Abstract
The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus–cell binding and virus–cell fusion, respectively4,5,6. Notably, Tmeff1−/− mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.
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Data availability
All data supporting the findings of this study are available in the paper and its Supplementary Information. Sequencing data from the CRISPR screens are available in the Gene Expression Omnibus (accession number GSE268182). Source data are provided with this paper.
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Acknowledgements
S.R.P. was supported by the European Research Council (786602), the Novo Nordisk Foundation (NNF18OC0030274 and NNF20OC0064301), the Lundbeck Foundation (R359-2020-2287), the Danish National Research Foundation (DNRF164) and the Swedish Research Council (2021-00942). Y.C. was supported by the National Natural Science Foundation of China (31971364 and 32370148), the National Key R&D Program of China (2022YFC3400205) and the Shanghai Jiao Tong University Scientific and Technological Innovation Funds Key forward-looking layout fund from Shanghai Jiao Tong University (number AF4150049). M.I. received funding from the Carlsberg Foundation (CF17-0687) and the Lundbeck Foundation (R251-2017-1124). T.H.M. was supported by the Independent Research Fund Denmark (0134-00006B), the Novo Nordisk Foundation (NNF21OC0067157 and NNF20OC0064890) and the Lundbeck Foundation (R268-2016-3927). M.D. was supported by the Lundbeck Foundation (DANDRITE-R248-2016-2518). J.G.M. was supported by the Independent Research Fund Denmark (9039-00173B), the Novo Nordisk Foundation (NNF17OC0029042) and the Carlsberg Foundation (CF21-0363). P.N. was supported by the Lundbeck Foundation (R310-2018-3713). AlphaFold calculations were carried out on a computer cluster established with support from the Department of Molecular Biology and Genetics, Aarhus University, and the Danish Research Agency through the Danish national research infrastructure for cryo-electron microscopy (EMBION 5072-00025B). The flow cytometry experiments carried out at Aarhus University were conducted at the FACS core facility.
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The study was conceived and designed by Y.C. and S.R.P. Cell experiments were designed, carried out and analysed by Y.D., M.I., M.C.S., X.P., E.A.T., R.N., M.M., X.Q., M.B.I., L.S.R., R.K.F., M.C., M.E.C.-T., X.D., B.-c.Z., Q.L., Z.J., Y.Z., S.Z., L.D., J.Z. and Y.C. (while he was a post-doctoral researcher at Aarhus university where TMEFF1 was identified). Animal experiments were designed, carried out and analysed by M.I. Data were analysed and interpreted by Y.D., M.I., P.N., T.H.M., J.G.M., S.-Y.Z., J.-L.C., Y.C. and S.R.P. Funding was acquired by M.I., M.D., T.H.M., J.G.M., Y.C. and S.R.P. The manuscript was drafted by Y.C. and S.R.P., and edited by all authors, who support the conclusions.
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Extended data figures and tables
Extended Data Fig. 1 Characterization of SK-N-SH cells and genome-wide CRISPR library fitness parameters.
a-b, SK-N-SH, SH-SY5Y, HaCaT, and THP1 cells were stimulated with polyIC in the culture medium (25 μg/mL) for 6 h, and total RNA was isolated for analysis of IFNB and MXA transcripts by RT-qPCR (n = 3 biological replicates). c, SK-N-SH, SH-SY5Y, HaCaT, and THP1 cells were stimulated with IFNβ (25 ng/mL) for 6 h, and total RNA was isolated for analysis of MXA transcripts by RT-qPCR (n = 3 biological replicates). d-e, SK-N-SH, and THP1 cells were infected with HSV-1 (MOI 1) for 8 h, and total RNA was isolated for analysis of IFNB and MXA transcripts by RT-qPCR (n = 3 biological replicates). f, The number of sgRNAs with zero read counts following mapping and quantification with MAGeCK, displayed as log10(number of zero read count sgRNAs) for each sample in the screens (n = 2 independent screens). g, Gini index of each sample as a measurement of the sgRNA read count distribution. h-i, Boxplot visualization of sgRNA read counts from mock and GFPhigh samples of each screen. Q1 (25th percentile), median, Q3 (75th percentile); Whiskers defines as minima Q1 −1.5* interquartile range and maxima Q3 + 1.5* interquartile range. The sgRNA read count average/depth is denoted for each sample. USC, unsorted controls. j, SK-N-SH cells were treated with Cas9-gRNA RNPs targeting TMEFF1, NKX2-8, CTXN1, or AAVS1. Total RNA was isolated 36 h later, and levels of TMEFF1, NKX2-8, and CTXN1 mRNA expression relative to AAVS1 controls were determined by RT-qPCR (n = 3 biological replicates). Expression of mRNA transcripts was measured by RT-qPCR (a-e + j) and data is represented as means ± s.d. and analysed using unpaired two-tailed t-test; exact P-value shown. Data are representative of 3 independent experiments.
