The research described in this paper complies with all relevant ethics regulations and was approved by UCSD Environment, Health and Safety (EH&S) under Biohazard Use Authorization No. 2215. Animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (Assurance number A3381-01).

Cell culture and transient transfection

HEK293T cells, BHK-21 [C-13] cells and H1-HeLa cells were obtained from ATCC (CRL-3216, CCL-10 and CRL-1958, respectively) and grown in complete medium containing DMEM medum (Gibco), 10% FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Gibco). For transient transfections, HEK293T, BHK-21 or H1-HeLa cells were seeded the day before transfection in a 24-well plate (Genesee) with 500 μl of complete media. Cells were transiently transfected with 500 ng of total DNA and 1.5 μl of TransIT-X2 (Mirus Bio) following manufacturer protocol.

For generation of inducible cell lines, sequences for mCherry, IFIT2, IFIT3, or IFIT2 and IFIT3 separated by a P2A site were cloned into the Flp-In vector pcDNA5/FRT/TO. Flp-In T-REx HEK293 cells (Invitrogen, R78007) maintained in 5 μg ml⁻¹ of blasticidin were transfected at 70% confluency with mCherry or IFIT constructs and the vector containing the Flp recombinase pOG44 in a 1:10 molar ratio using TransIT-X2 (Mirus Bio). After 1 day, cells were transferred to new dishes, and on the following day, hygromycin (100 μg ml⁻¹) was added to cells. Following selection, cells were maintained in 5 μg ml⁻¹ of blasticidin and 100 μg ml⁻¹ of hygromycin. For induction of mCherry or IFIT proteins, cells were treated with 500 ng ml⁻¹ of doxycycline for 24 h.

MEFs from Ifit2^−/−11 and ΔIfit3a/b mice were prepared from day 13.5–14.5 embryos according to published protocols⁵⁴. Isolated MEFs were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cytiva), 100 U ml⁻¹ penicillin–streptomycin (Invitrogen), non-essential amino acids (Cellgro) and Glutamax (Gibco). Passage 0 (P0 MEFs) were frozen or split 1:4 when ~80% confluent (~3 days). To generate transformed MEFs, 5 × 10⁶ P1 primary MEFs were transfected with 10 μg of a plasmid (SV2)⁵⁵ encoding the SV40 T antigen under control of a CMV promoter, using FuGene reagent (Promega) (3:1 μl FuGene to μg DNA ratio). Upon achieving confluence, MEF cultures were split 1:10. This process was repeated for ~10 passages, at which time the transformed MEFs were frozen or used for experiments.

IFIT small interfering (si)RNA knockdowns

Specific siRNAs against ifit1 (Integrated DNA Technologies; 5’-UAGACGAACCCAAGGAGGCUCAAGCUU-3’), ifit2 (Horizon Discovery, M-012582-01-0050) and ifit3 (Integrated DNA Technologies, TriFECTa RNAi kit – hs.Ri.IFIT3.13.1) were obtained from their respective manufacturers. A549 cells were seeded into 24-well plates. At 24 h after seeding, cells were transfected with 20 pmol of siRNA in Lipofectamine 2000 Transfection Reagent (Invitrogen) and allowed to incubate for 24 h before being used in subsequent infection experiments or being collected for western blot to validate knockdown efficiency.

Viral stocks and infections

VSV-GFP⁵⁶ was propagated in BHK cells. For siRNA experiments, siRNA-treated A549 cells in 24-well plates were induced with 500 U ml⁻¹ of IFNα for 24 h. Cells were then infected at a multiplicity of infection (MOI) of 0.01 for 16 h before collection, and virus was quantified by plaque assay. For ectopic overexpression experiments, Flp-In T-REx HEK293 cells in 24-well plates were induced with doxycycline for 24 h. Cells were then infected at an MOI of 0.01 for 16 h before collection, and virus was quantified by plaque assay. For high-MOI experiments, Flp-In 293 cells in 96-well (imaging) or 24-well (western blotting) plates were infected at an MOI of 3.0, and cells were collected or plates were imaged at 0, 2-, 4-, 6- and 8 h post infection. For evaluation of structure-guided mutations, 293T cells cultured in 24-well plates were transfected at 6 h post seeding with IFIT constructs. At 18 h post transfection, cells were infected at an MOI of 0.05 for 16 h before imaging.

