Human subjects
Informed consent was obtained in France in accordance with local regulations and a human-subjects research protocol approved by the institutional review board (IRB) of the Institut National de la Santé et de la Recherche Médicale (INSERM). Experiments were done in the United States and France in accordance with local regulations and with the approval of the IRB of The Rockefeller University and INSERM, respectively, to conduct human genetic and immunological studies, including those that use hPSC-derived tissue-specific cells for research purposes. Approval was obtained from the French Ethics Committee (Comité de Protection des Personnes), the French National Agency for Medicine and Health Product Safety, and INSERM in Paris (protocol C10-13) and the Rockefeller University Institutional Review Board in New York (protocol SHZ-0676). The hPSC-related work was approved by the Tri-Institutional Stem Cell Initiative Embryonic Stem Cell Research Oversight Committee (protocol 2021-05-003).
Cell culture
Primary human fibroblasts were obtained from skin biopsy specimens from controls, P1 and P2, and were cultured in DMEM (GIBCO BRL, Invitrogen) supplemented with 10% fetal calf serum (FCS) (GIBCO BRL, Invitrogen). Immortalized SV40-transformed fibroblast cell lines were created by using 4 mg of a plasmid containing T-antigen DNA to transfect about 5 million cells by electroporation. The cells were then placed in two fresh 75 cm2 flasks each containing 12 ml DMEM (GIBCO BRL, Invitrogen) supplemented with 10% FCS (GIBCO BRL, Invitrogen). SV40 fibroblast clones appeared after about 15 days. These clones were cultured and passaged for experimental use. HEK293T cells (ATCC) and HeLa cells (ATCC) were maintained in DMEM supplemented with 10% FCS. All cells were regularly checked to ensure they were negative for mycoplasma.
Whole-exome sequencing
Genomic DNA was isolated by phenol-chloroform extraction from peripheral blood cells or primary fibroblasts from the patient. DNA (3 µg) was sheared with a Covaris S2 Ultrasonicator (Covaris). An adapter-ligated library was prepared with the TruSeq DNA Sample Prep Kit (Illumina). Exome capture was done using the SureSelect Human All Exon 50 Mb kit (Agilent Technologies). Paired-end sequencing was done on an Illumina HiSeq 2000 (Illumina), generating 100-base reads. The sequences were aligned with the human genome reference sequence (hg19 build), with the Burrows-Wheeler aligner (v.0.7.12). Downstream processing was done using the Genome Analysis Toolkit (GATK, v.3.4), SAMtools (v.1.0) and Picard Tools (http://picard.sourceforge.net; v.1.92). Substitution and insertion or deletion (indel) calls were made using a GATK unified genotyper and GATK IndelGenotyperV2, respectively. All calls with a Phred-scaled single-nucleotide polymorphism quality of up to and including 20 and a read coverage of 2 or less were filtered out. All variants were annotated with annotation software developed in-house.
Mutation enrichment analysis of whole-exome sequencing data
We performed an enrichment analysis for homozygous non-synonymous and essential splice-site variants on our cohort of 319 HSE patients and 8,500 people with other infectious diseases (controls). We considered non-synonymous and essential splice-site variants with a MAF below 0.01. We searched for all variants present in the homozygous state in HSE patients, the 8,500 controls and the gnomAD database (v.2.1.1). We compared the proportions of patients and controls homozygous for variants in a logistic regression model, accounting for the ethnic heterogeneity of the cohorts by including the first five principal components of the principal component analysis (PCA). The PCA for ethnic heterogeneity was done using PLINK (v.1.9) on whole-exome sequencing and whole-genome sequence data, with the 1000 Genomes Project phase 3 public database as a reference, using more than 15,000 exonic variants with a MAF of more than 0.01 and a call rate greater than 0.99. We also performed Fisher’s exact test to compare the frequency of homozygous variants of TMEFF1 between our HSE cohort and gnomAD.