Extended Data Fig. 2 Expression of TMEFF1 in tissues and CNS cell types.
a, Domain structure of the TMEFF1 protein. A transmembrane protein with an EGF-like and two Follistatin-like domains in the extracellular region, a transmembrane region, and a cytoplasmic domain of unknown structure. SP signal peptide; FSLD: follistatin-like domain; EGF, EGF-like domain; TM, transmembrane domain; CTD, cytoplasmic domain. b, Expression of TMEFF1 mRNA in different human tissues. TPM, transcripts per million. The graph is based on publically available data from Proteinatlas.org (date of data retrieval: August 1, 2022). c, Expression of TMEFF1 mRNA relative to BACTIN in different cells representing cell types found in the human brain. Total RNA from iPSC-derived cortical neurons, astrocytes, microglia, and oligodendrocyte progenitor cells was analyzed for expression of TMEFF1 mRNA by RT-qPCR (n = 2). d, UMI data on TMEFF1 transcripts in annotated cell types in the cerebral cortex from previously published single-nucleus RNA sequencing data (ref. 50). VSMCs, Vascular smooth muscle cells. e, Total RNA was isolated from different regions of C57BL/6 mouse brain (n = 1), analyzed for Tmeff1 mRNA levels by RT-qPCR. Data were normalized against Bactin. f, HEK293T cells were transfected with increasing amounts of mouse Tmeff1 expression plasmid and infected with HSV-1-GFP. Percentage of GFP-positive cells was evaluated 12 h p.i (n = 3). Data are shown as means of biological sample replicates ± s.d. Statistical analysis was performed using a two-tailed one-way ANOVA test followed by unpaired t-tests of means indicated by P-values (c + e). P < 0.05 was considered statistically significant.
Extended Data Fig. 3 Generation of LUHMES- and stem cell derived neurons.
a, Schematic illustration of the protocol for generation of LUHMES-derived neuron-like cells. b, Immunoblotting for TMEFF1 and vinculin in LUHMES neurons and effect of TMEFF1 sgRNA treatment. c, Confocal microscopy image of differentiated neurons stained with MAP2, betaIII-Tubulin, and DAPI, following the protocol shown in panel a (n = 3). d, LUHMES-derived neurons treated with TMEFF1 sgRNA #2 (Fig. 2b: TMEFF1 sgRNA #1) and infected with HSV-1-K64GFP (MOI 5) (n = 3 biological replicates pr time point). Cells were isolated after 8, 12, and 24 h, and examined by flow cytometry for GFP+ signal. Data are presented as % GFP+ cells. Two-tailed two-way ANOVA test followed by unpaired t-test comparing ctrl. with TMEFF1 sgRNA #2, comparing means, ±s.d. indicated by exact P-values (d).; P < 0.05 was considered statistically significant. e, Schematic illustration of the protocol for generation of ESC-derived cortical neurons. f, Immunoblotting for TMEFF1 in hESC-derived neurons and effect of TMEFF1 sgRNA treatment. g, Confocal microscopy image of differentiated neurons stained with MAP2, betaIII-Tubulin, and DAPI, following the protocol shown in panel e. Scale bar, 50 µm. h, Percentage of MAP2-positive cells, normalized to DAPI, in control and TMEFF1 sgRNA-transfected hESC-derived neurons. Immunoblots (b + f) and confocal imaging data (g) are representative of at least 3 independent experiments. Data is represented as mean ± s.d. (n = 5 biological replicates), and analysed using an unpaired two-tailed t-test. Exact P-values shown. P-values < 0.5 were considered significant. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Generation and characterization of Tmeff1−/− mice.
