Abstract
With the near eradication of poliovirus due to global vaccination campaigns, attention has shifted to other enteroviruses that can cause polio-like paralysis syndrome (now termed acute flaccid myelitis)1,2,3. In particular, enterovirus D68 (EV-D68) is believed to be the main driver of epidemic outbreaks of acute flaccid myelitis in recent years4, yet not much is known about EV-D68 host interactions. EV-D68 is a respiratory virus5 but, in rare cases, can spread to the central nervous system to cause severe neuropathogenesis. Here we use genome-scale CRISPR screens to identify the poorly characterized multipass membrane transporter MFSD6 as a host entry factor for EV-D68. Knockout of MFSD6 expression abrogated EV-D68 infection in cell lines and primary cells corresponding to respiratory and neural cells. MFSD6 localized to the plasma membrane and was required for viral entry into host cells. MFSD6 bound directly to EV-D68 particles through its extracellular, third loop (L3). We determined the cryo-electron microscopy structure of EV-D68 in a complex with MFSD6 L3, revealing the interaction interface. A decoy receptor, engineered by fusing MFSD6 L3 to Fc, blocked EV-D68 infection of human primary lung epithelial cells and provided near-complete protection in a lethal mouse model of EV-D68 infection. Collectively, our results reveal MFSD6 as an entry receptor for EV-D68, and support the targeting of MFSD6 as a potential mechanism to combat infections by this emerging pathogen with pandemic potential.
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Data availability
The FASTQ files for CRISPR screens generated in this study have been deposited at Array Express (E-MTAB-14765). The MAGeCK analysis of the CRISPR screens and datasets analysed during the current study are appended as Supplementary Data. MFSD6 expression data (https://www.proteinatlas.org/ENSG00000151690-MFSD6/tissue) were retrieved from the Human Protein Atlas (v.24.0). The cryo-EM maps generated in this study have been deposited at the EMDB under accession numbers EMD-48705 and EMD-48713 for the EV-D68 apo and EV-D68 + Fc-MFSD6(L3) maps, respectively. The cryo-EM models generated in this study have been deposited at the PDB under accession numbers 9MWZ and 9MXC for the EV-D68 apo and EV-D68 + Fc-MFSD6(L3) models, respectively. Source MS data are available at the MassIVE repository under identifier MSV000096996 and the PRIDE repository via ProteomeXchange under identifier PXD060344. The default pGlyco human N-glycan database was used for proteomics data analysis53. Source data are provided with this paper.
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Acknowledgements
We thank the members of J.E.C.’s laboratory and the members of W.C.’s laboratory, especially A. Dupzyk, B. Waldman and F. Cooper, for their feedback and insights; I. L. Weissman, G. Tender and C. Xiao for their advice and feedback; M. Amieva and D. Monack for the use of their confocal microscope; D. Monack for the use of her tissue homogenizer; the members of the ISCRBM FACS Core for the use of their Sony MA900 system. Schematics in Figs. 1a and 2f,g were created using BioRender. This work was funded in part by the National Institutes of Health (NIH) T32G Stanford Cellular and Molecular Biology Training Program M007276 (to L.V.); NIH T32 AI00732 (to C.E.P.); NIH R01 CA200423 (to C.R.B.); NIH R01 AI169467 (to J.E.C.); NIH R01 AI153169 (to J.E.C.); NIH R01 AI140186 (to J.E.C.); National Science Foundation (NSF) GRFP DGE-1656518 (to L.X.), NSF DGE-1656518 (to C.E.P.) and NSF DGE-2146755 (to S.R.); Damon Runyon Cancer Research Foundation DRG-2526-24 (to D.S.R.); Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease (to J.E.C.); Stanford Maternal & Child Health Research Institute (to W.Q.); European Union’s Horizon Research and Innovation program PANVIPREP (grant number 101003627) (to F.J.M.v.K.); and EU Horizon ERC Advanced Grant program VIRLUMINOUS (grant number 01053576) (to F.J.M.v.K). Cell sorting was done through the Stanford Shared FACS Facility (RRID: SCR_017788) using either the BD FACSAria II (funded by NIH S10RR025518-01) or BD FACSFusion (purchased by the Parker Institute for Cancer Immunology) sorter. Cryo-EM data were collected at the Stanford-SLAC CryoEM Center (supported by the NIH Common Fund Transformative High-Resolution Cryo-Electron Microscopy program U24GM129541 to W.C.).