For siRNA experiments with PIV3-GFP and SeV-F1R-GFP⁵⁷, siRNA-treated A549 cells in 24-well plates were induced with 500 U ml⁻¹ of IFNα for 24 h. Cells were then infected at an MOI of 0.01, and supernatant was collected at 40 hpi. Virus was quantified using 50% tissue culture infectious dose (TCID₅₀) analysis. For ectopic expression experiments, Flp-In T-REx HEK293 cells in 24-well plates were induced with doxycycline for 24 h before being infected at an MOI of 0.1 for 48 h (PIV3) or 42 h (SeV). Supernatant was then collected, and virus was quantified using TCID₅₀.

CVB3 stocks were generated by co-transfection of CVB3-Nancy infectious clone plasmids with a plasmid expressing T7 RNA polymerase as previously described⁵⁸. For ectopic overexpression experiments, Flp-In T-REx HEK293 cells in 24-well plates were induced with doxycycline for 24 h before being infected at an MOI of 0.1. Supernatant was collected at 40 hpi and quantified by plaque assay.

Western blotting and antibodies

At 24 h post transfection, cells were resuspended in supernatant and collected, followed by centrifugation for 5 min at 587 × g. Cell pellets were washed with 1× PBS and lysed with 1× Bolt LDS sample buffer (Life Technologies) containing 5% β-mercaptoethanol at 98 °C for 7 min. The lysed samples were centrifuged at 21,130 × g for 2 min, followed by loading into 4–12% Bolt Bis-Tris Plus Mini Protein Gels (Life Technologies) with 1× Bolt MOPS SDS Running Buffer (Life Technologies). Following electrophoresis, gels were wet transferred onto nitrocellulose membranes using a Mini Blot Module (Life Technologies). Membranes were blocked with PBS-T containing 5% bovine serum albumin (BSA) (Spectrum), followed by incubation with primary antibodies (1:1,000) for VSV-G [8G5F11] and VSV-N [10G4] (Kerafast), V5 [D3H8Q] (Cell Signaling Technology), HA [3F10] (Roche), FLAG [M2] (Sigma), HaloTag (Promega), human IFIT1 [3G8] (Novus Biologicals) or GAPDH [14C10] (Cell Signaling Technology). Membranes were rinsed three times in PBS-T and then incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000; goat anti-rabbit IgG, Bio-Rad; goat anti-mouse IgG, Bio-Rad; goat anti-rat IgG, Invitrogen). Membranes were rinsed again three times in PBS-T and developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher). Blots were imaged on a Bio-Rad ChemiDoc MP using the Bio-Rad Image Lab Software suite (v.6.1.0).

Immunoprecipitations (IPs)

HEK293T cells were seeded in 6-well plates and transfected the next day with plasmids expressing the indicated IFITs (600 ng each). Cells were collected in PBS at 24 h post transfection and centrifuged at 8,500 × g for 5 min at room temperature. Cell pellets were then flash frozen in liquid nitrogen and stored at −80 °C. For immunoprecipitation, cells were thawed on ice and incubated for 15 min in 500 μl of lysis buffer [20 mM Tris-Cl (pH 8.0), 150 mM NaCl, 15 mM MgCl₂, 1% (v/v) Triton X-100, 1× Protease Inhibitor Mini Tablets (Thermo Scientific), 1 mM dithiothreitol and 4 U TURBO DNase (Invitrogen)]. Lysates were centrifuged at 500 × g for 5 min at 4 °C to pellet cell debris, and 440 μl of the supernatant was transferred to LoBind tubes (Eppendorf SE), while 50 μl of this supernatant was set aside to serve as ‘Input lysate’ control samples for immunoblots. Monoclonal anti-HA agarose beads (Sigma-Aldrich) were washed three times in 1 ml of lysis buffer, followed by centrifugation at 800 × g for 5 min at 4 °C and removal of the supernatant. A volume of 40 μl of a 1:1 agarose beads:lysis buffer mix was added to each LoBind tube containing lysate, followed by incubation on a rotator for 3.5 h at 4 °C. After incubation, samples were washed three times by centrifugation at 800 × g for 5 min 4 °C, discarding the supernatant and adding 1 ml of lysis buffer. After the final wash, the supernatant was discarded. To remove bound proteins from agarose beads, 100 μl of 2× LDS sample buffer (Life Technologies) containing 10% (v/v) 2-mercaptoethanol (Thermo Scientific) was added to the agarose beads. The same amount (100 μl) of 2× LDS sample buffer was added to input lysate control samples. Samples were heated at 98 °C for 7 min and centrifuged at maximum speed for 5 min. Samples were then loaded onto a 4–12% Bis-Tris gel (Invitrogen) and electrophoresed in 1× MOPS SDS running buffer (Invitrogen). Immunoblots for HA, FLAG, HaloTag, IFIT1 and GAPDH were performed as described above.