Sanger sequencing of genomic DNA
Genomic DNA samples from controls, the two patients and their parents were used as a template for the amplification of regions with 300–600 base pairs (bp) encompassing the mutation by PCR with site-specific oligonucleotide primers. The PCR products were purified by ultracentrifugation through Sephadex G-50 Superfine resin (Amersham-Pharmacia-Biotech) and sequenced with the Big Dye Terminator Cycle Sequencing Kit on an ABI Prism 3700 Genetic Analyzer (Applied Biosystems). The Applied Biosystems Foundation Data Collection (v.3.0) was used for the collection of Sanger sequencing data. SnapGene software (v.7) was used for sequence analysis.
Viral serological study by enzyme-linked immunosorbent assay and VirScan
Plasma was collected from P1 and P2 eight and ten years, respectively, after the HSE episode. Serological tests for a group of common viruses used conventional enzyme-linked immunosorbent assays to measure the antiviral IgG antibodies against various viruses, using the Liaison XL platform (Diasorin). The results were obtained in the form of relative light units but are presented here as units per millilitre of serum used. VirScan assays were used to assess antibody responses to a broader range of viruses or other pathogens on the basis of stringent cut-offs defined according to the number of non-overlapping enriched peptides, or seropositivity against a single recurrently targeted diagnostic peptide present in at least 30% of known positive samples, as previously described34. The integers on the VirScan heat map represent the number of significant non-overlapping enriched peptides of low homology for each virus recognized by the antiviral antibodies in each sample. Despite its high sensitivity and specificity, VirScan cannot detect epitopes that require post-translational modifications, or discontinuous sequences on protein fragments more than 56 amino acids in length. VirScan may also be less specific than certain nucleic acid-based tests in differentiating closely related virus strains34.
Plasmids
The TMEFF1 (accession number Q8IYR6) cDNA was inserted into the pDONOR cloning vector. Site-directed mutagenesis was used to obtain the mutant constructs indicated. All TMEFF1 and NECTIN-1 (Delta canonical isoform Q15223-1) constructs were then inserted into pTRIP for overexpression studies. For FRET assays61, CFP3A was inserted into the N-terminal region of TMEFF1 after the signal peptide, whereas pSYFP2-C1 was inserted into the N-terminal region of NECTIN-1, HVEM and PILRa after the signal peptide. The pSCFP3A-C1 and pSYFP2-C1 plasmids used for cloning were gifts from D. Gadella (Addgene plasmids #22879 and #22878). All primers used for subcloning or the production of truncated protein constructs were generated using SnapGene software (v.7). For lentiviral vector production, envelope plasmid pCMV-VSV-G, packaging plasmid PsPAX2 and transfer plasmid pTRIP were used. All constructs were resequenced to ensure that no adventitious mutations were generated during the cloning process.
Western blotting
HEK293T cells were lysed in NP-40 lysis buffer (280 mM NaCl, 50 mM Tris, pH 8, 0.2 mM EDTA, 2 mM EGTA, 10% glycerol, 0.5% NP-40) supplemented with 1 mM DTT, PhosSTOP (Roche) and cOmplete Protease Inhibitor Cocktail (Roche). The protein lysate was subjected to SDS–PAGE and the resulting bands were transferred to a nitrocellulose membrane, which was probed with unconjugated primary and secondary antibodies. An anti-GAPDH antibody (Santa Cruz Biotechnology) was used as a loading control. We used an antibody recognizing the N terminus of the TMEFF1 protein at a dilution of 1:500 (Santa Cruz Biotechnology, B4, sc-393457). This antibody, the anti-Flag (Sigma-Aldrich, A8592; 1:1,000 dilution) and anti-Myc (Cell Signaling Technology, 2040; 1:1,000 dilution) antibodies and GAPDH (sc-47724, Santa Cruz Biotechnology; 1:5,000 dilution) were purchased from commercial suppliers. The membrane was incubated overnight at 4 °C with the primary antibodies. SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher) was used to visualize HRP activity, and this signal was detected with an Amersham Imager 600 (GE Life Sciences).
