Sex as a biological variable. The sex of the mice used in this study was not considered as a biological variable.

Mice. C57BL/6 Zdhhc12–/– mice were generated via CRISPR/Cas9–mediated genome editing by Cyagen Biosciences, as described previously (18). C57BL/6 WT (GDMLAC-07) mice were obtained from Guangzhou Medical Laboratory Animal Center. In all experiments, 6- to 8-week-old mice were used. All the mice were housed in a specific pathogen–free animal facility with standard temperature conditions maintained at 20°C–26°C, relative humidity at 40%–70%, and a strict 12-hour light/12-hour dark cycle (lights on at 8 am and off at 8 pm) at Sun Yat-sen University.

Cell culture and transfection. The HEK293T cell line purchased from American Type Culture Collection (ATCC) was cultured in Dulbecco’s modified Eagle medium (DMEM; Corning, catalog 10-013-CVR) supplemented with 10% fetal bovine serum (FBS; Gibco, catalog 1099-141) and 1% l-glutamine (Gibco, catalog 25030-081). The MAVS-KO HEK293T cell line has been described previously (63). The human monocytic THP-1 cell line obtained from ATCC was cultured in RPMI 1640 medium (Gibco, catalog C22400500BT) supplemented with 10% FBS (Gibco) and 1% l-glutamine (Gibco). Monocytic THP-1 cells maintained in RPMI 1640 medium containing 100 ng/mL phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, catalog P1585) for 12 hours were differentiated into macrophages, and the macrophages were then treated with the indicated stimulus after a 24-hour resting period. PBMCs isolated from healthy donors were collected in BD Vacutainer CPT tubes. PBMCs were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco) and 1% l-glutamine (Gibco). Bone marrow cells were isolated from randomly chosen 6-week-old Zdhhhc12+/+ or Zdhhc12–/– mice and then differentiated into BMDMs cultured in 10 mL of conditioned medium (DMEM supplemented with 100 ng/mL macrophage colony-stimulating factor [PeproTech, catalog AF-315-02], 1% penicillin-streptomycin [Gibco, catalog 15140122], 10% FBS, and 1% l-glutamine), as described previously. HEK293T cells were transfected with the indicated plasmids or poly(I:C) (5 μg/mL; InvivoGen, catalog tlrl-picw) with Superluminal High-efficiency Transfection Reagent (MIKX, catalog 11231804). THP-1–derived macrophages and BMDMs were transfected with poly(I:C) (5 μg/mL) or the indicated plasmids with Lipofectamine 3000 reagent (Thermo Fisher Scientific, catalog L3000015) and jetPRIME (Polyplus-transfection, catalog 114-01), respectively. All the indicated cells were grown at 37°C in a 5% CO2 incubator.

Reagents and antibodies. Antibodies specific for TBK1 (1:1,000; catalog 3013), phospho-TBK1 (Ser172) (1:1,000; catalog 5483S), IRF3 (1:1,000; catalog 4302S), phospho-IRF3 (Ser396) (1:1,000; catalog 4947S), ubiquitin (1:1,000; catalog 58395S), ubiquitin K48 (1:1,000; catalog 4289S), and ubiquitin K63 (1:1,000; catalog 5621S) were purchased from Cell Signaling Technology. Horseradish peroxidase (HRP)–anti-FLAG (M2) (1:4,000; catalog A8592), anti–β-actin (1:5,000; catalog A1978), and FLAG beads (catalog A2220) were purchased from Sigma-Aldrich. Anti-MAVS (1:1,000; catalog sc-166583), anti-MAVS (1:1,000; catalog sc-365333), goat anti-rabbit (1:4,000; catalog sc-2004), and goat anti-mouse (1:4,000; catalog sc-2005) antibodies were purchased from Santa Cruz Biotechnology. Anti-TOMM20 (1:1,000; catalog 11802-1-AP) was purchased from the ProteinTech Group. Anti–c-Myc–HRP (1:2,000; catalog 11814150001) and anti-hemagglutinin (anti-HA)–HRP (1:3,000; catalog 12013819001) were purchased from Roche Applied Science. Alexa Fluor 488–conjugated goat anti-mouse (catalog A11029), Alexa Fluor 488–conjugated goat anti-rabbit (catalog A11034), Alexa Fluor 568–conjugated goat anti-mouse (catalog A11031), and Alexa Fluor 568–conjugated goat anti-rabbit (catalog A11036) antibodies were purchased from Invitrogen. Protein A–agarose (catalog 20333) and protein G–agarose (catalog 20399) were obtained from Pierce. EDTA-free protease inhibitor (catalog BL630B) and phosphatase inhibitor (catalog 04906837001) were purchased from Biosharp and Roche Applied Science, respectively.