Plasmids, constructs and molecular cloning

The coding sequences of mouse IFIT2 (NCBI accession BC050835) and mouse IFIT3 (NCBI accession BC089563) were cloned separately into the pcDNA5/FRT/TO backbone (Invitrogen) with an N-terminal 3×FLAG tag or the pQCXIP backbone (Takara Bio) with an N-terminal HA tag, respectively, and both were cloned into the pcDNA5/FRT/TO backbone (an N-terminal HA tag, followed by IFIT3, a P2A site, a 3×FLAG tag and IFIT2). IFIT2 and IFIT3 point mutants were generated using overlapping stitch PCR and cloned into their respective backbones. The 5’ UTR and coding sequence for each VSV gene (GenBank accession number NC_038236.1) was cloned into the pQCXIP backbone with a C-terminal V5 tag. Following 5’ RACE, ‘short’ 5’ UTR constructs were generated by truncating the pQCXIP 5’ UTR sequence. The ‘long’ and ‘short’ backbone constructs expressing VSV-P-V5 were used to further clone the VSV-P fluorescence reporter plasmids by subcloning GFP in between the VSV-P 5’ UTR and V5 tag. The mCherry normalization construct was generated by subcloning mCherry and a C-terminal 3×FLAG tag into the pcDNA5/FRT/TO backbone. All additional reporter constructs (including VSV-P truncations, UTR length constructs and viral 5’ UTRs [RABV (NCBI accession NC_001542)]; [PIV3 (NCBI accession NC_001796)]; [CVB3 (NCBI accession NC_038307)]; [SeV (NC_075392.1)]) were cloned using primers and inserted into the ‘short’ UTR reporter backbone upstream of GFP-V5. All generated plasmids were sequenced across the entire inserted region to verify that no mutations were introduced during the cloning process. Plasmids and primers used in this study can be found in Supplementary Table 3. Gene fragments were ordered from Twist Bioscience or Genscript. All newly created plasmids will be made available upon request.

Protein expression and purification

For expression of the IFIT2–IFIT3 complex, we cloned codon-optimized genes encoding M. musculus Ifit2 and Ifit3 into separate plasmid vectors for expression in E. coli, with Ifit2 cloned into UC Berkeley Macrolab vector 2-BT (Addgene, 29666; ampicillin resistant) encoding a TEV protease-cleavable His6-tag, and Ifit3 cloned into UC Berkeley Macrolab vector 13S-A (Addgene, 48323; spectinomycin resistant) with no tag.

Plasmids were co-transformed into E. coli Rosetta pLysS cells (EMD Millipore) and grown overnight at 37 °C in LB plus carbenicillin and spectinomycin. Saturated overnight cultures were used to inoculate six 1-l cultures of 2XYT media plus carbenicillin and spectinomycin, and cultures were grown at 37 °C with shaking at 180 r.p.m. to an optical density at 600 nm (OD₆₀₀) of 0.8. Protein expression was induced by the addition of 0.25 mM IPTG, then cultures were shifted to 20 °C and grown another 16 h with shaking. Cells were collected by centrifugation and resuspended in nickel wash buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole pH 8.0, 2 mM beta-mercaptoethanol and 10% glycerol).