Co-immunoprecipitation
Two million HEK293T cells were used to seed 6-cm dishes overnight. They were cotransfected with 2 µg empty vector or TMEFF1 plasmid and 2 µg of a Flag-tagged plasmid encoding an HSV-1 receptor (NECTIN-1, PILRa or HVEM) or an HSV-1 glycoprotein (gB, gD, gH, gL or gC). After 36–48 h, the cells were collected, washed with ice-cold PBS and lysed in IP buffer containing 1.0% (vol/vol) Triton X-100, 10% (vol/vol) glycerol, 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 75 mM NaCl and a protease-inhibitor cocktail (Roche). The crude whole-cell lysates were centrifuged and the supernatants were collected and incubated overnight at 4 °C with protein G magnetic beads (Bio-Rad) plus 2 µg anti-TMEFF1 antibodies (Santa Cruz Biotechnology, B4, sc-393457) or mouse IgG isotype control (Santa Cruz Biotechnology, sc-2025). The NECTIN-1 endogenous IP assays used 2 µg anti-NECTIN-1 antibodies (Thermo Fisher, 37-5900). For other co-IP assays, anti-Flag M2 affinity agarose beads (Sigma-Aldrich, F2426) or anti-Myc agarose beads (Sigma-Aldrich, A7470) were used in accordance with the manufacturer’s instructions. The beads were then washed three times with IP buffer and the corresponding immunoprecipitates were eluted with 1% SDS in protein loading buffer at 95 °C for 10 min. For immunoblotting, whole-cell lysates and immunoprecipitates were subjected to SDS–PAGE. The resulting bands were transferred onto PVDF membranes, which were then probed with anti-TMEFF1 (Santa Cruz Biotechnology, B4, sc-393457) at a dilution of 1:500, anti-Nectin-1 (Thermo Fisher, 37–5900; 1:250 dilution), anti-Flag M2 HRP (Sigma-Aldrich, A8592; 1:1,000 dilution), anti-c-Myc (9E10) HRP (Cell Signaling Technology, 2272 S) and anti-GAPDH mouse monoclonal (Proteintech, HRP-60004; 1:5,000 dilution) antibodies.
Immunostaining and confocal imaging
Cortical neurons were plated on MatTek 35 mm #1.5 glass coverslips (MatTek Corporation, P35G-1.5-14-C) at a density of 1.5 × 105 per cm2. The endogenous TMEFF1 in cortical neurons was stained by overnight incubation with rabbit polyclonal anti-TMEFF1 antibody (Biorbyt, orb325220) at 1:1,000 dilution, before counterstaining with goat anti-rabbit AF488 secondary antibody (Invitrogen, A11034; 1:500 dilution) for 1 h. Endogenous NECTIN-1 in HEK293T cells was stained by overnight incubation with mouse monoclonal anti-NECTIN-1 antibody (Novus Biologicals, NBP2-54643-0.1 mg) at 1:500 dilution, before counterstaining with goat anti-mouse AF647 polyclonal antibody (BioLegend, poly4053, 405322) at 1:500 dilution for 1 h. Cell surface staining was performed with the MemBrite Fix-ST 755/777 cell-surface staining kit (Biotium, 30104-T) or WGA Alexa Fluor Plus 770 (Thermo Fisher, W56134) according to the manufacturer’s protocol. AT-rich chromosomal DNA was stained with DAPI (Thermo Fisher, 62248) according to the manufacturer’s protocol. Cortical neurons were then imaged with an inverted LSM 980 laser scanning confocal microscope (Zeiss) equipped with a 60× 1.4 NA oil objective lens, 34 spectral detection channels (using a GaAsP PMT detector, two multialkali PMTs, one NIR GaAS PMT, one NIR GaAsP PMT and one Airyscan detector) and an Axiocan705 monochrome camera using Zeiss ZEN Blue acquisition software (v.3.5).