Bone marrow transplant. To perform bone marrow transplantation, recipient mice (8 weeks old) were given antibiotics (gentamicin, 1 mg/mL) in their drinking water for 2 days before being given 950 cGy irradiation. Donor bone marrow cells were obtained from donor mice (6–8 weeks old), and 5 × 106 cells were intravenously injected into the recipient mice (after irradiation), followed by antibiotic-containing drinking water for 1 week. The chimeric mice were housed in a specific pathogen–free animal facility and were used for related experiments 6 weeks later.

Virus infection. Sendai virus (SeV) and vesicular stomatitis virus (VSV) were provided by Xiaofeng Qin (Suzhou Institute of Systems Medicine, Suzhou, China). HSV-1 was provided by Guoying Zhou (Guangzhou Medical University, Guangzhou, China). The cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 1. Zdhhc12+/+ mice (8 weeks old), Zdhhc12–/– mice (8 weeks old), and the indicated chimeric mice (14 weeks old) were intravenously injected with VSV (1 × 108 PFU/mouse). Blood samples were collected from the angular vein at 24 hours after VSV injection, and serum samples were isolated by centrifugation at 500g at 4°C for 15 minutes and stored at –80°C until assayed. The concentrations of IFN-β and KL-6 in the serum samples were quantified by ELISA. Additionally, the levels of AST and ALT in the serum samples were assessed by Kingmed Diagnostics. All the mice were euthanized by CO2 exposure, and the lung and liver tissues were collected for immunoblotting, quantitative PCR (qPCR), and histological analyses. For the survival experiments, Zdhhc12+/+ mice (8 weeks old), Zdhhc12–/– mice (8 weeks old), and the indicated chimeric mice (14 weeks old) were monitored for survival after intravenous injection of VSV (1 × 108 PFU/mouse).

Histological assessment of acute injury. The indicated mice were euthanized by CO2 exposure. The isolated lung and liver tissues were fixed in 4% paraformaldehyde (Meilunbio, catalog MA0192-1), embedded in paraffin, sectioned (thickness, 6 μm), and stained with H&E (Servicebio). The H&E-stained sections were examined by microscopy (DMi8, Leica).

Small interfering RNA and single-guide RNA transfection. The chemically synthesized small interfering RNAs (siRNAs) of the target genes were generated by Sangon Biotech. The single-guide RNA (sgRNA) sequences were designed using the CRISPR Design Tool (http://chopchop.cbu.uib.no/), synthesized by Sangon Biotech, and cloned and inserted into the lenti-CRISPR v2 vector (Addgene plasmid 108100) to obtain the indicated gene-targeting vector. The siRNAs and sgRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen, catalog 13778100) and Superluminal High-efficiency Transfection Reagent (MIKX, catalog 11231804), respectively, according to the manufacturers’ instructions. The sequences of the siRNAs and sgRNAs used were as follows: control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′; human ZDHHC12 siRNA #1, 5′-GGUCAGUGGUUGCGGUCCATT-3′; human ZDHHC12 siRNA #2, 5′-AGGAGGAGCUCAAAGAGGATT-3′; human TRIM31 siRNA, 5′-GGACCACAAAUCCCAUAAUTT-3′; human ZDHHC4 siRNA, 5′-CCACCAACCAGACUACUAATT-3′; mouse Zdhhc12 siRNA, 5′-GGUCUGGCCUUCAGUUCUUTT-3′; mouse Zdhhc4 siRNA, 5′-GGCUAGUGUAUGCAGAAUATT-3′; ZDHHC3 sgRNA, 5′-TTGCACGCCCTCATCATGGT-3′; ZDHHC4 sgRNA, 5′-GAAGTATTTGGCTACTGTC-3′; ZDHHC5 sgRNA, 5′-ACGGGATTTCACGTGGTTC-3′; ZDHHC6 sgRNA, 5′-TTAGGAACAACCATAGCTGT-3′; ZDHHC7 sgRNA, 5′-GTCGCCTATGCAGACTTCG-3′; ZDHHC8 sgRNA, 5′-GCTCCGCTGTACAAGAACG-3′; ZDHHC9 sgRNA, 5′-CGCCCGAGGAATCACTCCA-3′; ZDHHC12 sgRNA, 5′-AGTATCTGCAGCGCCGAAGA-3′; ZDHHC13 sgRNA, 5′-CAGACCCCACTCTTATTGA-3′; ZDHHC16 sgRNA, 5′-TGGTGTGTTCGGGCTGGCTT-3′; ZDHHC17 sgRNA, 5′-GTGCTATTGTGGATCAACT-3′; ZDHHC18 sgRNA, 5′-CAGACAAGCTTCACCGACCC-3′; ZDHHC20 sgRNA, 5′-TAGGACCAGACGACCACGA-3′.