For protein purification, resuspended cells were lysed by sonication (Branson Sonifier), then cell debris was removed by centrifugation at 17,013 × g in a JA-17 rotor in an Avanti J-E centrifuge (Beckman Coulter) for 30 min. Clarified lysate was passed over a nickel column (5 ml HisTrap HP, Cytiva) in nickel wash buffer, then bound protein was eluted with nickel elution buffer (20 mM Tris-HCl pH 7.5, 75 mM NaCl, 250 mM imidazole pH 8.0, 2 mM beta-mercaptoethanol and 10% glycerol). Eluted protein was concentrated and buffer exchanged into nickel elution buffer containing 20 mM imidazole (Amicon Ultra, EMD Millipore), and the His₆-tag on IFIT2 was cleaved by addition of 1:10 w/w ratio of purified TEV protease (S219V mutant, purified in-house from expression vector pRK793; AddGene, 8827)⁵⁹, followed by incubation at 4 °C for 48 h. The reaction mixture was passed over a nickel column to remove cleaved His₆-tags, uncleaved IFIT2 and His₆-tagged TEV protease. The flow-through was concentrated, then passed over a size exclusion column (Superdex 200 Increase, Cytiva) in size exclusion buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1 mM dithiothreitol), and fractions containing both proteins were pooled and concentrated.

Cryo-EM grid preparation

Before use, UltrAuFoil 1.2/1.3 300 mesh grids were plasma cleaned for 12 s using a Solarus II plasma cleaner (Gatan). Purified IFIT2–IFIT3 at 3 mg ml⁻¹ was applied to the grid in a 3-μl drop within the environmental chamber adjusted to 4 °C temperature and ~95% humidity in a Vitrobot Mark IV system (Thermo Fisher). After a 4-s incubation, the grids were blotted with a blot force of 4 for 4 s; the sample was then plunged frozen into liquid nitrogen-cooled liquid ethane.

Cryo-EM data acquisition and image processing

All data were acquired at the UCSD Cryo-EM Facility on a Titan Krios G3 electron microscope (Thermo Fisher) operating at 300 kV and equipped with a Gatan BioContinuum energy filter. All images were collected at a nominal magnification of ×165,000 in EF-TEM mode (with a calibrated pixel size of 0.854 Å) on a Gatan K2 detector using a 20-eV slit width and a cumulative electron exposure of ~65 electrons Å⁻² over 50 frames (Supplementary Table 1). Data were collected automatically using EPU (Thermo Fisher) with aberration-free image shift with a defocus range of −1 to −2.5 µm. Data collection was monitored live using the cryoSPARC Live platform (Structura Bio)⁶⁰ where movies were patch motion corrected and patch contrast transfer function (CTF) estimated on the fly. Micrographs with a CTF estimation worse than 7 Å and/or a cumulative motion of more than 150 pixels were discarded.

An initial 484,841 particle picks were obtained with crYOLO⁶¹ picker using a general model, and these picks were imported into cryoSPARC⁶⁰. Particles were extracted with a box size of 384 pixels and Fourier cropped to 96 pixels at 3.34 Å per pixel. These particles were subjected to three rounds of two-dimensional (2D) classification, where classes with proteinaceous features were chosen to move forward. The selected particles were subjected to an ab initio reconstruction to generate a starting model and carried forward to a non-uniform (NU) refinement using C1 symmetry. These particles were then re-extracted at a box size of 384 pixels with a Fourier crop to 128 pixels (1.708 Å per pixel), and a second NU refinement was performed. The particle stack was then subjected two rounds of a 2-class heterogeneous refinement, with one volume being the volume from the previous NU refinement and the other volume from EMD-4877 (20S proteasome)⁶², followed by NU refinement. In each round, the particles that contributed to the best volume that resembled the IFIT2–IFIT3 dimer were selected. A final 3-class heterogenous refinement was performed using two IFIT2–IFIT3 volumes and a 20S proteasome volume. The particles associated with the volume that showed the best secondary structure features was selected and NU refinement was performed, resulting in a 3.51-Å-resolution map. These particles were then re-extracted at a box size of 384 pixels with no Fourier cropping (0.854 Å per pixel) and then NU refined, resulting in a 3.57-Å-resolution map. Due to heterogeneity that was observed in the map, particles were then exported from cryoSPARC into RELION⁶³, where they were extracted at a box size of 256 with a Fourier crop of 64 (3.34 Å per pixel). These particles were subjected to a round of 2D classification in which obvious junk classes were discarded. Selected particles were then re-extracted at a box size of 384 (0.854 Å per pixel) and subjected to 3D auto refinement, CTF refinement and a second 3D auto refinement⁶⁴. The particles were then Bayesian particle polished⁶⁵ before another round of two 3D auto refinements and a CTF refinement. This final particle stack was then imported back into cryoSPARC for a final NU refinement that resulted in a 3.22-Å-resolution map (Supplementary Table 1). 3DFSC was used to calculate directional Fourier shell correlation analysis for the final map⁶⁶.