HSV-1 gD cell-surface binding assay
WT or TMEFF1-KO HEK293T cells stably expressing NECTIN-1 were plated in 48-well plates at a density of 5 × 104 cells per well and treated with recombinant HSV-1 gD with a His tag (ACRO Biosystems Inc, GLD-V52H3) for 150 min at 37 °C. Cells were collected and fixed by incubation with 4% PFA for 15 min and blocked by incubation with 6% BSA in PBS for 1 h. Cells were then stained with PE-conjugated mouse anti-NECTIN-1 (BioLegend, R1.302, 340404) at a dilution of 1:2,000 and APC-conjugated mouse anti-His-Tag (BioLegend, J095G46, 362605) antibodies for 30 min. Cells were then washed twice with PBS and analysed by flow cytometry.
Flow cytometry
For assessments of the expression of TMEFF1 at the cell surface, cells were plated in six-well plates at a density of 5 × 105 cells per well and surface-stained with purified mouse anti-TMEFF1 antibody (Santa Cruz Biotechnology, B4, sc-393457) at a dilution of 1:1,000. They were then washed three times with PBS + 1% FCS and incubated for 30 min with AF488-conjugated anti-mouse antibody diluted 1:1,000 in PBS + 1% FCS (Invitrogen, A11001). The cells were then washed twice with PBS + 1% FCS and analysed by flow cytometry. Samples were acquired on a Gallios flow cytometer (Beckman Coulter) and the results were analysed using FlowJo software (Tree Star).
For assessments of the expression of endogenous NECTIN-1 at the cell surface, HEK293T cells were plated in 48-well plates at a density of 5 × 104 cells per well and surface-stained with PE-conjugated mouse anti-NECTIN-1 (BioLegend, R1.302, #340404) at a dilution of 1:2,000 for 1 h. Cells were washed twice with PBS and analysed by flow cytometry. Samples were acquired on an LSRII flow cytometer (BD Biosciences) and the results were analysed using FlowJo software (Tree Star).
In vitro cell stimulation
SV40 fibroblasts or hPSC-derived cortical neurons were used to coat a six-well plate at a density of 1.5 × 105 cells per cm2. For the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 µg ml−1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h). For the assessment of HSV-1-induced responses, cells were infected with HSV-1 (MOI 1) and collected for assessment by RT–qPCR 24 h after infection. To assess type I interferon responses, cells were stimulated with either IFNα2b (Schering, NDC-0085-0120-02; 1,000 units per ml) or IFNβ (PBL Assay Science 11415-1; 100 units per ml) and collected for assessment by RT–qPCR 8 h after stimulation.
RT–qPCR
RNA was isolated from commercially available human tissues (Clonetech, 636643), peripheral blood mononuclear cells, fibroblasts, hPSC-derived cortical neurons, HeLa or HEK293T cells with and without plasmid transfection using the Quick-RNA MicroPrep Kit and Zymo-Spin IC Columns (R1051, Zymo Research), according to the manufacturer’s protocol. We extracted mRNA from the cells with a cell-to-CT kit (AM1729, Thermo Fisher), according to the manufacturer’s instructions. RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization. Results were analysed using Applied Biosystems 7500 software (v.2.0.6) and are expressed according to the ΔΔCt method, as described in the manufacturer’s kit.
Bulk RNA-seq and analysis
RNA was extracted from hPSC-derived cortical neurons using the Quick-RNA MicroPre Kit (R1051, Zymo Research). RNA-seq libraries were prepared with the Illumina RiboZero TruSeq Stranded Total RNA Library Prep Kit (Illumina) and RNA-seq was done on the Illumina NovaSeq platform, with a read length of 100 bp and a read depth of around 40 million reads. All samples were sequenced in technical duplicates. All FASTQ files passed quality control and were aligned with the GRCh38 reference genome with STAR (2.6.1d). Gene-level features were quantified using featureCounts v.1.6.0 based on GRCh38 gene annotation. Count data were normalized using counts per million in the EdgeR package (v.3.40.2)62, dimension-reduced through PCA and subjected to heat-map analysis using ComplexHeatmap (v.2.14.0)63. Differential expression analysis was done using DESeq2 (v.1.38.3)64. For the isoform-level analysis of NECTIN1, RNA-seq FASTQ files were pseudo-aligned with transcriptome indices (Ensembl release 110) with Kallisto (v.0.48.0)65. Transcriptomics-level features were quantified and normalized as transcripts per million. Duplicates were studied for each set of conditions and mean gene expression levels were used for subsequent analyses.