RNA extraction and real-time qPCR. Total RNA was isolated from the indicated cells or tissues with TRIzol reagent (Invitrogen, catalog 10296010). cDNA was subsequently generated using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, catalog R323) according to the manufacturer’s instructions. Real-time qPCR analysis was performed using 2× PolarSignal SYBR Green mix Taq (with Tli RNase H) (MIKX, catalog MKG900). All the data were normalized to the expression of human GAPDH or mouse Gapdh. The following primers were used: human IFNB forward, 5′-CCTACAAAGAAGCAGCAA-3′; human IFNB reverse, 5′-TCCTCAGGGATGTCAAAG-3′; human ISG15 forward, 5′-CGCAGATCACCCAGAAGATCG-3′; human ISG15 reverse, 5′-TTCGTCGCATTTGTCCACCA-3′; human GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′; human GAPDH reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′; human ZDHHC3 forward, 5′-GCTCTCATTTCCTTGCACGC-3′; human ZDHHC3 reverse, 5′-AGTCTCTGCCATTTCTTCCCTG-3′; human ZDHHC4 forward, 5′-TGGTCTTGCAAGGGATGGTTT-3′; human ZDHHC4 reverse, 5′-GCAGCAGATAGGGCAGAAGAA-3′; human ZDHHC5 forward, 5′-TGGCAGTAATGTGTGTGGCT-3′; human ZDHHC5 reverse, 5′-GTTCATTGGTTGTGCGTCCC-3′; human ZDHHC6 forward, 5′-AGCTGCATTTGCTACCACCT-3′; human ZDHHC6 reverse, 5′-AGCCTTCTCTTCAATCCATGACT-3′; human ZDHHC7 forward, 5′-TCATGACGTGGCTTCTGGTC-3′; human ZDHHC7 reverse, 5′-CCCGTTGACCACAGAGTACC-3′; human ZDHHC8 forward, 5′-ATGGACCCTGGTGTTTTCCC-3′; human ZDHHC8 reverse, 5′-CATGCGGACCTGGATACCTC-3′; human ZDHHC9 forward, 5′-CCTGCCATCCCTGTATTTGCT-3′; human ZDHHC9 reverse, 5′-TAGCGCCCGAGGAATCACT-3′; human ZDHHC12 forward, 5′-ACCCTGGCTACGTGAATGTG-3′; human ZDHHC12 reverse, 5′-TGCAGCACCAGGCAGTATC-3′; human ZDHHC13 forward, 5′-TCCTCTTCACTGGGCCATCC-3′; human ZDHHC13 reverse, 5′-AATACTGCCAGGTGGATGCT-3′; human ZDHHC16 forward, 5′-ACCCCAGGGCAGGAATGAT-3′; human ZDHHC16 reverse, 5′-CCCACACAATTGTTTAGCCAG-3′; human ZDHHC17 forward, 5′-TACGGCAACCGGACAAAGAA-3′; human ZDHHC17 reverse, 5′-GGTCCCCTCCAAGTTGATCC-3′; human ZDHHC18 forward, 5′-TGACTGTCCCTACCTGGCTC-3′; human ZDHHC18 reverse, 5′-AGGCAGCTCATGACGAAGAA-3′; human ZDHHC20 forward, 5′-TGGAGCTCTGCGTGTTTACT-3′; human ZDHHC20 reverse, 5′-GAACAGATGGAAAGCCACAAGG-3′; mouse Ifnb forward, 5′-AGATCAACCTCACCTACAGG -3′; mouse Ifnb reverse, 5′-TCAGAAACACTGTCTGCTGG-3′; mouse Isg15 forward, 5′-TCCATGACGGTGTCAGAACT-3′; mouse Isg15 reverse, 5′-GACCCAGACTGGAAAGGGTA-3′; mouse Zdhhc12 forward, 5′-CCCTGCTGCTTTATCTGGCT-3′; mouse Zdhhc12 reverse, 5′-TGCGTTCACCCACACAGTTC-3′; mouse Zdhhc4 forward, 5′-TGATTTGTGTTGTCCTGATCTGC-3′; mouse Zdhhc4 reverse, 5′-GTGCCCCACTTGCGATTTAAG-3′; mouse Gapdh forward, 5′-GAAGGGCTCATGACCACAGT-3′; mouse Gapdh reverse, 5′-GGATGCAGGGATGATGTTCT-3′; VSV-G forward, 5′-CAAGTCAAAATGCCCAAGAGTCACA-3′; VSV-G reverse, 5′-TTTCCTTGCATTGTTCTACAGATGG-3′.

