system. Cell lysates were immunoprecipitated with anti-IRF3 antibodies or anti-p300 antibodies. c. Co-IPs of Flag-IRF3 and endogenous p300 in IRF3−/− HEK293T cells reconstituted with Flag-IRF3-WT, Flag-IRF3-K193R or Flag-IRF3-NES*. Cells were infected with SeV (MOI = 1) for the indicated time, and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. d. Immunoblotting of the K63-ubiquitylation of p-IRF3, p-IRF7, p-IRF5 or p-p65 in WT or Rad18−/− BMDMs. Cells were infected with VSV (MOI = 10) for 8 hr, and BafA1 (200 nM) was added 6 hr before cell collection. e. Immunoblotting of p-IRF3 and IRF3 in BMDMs isolated from WT and Rad18 KO mice. Cells were infected with low-titer VSV (MOI = 0.1) for the indicated time. f. Immunoblotting of p-IRF3 and IRF3 in WT or RAD18−/− HEK293T cells that were infected with low-titer SeV (MOI = 0.01) for the indicated time. g. ChIP-PCR analysis of IRF3 binding to the IFNB1 promoter in WT or Rad18−/− BMDMs infected with low-titer VSV (MOI = 0.1) for the indicated time. h. ChIP-PCR analysis of IRF3 binding to the IFNB1 promoter in WT or RAD18−/− HEK293T cells infected with low-titer SeV (MOI = 0.01) for the indicated time. i. The relative Ifnb1 mRNA levels in BMDMs isolated from WT and Rad18 KO mice. Cells were infected with low-titer VSV (MOI = 0.1) for the indicated time. j. The relative IFNB1 mRNA levels in WT or RAD18−/− HEK293T cells that were infected with low-titer SeV (MOI = 0.01) for the indicated time. Data in c-f are shown as mean ± s.e.m. and represent three biological replicates. These data were analyzed using one-way ANOVA followed by a Tukey test for multiple-group comparisons.
Extended Data Fig. 9 OPTN delivers ubiquitylated p-IRF3 to autophagosomes.
a, b. Two round co-IPs of p-IRF3 or unphosphorylated IRF3 with Myc-NDP52 (a) or Myc-NBR1 (b) in HEK293T cells. Cells were infected with SeV (MOI = 1) for 36 hr and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were sequentially immunoprecipitated with anti-p-IRF3 antibodies or anti-IRF3 antibodies. c, d. Co-IPs of Flag-NDP52 (c) or Flag-NBR1 (d) with HA-IRF3-WT, HA-IRF3-5A, or HA-IRF3-5D mutant in HEK293T cells that were transfected with IKKε and infected with SeV (MOI = 1) for 36 hr. BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-HA antibodies. e. Knockout of OPTN, NDP52 or NBR1 in HEK293T cells using the CRISP-cas9 system, was detected by immunoblotting. f. Immunoblotting of p-IRF3 and IRF3 in BMDMs transfected with a scrambled control siRNA (siCtrl) or the siRNA targeting Optn. Cells were infected with VSV (MOI = 10), and 4 hr later CHX (50 μg/ml) was added and incubated for the indicated time. g. Representative IF images of co-localization among GFP-IRF3, Flag-OPTN and mCherry-LC3B in HEK293T cells without infection (upper panel). To activate GFP-LC3, cells were transfected with IKKε and infected with SeV (MOI = 1) for 48 hr (lower panel). BafA1 (200 nM) was added 16 hr before cell collection. Scale bar, 10 μm. h. Immunoblotting of IRF3 K63-ubiquitylation in BMDMs from Atg7f/f or Atg7f/f Lysm-Cre mice, which were infected with VSV (MOI = 10) for the indicated time. i. Co-IPs of GFP-IRF3 and Flag-OPTN in HEK293T cells expressing IKKε, and DMSO or BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. j. Immunoblotting of p-IRF3, IRF3, and Flag-fused protein in HEK293T cells transfected Flag-OPTN or empty vector. Twenty-four hours after transfection, cells were infected with SeV (MOI = 1) for 36 hr, and DMSO, 3-MA (2 mM), CQ (50 μM), BafA1 (200 nM), MG132 (10 μM) or PS-341 (25 nM) was added 24 hr before cell collection. k. Schematic structure of OPTN protein, its truncated mutant, and point mutant. l. Co-IPs of GFP-IRF3 