hPSC culture and characterization
Patient-specific iPSCs were obtained by reprogramming the patients’ primary fibroblasts by infection with the non-integrating CytoTune Sendai viral vector kit (Life Technologies). Human embryonic stem cell (hESC) or iPSC (together referred to as hPSC) cultures were maintained in Essential 8 medium (Life Technologies, A1517001). We used one healthy control hESC line (H9, which we were allowed to use following the Materials Transfer Agreement from WiCell for the described experiments), one healthy control iPSC line (BJ1) and iPSCs from other HSE patients with autosomal recessive TLR3 or IFNAR1 deficiencies (TLR3−/− and IFNAR1−/−) that were made available from our previous studies22,24. We also used two gene-edited TMEFF1-KO iPSC lines and two patient-specific TMEFF1-mutated iPSC lines that were made available through this study. The newly derived iPSC lines were verified for the elimination of transgene expression before biobanking and experimental use24. Patient-specific TMEFF1 mutations were confirmed by the Sanger sequencing of genomic DNA extracted from patient iPSC lines. All hPSCs were karyotyped to ensure that the genome was intact.
Differentiation of cortical neurons from hPSCs
Cortical neurons were differentiated from hPSCs grown in E8 essential medium on 10-cm plates coated with VTN-N (Thermo Fisher). Cells were maintained at 37 °C in an atmosphere containing 5% CO2. The hPSCs were differentiated into cortical neurons according to a published protocol37. In brief, hPSCs were dissociated by Accutase (Innovative Cell Technologies, AT-104) treatment to obtain a single-cell suspension, which was plated at a density of 300,000 cells per cm2 in Essential 8 medium supplemented with ROCK inhibitor (Y-27632 dihydrochloride, 10 µM; Tocris, 1254) on Matrigel-coated plates. Cells were cultured in Essential 8 medium containing LDN193189 (100 nM) (Stemgent, 04-0074) and SB431542 (10 µM) (STEMCELL Technologies, 72234) for 10 days, with the addition of XAV939 (2 µM; Tocris, 3748/10) for the first three days of differentiation. Cells were allowed to differentiate for 11–20 days in N2-based medium (Gibco, 17502048) supplemented 1:1,000 with B27 (Gibco, 12587010) to promote the development of neural progenitor cells. These cells were then dissociated and replated on polyornithine/fibronectin/laminin-coated plates and maintained in Neurobasal medium supplemented with BDNF (R&D Systems, 248-BD), ascorbic acid (L-AA, Sigma-Aldrich, A4034), GDNF (Peprotech, 450-10), cAMP (Sigma-Aldrich, D0627), l-glutamine (Gibco, 35050061) and B27 (Gibco, 12587010) to promote neuronal differentiation and maturation. All experiments were performed on hPSC-derived cortical neurons at DIV 50.
TOPO cloning and sequencing of cDNAs from patient cells
Total RNA was extracted using the RNeasy mini kit (Qiagen) from SV40-transformed fibroblasts and hPSC-derived cortical neurons. The RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System (Thermo Fisher), according to the manufacturer’s instructions. PCR was performed with 2× Taq PCR master mix (APExBIO) and the TMEFF1 primers (forward, 5′-GCCTTGCCCTGAAAACCTCA-3′; reverse, 5′- GTTCATGCGATAGGCAGTGTC-3′). The PCR products were inserted into the pCR2.1-TOPO vector (Life Technologies) and used to transform Stellar competent cells (Takara Bio). At least 100 colonies per subject were picked for P1 and a healthy control. Finally, we performed PCR on these colonies and sequenced them with TMEFF1 primers (forward, 5′-GTAAAACGACGGCCAG-3′; reverse: 5′-CAGGAAACAGCTATGAC-3′).