a, Illustration of the targeting region of the gene trapping cassette used to generate Tmeff1−/− mice. The arrows in red represent the primers used for analysis og mouse genotypes. b, PCR analysis of DNA from Tmeff1+/+ (WT), Tmeff1+/− (HET), and Tmeff1−/− (HOM) mice (littermates) using the primers pairs 1 and 2 versus 1 and 3 (Gel image representative of the standard genotyping of all HET x HET litters). c, Genomic sequences from WT and HOM mice around the genomic region targeted by the gene trapping cassette. d, Total RNA was isolated from the brains of C57BL/6N and HOM mice, analyzed for Tmeff1 mRNA levels by RT-qPCR. Data were normalized against Bactin levels (n = 3 mice /genotype). Tmeff1 expression in WT and HOM mice were compared using an unpaired two-tailed t-test. P < 0.05 was considered significant. e, Brain lysates from WT and HOM mice were immunoblotted for TMEFF1 (representative of 2 individual repeats). f-g, WT, Irf3−/−, Ifnar1−/−, and Tmeff1−/− mice were infected in the cornea with 2 × 106 PFU/eye HSV-1 (Mckrae) and monitored for f, Weight change (day 5 p.i.), and g, Disease symptoms (day 5 p.i.). Each dot represents one animal (Mock infected, WT n = 5; Infected WT n = 12, Tmeff1−/− n = 7, Irf3−/− n = 5, Ifnar1−/− n = 4). Two-tailed one-way ANOVA test for difference of means ± s.d., followed by unpaired two-tailed t-tests of means of groups indicated by P-values. P-values < 0.05 were considered statistically significant. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Immunohistochemical staining of HSV-1-infected brainstems.
a, Total RNA was isolated from microglia, neurons, and astrocytes obtained from newborn C57BL/6N mice, and analyzed for Tmeff1 mRNA levels relative to Bactin by RT-qPCR. Each dot represents one animal (n = 3). Two-tailed one-way ANOVA test for difference of means, followed by unpaired two-tailed t-tests of means of groups indicated by P-values. Error bars, ± s.d. P-values < 0.05 were considered statistically significant. b-c, Select examples of stainings visualized by fluorescence microscopy of brainstem sections from HSV-1-infected WT and Tmeff1−/− littermates (5 days p.i.) stained with DAPI, HSV-1 and, NeuN (neuron-marker) or DAPI, HSV-1, and S100 (astrocyte marker). Scale bar 10×, 50 μm; Scale bar 40×, 20 μm. Each brainstem (WT, n = 4 mice; Tmeff1−/−, n = 5 mice.) is represented by images of 3 different anatomic bregma (−5.80, −6.72, −7.20 mm) and images of left and right side HSV-1+ foci, pr. bregma site. d, Primary mouse microglia infected with HSV-1 (McKrea) at increasing MOI. Viral HSV-1 gB mRNA transcripts were evaluated by RT-qPCR and shown relative to Bactin, 24 h post infection. Each dot represents biologically independent samples (n = 3 pr genotype at different MOIs). e-g, Tmeff1−/− mice fully backcrossed for 10 generations to C57BL/6N background were infected in the cornea with HSV-1 (2 × 106 pfu/eye) and followed over time until reaching humane endpoint or recovering 100% of starting weight. e, % Weight change and f, Symptom scores. g, Survival curve (Uninfected, UI, n = 7; WT, n = 19, Tmeff1−/−, n = 17). Dead animals were censored in the graphs, and thus represented in the graphs with the weight and symptom score at the time of death. Survival was analyzed using a Log-Rank Mantel-Cox test, and a comparison of disease development (e + f, weight change and symptom score) between the groups were compared with a Mixed-Effect Analysis with Geisser-Greenhouse Correction for comparison of multiple interacting variables (time and genotype). Error bars; S.E.M., P-values < 0.05 were considered significant.
Extended Data Fig. 6 Characterization of TMEFF1 expression and function.