Enzyme-linked immunosorbent assay. The secretion levels of IFN-β or KL-6 in the cell supernatants or serum samples were determined by enzyme-linked immunosorbent assay (ELISA) using a human IFN-β kit (InvivoGen, catalog luex-hifnbv2), mouse IFN-β kit (InvivoGen, catalog luex-mifnbv2), or mouse KL-6 kit (JINGMEI, catalog JM-11515M1), respectively, according to the manufacturers’ protocols.

RNA sequencing analysis. Total RNA was extracted from BMDMs using TRIzol, and high-throughput sequencing was performed by Sangon Biotech Co. using the Illumina platform. Quality control of the FASTQ data was assessed with FastQC. HISAT2 was used to align the high-quality reads to the mouse reference genome (mm10) (64). The BAM files were sorted using SAMtools (https://www.htslib.org/), and the total number of reads that mapped to the genome was determined using the HTSeq-count program. The differential gene expression analysis was performed with the DESeq2 R package, and a P less than 0.05 and a fold change greater than 4 were used as the thresholds. Gene enrichment was analyzed using the clusterProfiler R package. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA) were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool, KEGG Automatic Annotation Server, and GSEA software, respectively.

Immunoblot analysis. HEK293T cells, THP-1–derived macrophages, and BMDMs were transfected or stimulated as indicated. The cells were subsequently harvested in low-salt lysis buffer (50 mM HEPES [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, and 1% Triton X-100) containing phosphatase inhibitor (Roche) and EDTA-free protease inhibitor (Biosharp), incubated on a rocker with ice for 30 minutes, and then centrifuged at 12,000g at 4°C for 15 minutes to obtain whole-cell lysates. The cell lysates were boiled with 5× SDS loading buffer at 95°C for 5 minutes and resolved by SDS-PAGE. For immunoprecipitation, the indicated whole-cell lysates were incubated with anti-FLAG beads (Sigma-Aldrich) or the appropriate antibodies plus protein A/G beads (Pierce) on a 3D shaker at 4°C overnight. The beads were washed 3 to 5 times with low-salt lysis buffer, and the immunoprecipitates were eluted with 2× SDS loading buffer for subsequent SDS-PAGE. For the MAVS ubiquitination assay, 1% SDS was added to the low-salt lysis buffer, and the cell lysates were denatured at 95°C for 5 minutes. Then, the denatured lysates were diluted with 0.1% SDS for immunoprecipitation with the indicated antibodies. The beads were washed several times by full immersion in low-salt lysis buffer, eluted with 2× SDS loading buffer, and resolved by SDS-PAGE. Proteins were transferred to Immobilon-PSQ Transfer Membranes (Millipore, catalog ISEQ00010) and incubated with the appropriate antibodies. Protein detection was performed using Immobilon-Western Chemiluminescent HRP Substrate (Millipore). Images were assayed using ChemiDoc MP System (Bio-Rad Laboratories Inc.) and Image Lab version 6.0 (Bio-Rad Laboratories Inc.).

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE). The cell lysates were harvested as described above in the Immunoblot analysis section. Then, the cell lysates were resuspended in 1× sample buffer (0.5× TBE buffer [Sigma-Aldrich, catalog T3913], 10% glycerol [Vetec, catalog V900122], 2% SDS [Ruishu Biotechnology, catalog 151-21-3], and 0.0025% bromophenol blue [Beyotime, catalog ST2258]) and loaded into a 1.5% agarose gel (containing 10% SDS). After electrophoresis in running buffer (1× TBE buffer containing 0.1% SDS) for 40 minutes at a constant voltage of 100 V at 4°C, the proteins were transferred to Immobilon-PSQ transfer membranes (Millipore, catalog ISEQ00010) for immunoblotting.

Flow cytometry. HEK293T cells were transfected with the indicated plasmids for 24 hours and then infected with VSV-eGFP for 12 hours. VSV-eGFP replication was examined by microscopy (DMi8, Leica). After trypsin digestion, the cells were washed with PBS twice and analyzed by flow cytometry using an LSR Fortessa Cell Analyzer (BD Biosciences) and FlowJo version 10.0 (Tree Star Software).