CRISPR–Cas9-mediated gene knockout
Gene-editing experiments were performed as previously described36. In brief, guide RNA sequences were generated using the CRISPR design tool (http://crispr.mit.edu/). The Cas9 target site for human TMEFF1 is 5′-GATGAGTCATCATGTAAATA-3′. The guide RNA was inserted into the pSpCas9(BB)-2A-EGFP vector, which was then used to transfect BJ1 iPSC cells in the presence of Lipofectamine LTX (Invitrogen); 24 h later, EGFP signals were sorted by flow cytometry on cells in a 96-well plate. The single-cell clones were genotyped by Sanger sequencing. For BJ1 TMEFF1-KO genotyping, the following primers were used in this study (forward, 5′-GCAGGATTCTGTTTGGGGAATAC-3′; reverse, 5′-CCTCTCAATTCCATGCAAGCAG-3′).
Gene editing was performed using HEK293T or HeLa cells by electroporation with the Synthego CRISPR Gene Knockout Kit v.2. The Cas9 target site for human TMEFF1 is 5′-CAGCCGGAGCGGCGCCTCAG-3′, 5′-CGCGCGTCCAACCAGCCCCC-3′ and 5′-ATGCTCTTGCCTTTGCCGCC-3′, and the target site for human NECTIN1 is 5′-GCAGGAATTCCACACGCTCG-3′, 5′-GTAGATGGCCACGTTCTGCT-3′ and 5′-GTGATCTTCACGCTGGGAAG-3′. Bulk or single-cell clones were genotyped by Sanger sequencing. For HEK293T and HeLa TMEFF1-KO genotyping, the following primers were used: forward, 5′-CACAAAGGGAAGGCGAGGA-3’; reverse, 5′-CCACGACGGGGTCTTTCC-3′. For HEK293T and HeLa NECTIN1-KO genotyping, the following primers were used: forward, 5′-TCTGGATGAACAGGGAGGGG-3′; reverse, 5′-AACTGTGTGGGTGGGGG-3′.
HSV-1 entry assay
We cultured iPSC-derived cortical neurons as described above. The delivery of β-lactamase to cells through HSV-1 entry was assessed in the CCF2 assay, as previously described47. The medium was replaced with 0.6 µl CCF2-AM, 5.4 µl solution B, 79 µl solution C (CCF2-AM Live-Blazer dye solution; Invitrogen, K1032), 15 µl probenecid and 500 µl of the initial medium. Cells were incubated for 40 min at 37 °C under an atmosphere containing 5% CO2 to load the cytosol with the CCF2 substrate. The cells were then washed three times with medium and infected at a MOI of 100 with HSV-1 in which β-lactamase was fused to the C terminus of pUL47 (HSVF-GS#6389) or WT HSV-1 (GS#2695) as a negative control. One hour after infection, the cells were imaged using an Olympus IX-70 inverted microscope equipped with a 40×, 1.3 NA oil objective lens, DAPI (excitation filter 381–399 nm, emission filter 435/48 nm) and FITC (emission filter 525/50 nm) filter sets and a pco.edge scientific complementary metal–oxide–semiconductor (sCMOS) camera using the SoftWoRx acquisition software (V6.5.2). Three sequential images of a single field were captured, the first being a fluorescence image obtained with a 381–399 nm excitation filter and a 435/48 nm emission filter. The second image was obtained with a 381–399 nm excitation filter and a 525/50 nm emission filter, and the final image was a bright-field image. CCF2 cleavage was quantified by drawing a region of interest (ROI) in the centre of the cell on the bright-field image and then determining the mean fluorescence intensity in the ROI for the 435 nm and 525 nm images. Ratiometric values were calculated by dividing the mean fluorescence intensity of the 435 nm ROI by the corresponding mean fluorescence intensity of the 525 nm ROI. Ratiometric values were obtained for three independent experiments with at least 30 ROIs recorded for each set of conditions in each experiment. Each dot shown in the results represents a measurement of intracellular CCF2 cleavage from one cell. Image analysis was performed with ImageJ software (v.2.3.0/1.53f). Statistical analysis was done using GraphPad Prism 9 (v.9.2.0).