Expression of TMEFF1, MXA, and CCL2 in hESC-derived cortical neurons (a-c, n = 3 biological replicates) and SK-N-SH cells (d-f, n = 3 biological replicates) stimulated with IFNα (50 U/mL), IFNβ (100 U/mL), IFNγ (100 U/mL), or TNFα (50 ng/mL). for 6 h. Total RNA was isolated and examined for levels of mRNA transcripts by RT-qPCR. g, HEK293T cells were transfected with TMEFF1 or empty vector and stimulated with IFNβ at increasing concentrations (1, 3, 10, 30 U/mL). Total RNA was isolated 6 h later and examined for levels of mRNA transcripts for MXA (n = 3 biological replicates). h, HEK293T cells transfected with TMEFF1 or empty vector were treated with HSV-1 (MOI 100) at 4 °C, and stained for VP5 (HSV-1), and DAPI. Representative images are shown, scale bar = 10 um. i, HEK293T cells transfected with TMEFF1 or empty vector were treated with HSV-1 WT, ΔgD, and ΔgB (MOI 100) at 4 oC as illustrated in panel h. Following adsorption, cells were washed 3 time with ice-cold PBS. Cell-associated HSV-1 was quantified, and presented with each dot representing one cell (n = 10). j, DNA from cytoplasm/membrane or nuclear fractions of HEK293T cells transfected as indicated and infected with HSV-1 (MOI 1, 6 h). qPCR. data are presented as % viral DNA relative to infection doses (n = 10 biological replicates pr group, 4 individual experiments). k, HEK293T cells transfected with TMEFF1 or empty vector were infected with HSV-1 (MOI 1, 10, 30, 100) for 4 h. The cells were washed extensively, and cytoplasmic lysates were isolated and immunoblotted for VP5 and Vinculin (n = 4). l, HEK293T cells transfected with empty or TMEFF1 vector before infection with HSV-1 (MOI 15) for 12 h. The cells were fixed and visualized using electron microscopy. Scale bar, 1 μm. m, Quantification of virus capsids from the EM data, represented in panel k. Each data point represents one cell (n = 7). n, HEK293T cells were transfected with TMEFF1 or empty vector. Twenty four hours later, the cells were infected with HSV-1 (MOI 1) or transfected with HSV-1 DNA (500 ng/106 cells). Culture supernatants were isolated 24 h later, and viral yield was quantified by plaque assay (n = 5 biological replicates). o, LUHMES-derived WT and TMEFF1-deficient neurons were infected with HSV-1-K26GFP (MOI 50) in the presence of acyclovir. Cells were fixed after 2 h and subjected to confocal microscopy. GFP+ spots were quantified in a blinded fashion. Data are presented as capsids per nuclei (DAPI, n = 100 nuclei). P value shown in graph is for comparisons between TMEFF1 and Ctrl. p, HEK293T cells, SK-N-SH, LUHMES-derived neurons, and ESC-derived neurons were lysed and subjected to immunoblotting for TMEFF1 and the indicated cellular proteins involved in HSV-1 entry. Vinculin was included as a control (n = 2). Data are shown as means of biological sample replicates ± s.d. Statistical analysis was performed using a two-tailed one-way ANOVA test followed by unpaired two-tailed t-tests of means indicated by P-values (a-f, i-j, m-o). P < 0.05 was considered statistically significant. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 Identification and characterization of TMEFF1-interacting proteins.