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Contributions
C.E.P., Y.S.O. and J.D. contributed to project conception. L.V., L.X. and J.E.C. contributed to experimental design. C.E.P., Y.S.O., J.D., W.Q. and C.M.R. contributed to CRISPR library construction. L.V. and C.E.P. performed CRISPR–Cas9 knockout screens and L.V. and W.Q. performed data analysis. L.V. engineered cell lines, propagated viruses, generated the replicon and cloned Fc constructs. L.V. conducted the replicon assay, IP assay, cell inhibition assay, RT–qPCR and western blots. L.V. and L.X. generated knockout cells and performed PFU production assays. L.X. purified virus and Fc constructs, conducted cell surface biotinylation, and viral binding and internalization assays. S.R., L.X. and L.V. engineered iPS cells and differentiated iPS cells. L.V., L.X., C.E.P., S.R. and J.C. performed immunofluorescence imaging and L.X., C.E.P. and S.J. performed confocal data analysis. L.X. conducted cryo-EM imaging and data processing. G.P. conducted modelling and generated the interaction video. W.C. provided structural insights. L.V., L.X. and C.M.N. performed in vivo mouse experiments. D.S.R. conducted MS and analysis. C.R.B. provided insights on glycobiology. M.L., E.d.V. and F.J.M.v.K. conducted BLI and analysis. L.V., L.X. and J.E.C. interpreted the experimental data and wrote the manuscript. L.V., L.X. and G.P. generated figures. W.C. and J.E.C. supervised the research. All of the authors read and approved the final version of this manuscript before submission.
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J.E.C. consulted for Janssen BioPharma on topics unrelated to this study. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Expression of MFSD6 and characterization of ΔMFSD6 cells.
(a) RNA expression of MFSD6 from Human Protein Atlas80,81 using the GTEx dataset. nTPM is the normalized protein-coding transcripts per million. (b) Percentage of edited DNA harvested from pooled knockouts (KOs). Analysis was done with CRISPResso2. (c) Detection of MFSD6 by western blot in A549 cells (top) and U87-MG cells (bottom) using an endogenous MFSD6 antibody. The black triangle points to MFSD6. (d) Representative immunofluorescence images, zoomed in, from two independent experiments for A549 wild-type (left), A549 ΔMFSD6 cells (middle left), U87-MG wild-type (middle right), and U87-MG ΔMFSD6 cells (right) infected with EV-D68 (MOI = 5) for 24 h and stained for EV-D68 VP1 (green). Nuclei are labelled with Hoechst (blue). Percentages indicate the mean with s.e.m. (n = 5 images). Over 500 cells were imaged for each condition. Scale bar, 25 μm.
Extended Data Fig. 2 Characterization of MFSD6 clonal knockouts made in iPSC-derived neurons.
(a) Sanger sequencing of wild-type cells and MFSD6 knockout clones in iPSC-derived neurons. Two ΔMFSD6 clones were generated. ΔMFSD6 1 has mixed sequencing and ΔMFSD6 2 has a 99 bp deletion. The black underline refers to the guide sequence, the red underline is the PAM site, and the black dash is the cleavage site. Sequences were analysed using CRISP-ID and Synthego Performance Analysis, ICE Analysis. (b) Representative immunofluorescence images, zoomed in, from two independent experiments for non-target cells and ΔMFSD6 cells. Cells are co-stained with the neuronal marker TUJ1 (green) and DAPI (blue). A full 96-well scan of images containing over 500 cells was taken for each condition. Scale bars, 5 μm.
Extended Data Fig. 3 Characterization of MFSD6 and SLC35A1 clonal knockout cell lines.