HSV-1 nuclear translocation reporter assay in cortical neurons
The iPSC-derived cortical neurons were infected with HSV-1 fused to the immediate–early RFP reporter (HSVF-GS#3217)66,67,68,69. Cells were fixed with 4% paraformaldehyde in PBS 10 h after infection. Cortical neurons were stained with anti-MAP2 antibody at a dilution of 1:1,000 (Abcam, ab11267) and were then counterstained with anti-mouse IgG Alexa Fluor 488 at a dilution of 1:500 (Invitrogen, A11001) antibody. Nuclei were labelled with DAPI (Thermo Fisher, 62248). Images were captured with an Olympus IX-70 inverted microscope equipped with a 40×, 1.3 NA oil objective lens and a pco.edge scientific complementary metal–oxide semiconductor camera. A ROI was drawn at the centre of the nucleus of cortical neurons expressing MAP2, and RFP intensity was measured in it. Cells were considered to be infected if their RFP emissions were more than 3.1 times higher than the background of the field. We analysed a minimum of 50 cells per condition. Images were acquired for at least three independent experiments. Image analysis was performed with ImageJ software (v.2.3.0/1.53f). Statistical analysis was performed using GraphPad Prism 9 (v.9.2.0).
HSV-1 nuclear translocation reporter assay in HEK293T and HeLa cells overexpressing TMEFF1
HEK293T and HeLa cells transiently transfected with TMEFF1 variants were infected with HSVF-GS#3217. Cells were fixed with 4% paraformaldehyde 10 hours after infection. Cells were permeabilized with 0.1% Triton-X100 (Sigma-Aldrich) in PBS (PBST) and blocked by incubation with 6% goat serum in PBST for one hour. Cells were then stained by overnight incubation with mouse anti-TMEFF1 antibody at a dilution of 1:250 (Santa Cruz Biotechnology, B4, sc-393457) and counterstained by incubation with anti-mouse IgG Alexa Fluor 488 antibodies at a dilution of 1:500 (Invitrogen, A11001). Nuclei were labelled with DAPI (Thermo Fisher, 62248). Images were captured with an LSM 880 Airyscan NLO inverted laser scanning confocal and multiphoton microscope equipped with a 63×, 1.4 NA oil objective lens using Zeiss ZEN Black acquisition software (v.2.3 SP1). A ROI was drawn at the centre of the nucleus of cells overexpressing TMEFF1, and RFP intensity was measured in it.
HSV-1 infection and quantification of viral replication
For WT HSV-1 (KOS strain, ATCC) infection, 1.75 × 105 cortical neurons per well were used to seed 48-well plates. The cells were infected at an MOI of 0.001 in neuron culture medium. After 2 h, the cells were washed and 250 μl fresh medium was added to each well. Both cells and supernatants were collected at various time points after HSV-1 infection and frozen. HSV-1 titres were determined by calculating the TCID50 per ml on Vero cells (ATCC), as previously described21.
Statistical analysis
Where applicable, results are presented as mean ± s.e.m. or median ± interquartile range. Mean values were compared between control cells and cells from the patients in one-way ANOVA with Tukey tests for multiple comparisons. Where indicated, linear mixed models were used for log-transformed relative values to account for repeated measurements. Kruskal–Wallis tests with Dunn’s test for multiple comparisons were used if the data were found to follow a non-normal distribution. Two-tailed Mann–Whitney U tests were used for comparisons between two groups. No blinding or randomization were used in this study. Statistical analysis was performed using SPSS 19.0 and GraphPad Prism 9 (v.9.2.0). Statistical significance is denoted as follows: NS, not significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