a, Lysates from HEK293T cells transfected with TMEFF1-HA were subjected to immunoprecipitation with anti-HA and anti-IgG beads, and visualized by silver staining of the SDS-PAGE gel (representative of n = 2 individual experiments). A > 170 kDa band highly abundant in the anti-HA precipitate (red box) was excised for mass spectrometry analysis. b, Data from the mass spectrometry-based identification of NMHC-IIA/IIB in the anti-HA-immunoprecipitate from TMEFF1-HA-transfected cells. c, Illustration of the principle behind split-GFP, allowing identification of in situ proximity of two fusion-proteins. d, Images of GFP signals in HEK293T cells expressing GFP1-10-fused NMHC-IIA (upper row), and NMHC-IIB (lower low) in combination with GFP11-fused TMEFF1 and SARS-CoV-2 spike as control. Scale bar, 20 μm (n = 3). e, Lysates from HEK293T cells transfected with TMEFF1-HA were subjected to immunoprecipitation with anti-HA and anti-IgG beads, and immunoblotted for NMHC-IIB, NMHC-IIA, Nectin-1, and TMEFF1. f-g, Quantification of the split-GFP signals by flow cytometry. Representation of the colocalization of NMHC-IIA-GFP1-10 or NMHC-IIB-GFP1-10 with TMEFF1-GFP11 and SARS-CoV-2 Spike GFP11, respectively. The data are shown as percentage of GFP-positive cells in individual biological samples (n = 3). h-i, Immunoblots for NMHC-IIA, NMHC-IIB, Nectin-1, and Actin in lysates from parental HEK293T or SK-N-SH cells or pools of cells treated for 48 h with gRNAs/Cas9 complexes targeting MYH9, MYH10 and NECTIN1 as indicated (n = 3). j-m, Parental HEK293T cells or pools of SK-N-SH cells treated with gRNAs/Cas9 complexes targeting MYH9, MYH10, MYH9/10 DKO or NECTIN1 were infected with HSV-1 (MOI 0.5). DKO; Double Knock Out. Supernatants were isolated 20 h later and viral yield was determined by plaque assay (n = 3 biological replicates). n, Percentage identity of the amino acid sequences between human and mouse TMEFF1, Nectin-1, NMHC-IIA, and NMHC-IIB. Alignment was performed using Uniprot. o, Parental HEK293T cells or pools of cells treated with gRNAs/Cas9 complexes targeting MYH9 and MYH10 were transfected with TMEFF1 and infected with HSV-1(MOI 0.5). Supernatants were isolated 20 h later and viral yield was determined by plaque assay (n = 8 biological replicates). p, NECTIN1 deficient HEK293T cells were transfected with Nectin-1 (Nec1), HVEM and TMEFF1 as indicated and infected with HSV-1-GFP (MOI 0.5). The percentage of GFP+ cells was analyzed 12 h post infection by flow cytometry (n = 5 biological replicates). q, The GFP ratio between the TMEFF1+ and TMEFF1÷ cells within the groups expressing Nectin-1 and HVEM was calculated (for each, n = 5 biological replicates). Data are ratios for individual data points normalized to means from the relevant TMEFF1÷ group. Statistical analysis was performed using an unpaired two-tailed t-test (f, g, q) and two-tailed one-way ANOVA test for difference of means (j-m, o, p), followed by unpaired two-tailed t-tests of means ± s.d. indicated by P-values (h-j). P < 0.05 was considered statistically significant. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 8 TMEFF1 associates with NMHC-IIA and Nectin-1.
a, Illustration of the domain organization of TMEFF1 and NMHC-IIA, and Nectin-1. b, HEK293T cells were transfected with TMEFF1 and the indicated NMHC-IIA domains (Flag-tagged). Lysates were subjected to IP with anti-Flag, and immuno-blotted for TMEFF1 and Flag. EV, empty vector; CC, coiled coil. Data isrepresentative of 3 independent experiments. c, The cytoplasmic C-terminal domain of TMEFF1 (TMEFF1-C) was expressed in bacteria fused to Glutathione-S-transferase (GST). GST pull-down was mixed with cell lysates from HEK293T cells, washed, and immunoblotted for NMHC-IIA and GST. Data representative of 2 independent experiments. d, Lysates from HEK293T cells transfected with Flag-tagged NMHC-IIA Coiled coil (CC) domain (IIA-CC-Flag) and HA-tagged N and C terminal fragments of TMEFF1 were subjected to IP with anti-HA, and immunoblotted as indicated. EV, empty vector. e, Lysates from HEK293T cells transfected with Flag-tagged Nectin-1 and HA-tagged N terminal fragment of TMEFF1 were subjected to IP with anti-HA, and immunoblotted as indicated.