To generate a starting model, we used ModelAngelo⁶⁷ and supplied our final 3.22 Å map and sequence files for IFIT2 and IFIT3. This resulting model was then iteratively real-space refined using Phenix⁶⁸ and manually adjusted in COOT⁶⁹. After the final refinement, the model was checked for accuracy in COOT (Supplementary Table 1).

Evolutionary analyses

For evolutionary analyses of primate and rodent IFIT2 and IFIT3, Uniprot reference protein sequences for human IFIT2, human IFIT3, mouse IFIT2 and mouse IFIT3 were used as a search query against NCBI’s non-redundant (NR) database using tBLASTn⁷⁰. Searches were restricted to simian primates and the Muroidea superfamily of rodents respectively. For each species, the nucleotide sequence with the highest bit score was downloaded and aligned to the human or mouse ORF nucleotide sequence using MAFFT⁷¹ implemented in Geneious software (Dotmatics; geneious.com). Poorly aligning sequences or regions were removed from subsequent analyses. Accession numbers of final sequences used for analyses are provided in Supplementary Table 2. Using these aligned sequences, FUBAR⁷² was performed on Datamonkey.org using 50 grid points and a 0.5 concentration parameter of the Dirichlet prior to infer codons evolving under positive and negative selection. Codons with a posterior probability of 0.9 or higher are given in Supplementary Table 2. PAML⁷³ was used to infer gene-wide positive selection, as well as codon-based estimates of positive selection. Aligned sequences were analysed using the NS sites models, disallowing (M7) or allowing (M8) positive selection. The P value reported is the result of a chi-squared test on twice the difference of the log likelihood (lnL) values between the two models using two degrees of freedom. Analyses were performed using two models of frequency (F61 and F3×4), and both sets of values are reported in Supplementary Table 2. For each codon model, we confirmed convergence of lnL values by performing each analysis using two starting omega (dN/dS) values (0.4 and 1.5). Positively selected codons with a posterior probability >0.90 using a Bayes empirical Bayes (BEB) analysis and the F61 codon frequency model are provided in Supplementary Table 2.

eCLIP experimental methods

Flp-In T-REx HEK293 cells in 10-cm culture dishes were induced with doxycycline for 24 h. Cells were then infected at an MOI of 3.0, and dishes were crosslinked using a UV crosslinker (254 nm; CL-1000 from UVP/Analytik Jena) at 400 mJ cm⁻². Downstream sample processing and eCLIP were performed as previously described⁷⁴, using antibodies against FLAG (M2/F1804, Sigma) and HA (HA.11/901502, Biolegend) for 293T experiments, and IFIT2 (PA3-845, Thermo Fisher) and IFIT3 (ABF1048, Millipore) for MEF experiments. Most experiments were performed in biological duplicate, except for the uninfected 293T samples (which were single replicates).

eCLIP computational analysis

Standard processing of eCLIP data was performed as previously described⁷⁴, with mapping performed to a custom genome index that included both the VSV genome and either hg19 (for 293T experiments) or mm10 (for MEF experiments). Data (Fig. 3 and Extended Data Fig. 4) are plotted as normalized reads per million (RPM), where reads per million are normalized to density of reads mapped to viral and human genomes.

Generation of Ifit3a/b DKO mice

Wild-type C57BL/6J were commercially obtained from Jackson Laboratories (Strain 000664). To generate Ifit3-deficient mice, single guide RNAs (sgRNAs) were designed to target exon two in Ifit3a and Ifit3b. Two sgRNAs were chosen that target conserved sequences between Ifit3a and Ifit3b: sgRNA-4, 5’-ATTTCACCTGGAATTTATTCNGG-3’; and sgRNA-30, 5’-AATGGCACTTCAGCTGTGGANGG-3’. Two additional sgRNAs were chosen that target only Ifit3a due to polymorphisms: sgRNA-21, 5’-AATTCGTCGACTGGTCACCTNGG-3’; and sgRNA-22, 5’-ATTCGTCGACTGGTCACCTGNGG-3’. The sgRNAs were selected on the basis of their low off-target profile. Guide RNAs and Cas9 protein were complexed and electroporated concurrently into C57BL/6J zygotes. Using this approach, we identified a mouse that had both Ifit3a and Ifit3b targeted (22-nt and 20-nt frameshift deletions, respectively), two mice in which only Ifit3a was targeted (2-nt and 119-nt frameshift deletions), and two mice in which only Ifit3b was targeted (1-nt and 2-nt frameshift insertions). After genotyping and two rounds of backcrossing, five founder lines (Ifit3a del22/Ifit3b del20, Ifit3a del2, Ifit3a del119, Ifit3b ins1 and Ifit3b ins2) were generated. The generation of gene-edited mice was accomplished with the aid of the Genome Engineering and iPSC Center, and the Department of Pathology Micro-Injection Core (Washington University School of Medicine).