Confocal analysis. HEK293T cells (2 × 104/mL) were cultured in glass-bottom culture dishes (Nest Scientific, catalog 801002) and transfected/treated as indicated. The culture medium was subsequently gently removed. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes. After the cells were washed 3 times, precooled methyl alcohol at –20°C was added to the dishes for 20 minutes, followed by 3 washes with PBS. The cells were blocked in 6% goat serum (Boster Biological, catalog AR1009) at room temperature for 1 hour and incubated with primary antibodies diluted in 6% goat serum at 4°C overnight. After washing with PBS 3 more times, the cells were incubated with fluorescently labeled secondary antibodies (Alexa Fluor 488– or Alexa Fluor 568–conjugated antibodies against mouse or rabbit) for 1 hours. Nuclei were counterstained with DAPI (Sigma-Aldrich, catalog D9542) for 5 minutes. Confocal images were obtained using a microscope (TCS-SP8, Leica) equipped with 100×1.40 NA oil objectives and processed for gamma adjustments using Leica AS Lite. Colocalization was quantified (quantification of >12 cells) by calculation of Pearson’s correlation coefficient using ImageJ software (National Institutes of Health).

Fluorescence resonance energy transfer analysis. HEK293T cells were seeded in glass-bottom culture dishes and transfected/treated as indicated. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and mounted in PBS. A fluorescence resonance energy transfer (FRET) assay was performed with a Leica TCS SP8 confocal microscopy system using FRET SE (sensitized emission) Leica software, as described previously (18).

Acyl-biotin exchange assay. The indicated cells were collected in low-salt lysis buffer, incubated on a rocker with ice for 30 minutes, and then centrifuged at 12,000g at 4°C for 15 minutes. The cell lysates were incubated with TCEP (500 mM; Sangon Biotech, catalog 51805-45-9) at 55°C for 1 hour and then incubated with N-ethylmaleimide (NEM) (50 mM; Thermo Fisher Scientific, catalog 23030) at 4°C overnight. The NEM was then removed from the cell lysates by repeated methanol-chloroform precipitation. The protein pellets were dried at room temperature and dissolved in SDS buffer containing 4% SDS, 5 mM EDTA, 50 mM Tris (pH 8.0), and protease inhibitor at 37°C. Each sample was divided into 2 parts and mixed with the thiol-reactive biotin molecule HPDP-biotin (0.4 mM; Thermo Fisher Scientific, catalog 21341). The part for which the HAM (Sigma-Aldrich, catalog 379921) cleavage step was omitted (– HAM) was supplemented with lysis buffer, and the part for which the HAM step was included (+ HAM) was supplemented with 1 M HAM. After 5 hours of incubation at 4°C, the samples were pelleted by methanol-chloroform precipitation and resuspended in 4% SDS buffer. After dissolution, one-fifth of the samples were eluted with 5× SDS loading buffer and boiled at 95°C for 5 minutes for “input,” and the remaining samples were incubated with neutravidin agarose beads (Thermo Fisher Scientific, catalog 29200) at 4°C overnight. The beads were washed 4 times with lysis buffer, eluted with 2× SDS loading buffer, and subsequently resolved by SDS-PAGE. Quantification of the relative palmitoylation (Rel. palm) level of MAVS (normalized to “input” MAVS) was determined by ImageJ software.

Luciferase reporter assays. HEK293T cells were plated in 24-well plates and transfected with plasmids encoding the IFN-β/ISRE luciferase reporter (firefly luciferase) and pRL-TK (Renilla luciferase), together with the indicated plasmids, using Lipofectamine 2000. After 24 hours, the cells were infected or not infected with SeV (MOI = 1) for 12 hours. The cells were subsequently collected and lysed in Passive Lysis Buffer (Promega, catalog E1941). The luciferase activity was measured with a Dual-Luciferase Assay (Promega, catalog E1910) with a Luminoskan Ascent luminometer (Thermo Fisher Scientific, catalog 2805621) according to the manufacturer’s protocol. Induction firefly luciferase activity was normalized to Renilla luciferase activity.

Statistics. All the quantitative data were analyzed using GraphPad Prism (GraphPad software Inc.). Data are presented as the mean value ± SEM or ± SD, as indicated in the figure legends. Data were analyzed by 2-tailed Student’s t test for 2-group comparisons or by 1-way ANOVA for multiple comparisons. The log-rank (Mantel-Cox) test was performed for survival studies. P less than 0.05 was considered statistically significant.

Study approval. All animal experimental protocols were approved by the Animal Care Committee of Sun Yat-sen University [authorization number: SYXK (YUE) 2023-0313; Guangzhou, China]. The mice were euthanized with CO2 from compressed gas cylinders in compliance with all ethical regulations.

Data availability. The RNA sequencing data were submitted to the Sequence Read Archive database under accession number PRJNA1144358. Values for all data points in graphs are shown in the Supporting Data Values file. See complete unedited blots in the supplemental material.