with Flag-OPTN WT, or Flag-OPTN-S473A in HEK293T cells expressing IKKε. BafA1 (200 nM) was added at 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-Flag antibodies. m. Reciprocal Co-IPs of endogenous IRF3 and OPTN in WT or RAD18−/− HEK293T cells. Cells were infected with SeV (MOI = 1) for the indicated time and BafA1 (200 nM) was added 12 hr before cell collection. Cell lysates were immunoprecipitated with anti-IRF3 antibodies and anti-OPTN antibodies. n-p. Representative IF images of co-localization among p-IRF3 and mCherry-OPTN and HA-Ub-K63(n), co-localization among GFP-IRF3 and mCherry-OPTN and p-IRF3 (o), or co-localization among GFP-IRF3 and mCherry-OPTN and HA-Ub-K63 (p) in HEK293T cells without infection (upper panel). To activate GFP-LC3, cells were transfected with IKKε and infected with SeV (MOI = 1) for 48 hr (lower panel). BafA1 (200 nM) was added 24 hr before cell collection. Scale bar, 10 μm. q. Immunoblotting of IRF3 K63-ubiquitylation in IRF3−/− HEK293T cells that were reconstituted with IRF3 WT or its indicated mutants. Cells were co-transfected with IKKε and V5-RAD18 or empty vector, and BafA1 (200 nM) was added 12 hr before cell collection.
Extended Data Fig. 10 RAD18 deficiency and Irf3-K188R mutation result in the resistance to RNA virus infection or EAE induction.
a-f. The VSV titers (a, c and e) and relative mRNA levels of Ifnb1, Ifna, and Tnf (b, d and f) in the lung (a and b), spleen (c and d), liver (e and f) from Rad18f/f or Rad18f/f Lysm-Cre mice (n = 8) at 12 hr after non-lethal dose VSV (2×107 pfu/g, i.p.) infection, were determined by TCID50 assay and qPCR assay. g. Immunoblotting of RAD18 in human monocyte-derived macrophages (MDMs) isolated from mild subjects (n = 24) and severe subjects (n = 24) recovered from COVID-19 infection before H1N1 (MOI = 10) infection. Quantification data is shown in Fig. 7d. h. The relative RAD18 mRNA levels in MDMs shown as in g, were determined by qPCR assay. i. Immunoblotting of p-IRF3 and IRF3 in MDMs after H1N1 (MOI = 10) infection in vitro for indicated time points. j. Immunoblotting of p-IRF3 in MDMs isolated from mild subjects (n = 24) and severe subjects (n = 24) at 12 hr after in vitro H1N1 (MOI = 10) infection. Quantification data is shown in Fig. 7e. k. The relative IFNA mRNA levels in MDMs from mild subjects (n = 24) and severe subjects (n = 24) at 12 hr after H1N1 (MOI = 10) infection. l. The correlations between RAD18 protein levels and IFNA mRNA levels in MDMs from mild subjects (n = 24) and severe subjects (n = 24) (Data from k and Fig. 7d). m, n. The relative mRNA levels of Isg15, Ifit1, Rantes, and Cxcl10 (m) and VSV gRNA levels (n), in BMDMs derived from WT and Irf3K188R/K188R mice. BMDMs were infected with VSV (MOI = 10) in vitro for the indicated time. o, p. Representative histological images of spinal cord sections from WT and Irf3K188R/K188R mice on day 21 after EAE induction. The sections were stained with H&E (o) for the infiltration of inflammatory cells, and stained with luxol fast blue (LFB, p) for demyelination. Scale bar, 100μm. Data in a-f, h, k, m, n are shown as mean ± s.e.m. and represent three biological replicates. These data were analyzed using unpaired two-sided Student’s t-tests.
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Supplementary Figs. 1–7 with uncropped blots.
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Cai, Y., Zheng, J., Zhao, L. et al. E3 ligase RAD18 targets phosphorylated IRF3 to terminate IFNB1 transcription. Nat Immunol 26, 1581–1595 (2025). https://doi.org/10.1038/s41590-025-02256-x
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DOI: https://doi.org/10.1038/s41590-025-02256-x