5’ RACE

HEK293T cells were maintained as described above and subcultured into 6-well plates (Genesee) in 2 ml of complete media at 24 h before transfection. Cells were transiently transfected with 2,500 ng of total DNA and 7.5 μl of TransIT-X2 (Mirus Bio) following manufacturer protocol. At 24 h post transfection, cells were collected and pelleted; cell pellets were washed with 1× PBS, pelleted again, and supernatant was aspirated. RNA was extracted from pellets using the Takara Bio NucleoSpin RNA Plus kit following manufacturer protocol. Downstream processing of RNA was performed using the Takara Bio SMARTer RACE 5’/3’ kit according to manufacturer protocol.

Fluorescent reporter assay

Cells (HEK293T or inducible Flp-In lines) were maintained as described above and subcultured into 24-well plates for transfection. Transfections were performed as described above except for the addition of 100 ng of an mCherry-expressing DNA plasmid, resulting in 600 ng of total DNA transfected along with 1.8 μl of TransIT-X2. At 24 h post transfection, cells were imaged using the BioTek Cytation 5 cell imaging multimode reader. Four images were taken at fixed positions in each well using a ×20 objective lens, with each condition in two replicates; both GFP and RFP images were collected for each well position. Non-transfected wells were imaged for use in background subtraction. Images were preprocessed in the BioTek Gen5 Image Prime 3.1 software package (v.3.1.06) using the default rolling ball algorithm settings, and mean fluorescence values for GFP and RFP were quantified using the Gen5 software before being exported to Microsoft Excel 2019 (v.2507).

Image analyses

Normalized GFP intensity for each image was calculated in Microsoft Excel as follows: (experimental GFP signal−background GFP signal)/(experimental RFP signal−background RFP signal). Background GFP and RFP signals are the average of the quantified preprocessed values from 8 total images taken of non-transfected wells. Once the normalized GFP intensity was calculated for each image, we averaged the normalized GFP intensity of each set of four images (that is, for each well). Finally, we calculated the relative GFP intensity for each well by dividing the average normalized GFP intensity from each well by the average of the two IFIT-untreated wells, thus representing each data point as relative to 100%.

RT–qPCR

HEK293T cells were maintained as described above in 24-well plates. Cells were collected in 1× PBS and pelleted at 500 × g for 1 min. RNA was then extracted using the New England BioLabs Monarch Total RNA Miniprep kit according to manufacturer protocol. RNA (100 ng) was then subjected to reverse transcription using the Applied Biosystems High-Capacity cDNA Reverse Transcription kit according to manufacturer protocol. The resulting cDNA was then used to conduct quantitative PCR on the Applied Biosystems StepOnePlus machine with gene-specific primers (GFP-F, 5’-CCGACCACTACCAGCAGAACAC-3’; GFP-R, 5’-GGACCATGTGATCGCGCTTCTC-3’; 18S-F, 5’- TCGCTCGCTCCTCTCCTACTTG-3’; 18S-R, 5’- GCTGACCGGGTTGGTTTTGATCTG-3’) and the Luna Universal qPCR Master Mix according to manufacturer protocol.

Statistics and reproducibility

Statistical analyses and data visualization were performed using GraphPad Prism 10 (v.10.4.1). Tests were performed as indicated in figure legends. All error bars represent s.e.m. No statistical method was used to predetermine sample size, but our sample sizes are similar to those reported in previous publications^75,76. No data were excluded from the analyses. The experiments were not randomized. Data distribution was assumed to be normal, but this was not formally tested. Data collection and analysis were not performed blind to the conditions of the experiments, and the investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.