Extended Data Fig. 9 TMEFF1 interacts with NMHC-IIA and Nectin-1 to exert antiviral activity.
a, Model from AlphaFold prediction of TMEFF1 C-terminal domain (gold) with NMHC-II coiled-coil domain (olive green). Window: enlarged view showing the TMEFF1 residues in closest proximity to NMHC-II residues to interact. The four TMEFF1 residues labeled were selected as candidates for mutational studies. Insert lower right, plot of model predicted aligned error scores. b, Model from AlphaFold prediction of TMEFF1 ectodomain (gold) with Nectin-1 ectodomain (orange). Window: enlarged view showing the TMEFF1 residues in closest proximity to NMHC-II residues to interact. The five TMEFF1 residues labeled were selected as candidates for mutational studies. Insert lower right, plot of model predicted aligned error scores. c, Illustration of the TMEFF1-C peptide consisting of the HIV1 TAT cell-penetrating peptide fused to the peptide corresponding to the cytoplasmic domain of TMEFF1. d, Viability of HEK293T cells after treatment with increasing amounts of TMEFF1-C peptide measured by LDH assay (n = 3 biological replicates). e-f Percentage of GFP+ cells post HSV-1 infection following treatment with TMEFF1-C and different control peptides. HEK293T cells were pretreated with peptides 6 h prior to infection with HSV-1-GFP (MOI 1). Cells were analyzed for GFP 17 h post infection by flow cytometry. Ctrl, control peptide; HA, influenza hemagglutinin (n = 3 biological replicates). g, Cells were treated as in panel f, and evaluated for HSV-1 replication by immunoblotting for HSV-1 VP5 in lysates isolated 17 h post infection. Data representative of 2 independent experiments. h, Parental HEK293T cells or pools of cells treated with gRNAs/Cas9 complexes targeting MYH9 and MYH10 were treated with TMEFF1-C peptide (40 μM) and infected with HSV-1(MOI 0.5). Nuclear DNA was isolated 3 h post infection, and HSV-1 DNA was measured by PCR (n = 10 biological replicates). i, LUHMES-derived neurons were treated with TMEFF1-C or HIV-Tat peptide (40 μM) and infected with HSV-1-K26GFP (MOI 50) in the presence of acyclovir. Cells were fixed after 2 h and subjected to confocal microscopy. GFP+ spots were quantified in a blinded fashion (300 cells per group). Data are presented as capsids per nuclei (DAPI). j, C57BL/6 mice were treated with TMEFF1-C or control peptide (8 µL, 80 µM) or acyclovir (8 µL, 40 µM) and infected in the cornea with HSV-1 (2.6 × 106 PFU/eye). HSV-1 DNA levels in eye homogenates was determined on material isolated 2 days post infection (HIV-TAT and TMEFF1-C n = 8; ACV n = 10). k, Mouse brains were imaged by confocal microscope six days post infection. HSV-1 VP5 is shown in red and cell nuclei in blue. Scale bar: 1 mm (upper), 100 µm (lower). Data is represented as mean mean ± s.d. Statistical analysis was performed using two-tailed one-way ANOVA test followed by unpaired t-tests of means indicated by P-values (d-f, h-j). P < 0.05 was considered statistically significant. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 10 Model for restriction of HSV-1 replication in CNS neurons by TMEFF1.
Following HSV-1 infection at mucosal surfaces, the virus transports to the trigeminal ganglia for establishment of latency. In rare cases, the virus enters into the CNS where it can cause encephalitis. TMEFF1 is expressed preferentially in CNS neurons, and restricts HSV-1 replication by inhibiting viral entry. This involves interaction with the virus entry receptor Nectin-1 and the entry mediators NMHC-IIA/B. Created with BioRender.com.
Supplementary information
Supplementary Figures
Supplementary Figs. 1 and 2.
Supplementary Table 1
List of genes enriched in CRISPR–Cas9 screen for HSV-1 replication.
Supplementary Table 2
Guide RNAs used in the work.
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Dai, Y., Idorn, M., Serrero, M.C. et al. TMEFF1 is a neuron-specific restriction factor for herpes simplex virus. Nature 632, 383–389 (2024). https://doi.org/10.1038/s41586-024-07670-z
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DOI: https://doi.org/10.1038/s41586-024-07670-z
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