(a) Sanger sequencing of wild-type cells and MFSD6 knockout clones in A549 and U87-MG cells. The A549 clone has a one base pair insertion. The U87-MG clone has a one base pair and two base pair insertion. The black underline refers to the guide sequence, the red underline is the PAM site, and the black dash is the cleavage site. Sequences were analysed using CRISP-ID and Synthego Performance Analysis, ICE Analysis. (b) Sanger sequencing of wild-type cells and SLC35A1 knockout clones in A549 and U87-MG cells. The A549 clone has a two base pair deletion. The U87-MG clone has a two base pair insertion and other mixed sequencing. The black underline refers to the guide sequence, the red underline is the PAM site, and the black dash is the cleavage site. Sequences were analysed using CRISP-ID and Synthego Performance Analysis, ICE Analysis. (c) Representative immunofluorescence images, zoomed in, from two independent experiments for WT, MFSD6 knockout (KO), MFSD6 add back (AB) , and SLC35A1 knockout cells in A549 and U87-MG cell lines (n = 5 images for each cell line). Cells are co-stained with SNA-FITC (green) and nuclei are stained with Hoechst (blue). Over 50 cells were imaged for each condition. Scale bar for A549 cells, 10 μm. Scale bar for U87-MG cells, 20 μm.
Extended Data Fig. 4 Characterization of other enteroviruses and other EV-D68 strains in ΔMFSD6 cells.
(a) Dendrogram of different strains of EV-D68 used for infection. Dendrogram was based on Nextstrain82,83. (b-c) Wild-type, ΔMFSD6, and ΔMFSD6 + A549 cells were infected (MOI = 1), and harvested for PFU production after 24 h (n = 3, biological replicates) (b) or stained for crystal violet after 5 days (c). (d) RT–qPCR analysis of various enteroviruses in U87-MG cells. Cells were infected (MOI = 5), and harvested 24 h post-infection. Samples were normalized to WT as 100% infection (n = 3, biological replicates). (e) Crystal violet stains of wild-type, ΔMFSD6, ΔMFSD6 + , and ΔMFSD6 + MFSD6-mNeon A549 cells infected with EV-D68 (MOI = 1) and fixed after 5 days. (f) Schematic of EV-D68 replicon. Renilla luciferase replaces VP4, VP2, and part of VP3. Datasets show mean and error bars show s.e.m. All P values are indicated and were determined by one-way ANOVA (Holm–Sidak corrected) on log-transformed data.
Extended Data Fig. 5 Characterization and validation of A549 ΔMFSD6+ overexpression constructs, Fc constructs, and purified EV-D68.
(a) Western blot for MFSD6 and MFSD8 in A549 wild-type, ΔMFSD6, ΔMFSD6+ , ΔMFSD6 + MFSD6ΔL3, MFSD6 + MFSD8, ΔMFSD6 + MFSD8(LS), and ΔMFSD6 + MFSD6-mNeon cells. The black triangle indicates MFSD6 or MFSD8. (b) Coomassie stain of purified Fc constructs (left) and purified EV-D68 (right). (c) Western blot of immunoprecipitation (IP) of Fc-MFSD6(L3) and Fc-MFSD8(L9) with EV-D68 or EV-A71. (d) Biolayer interferometry (BLI) assay. The biosensor was coupled with Fc-MFSD6(L3) (green), Fc-MFSD8(L9) (grey), or PBS (blue) and binding with 2 * 109 PFU ml−1 CVB3 was determined. Fc constructs were loaded at 0.2 μg μl−1 and reached saturation. (e) Gradient banding pattern from 10–50% (w/v) iodixanol gradient and corresponding fractions taken for (f) western blotting against EV-D68 viral capsid proteins and plaque assay (g). Fractions I and J contained bands corresponding to the mature virus, which were combined and used for cryo-EM, with representative micrographs shown for (h) apo EV-D68 (from 10,400 micrographs) and (i) EV-D68 bound to Fc-MFSD6(L3) (from 11,869 micrographs). Scale bars, 20 nm.
Extended Data Fig. 6 Top-down and bottom-up glycoproteomics characterizing Fc-MFSD6(L3) glycoforms.
(a) Protein sequence table showing all identified N- and O-glycosites found for the Fc-MFSD6(L3) construct. (b) Top-down MS analysis of Fc-MFSD6(L3) construct expressed from wild-type cells (HEK293FT) (bottom) and cells deficient in O-linked glycosylation machinery (HEK293T ΔGALE/ΔGALK2 knockout) (top). Heterogeneity, imparted by the various O-glycosylation sites and structures, is observed in the wild-type Fc-MFSD6(L3). These O-glycan modifications are ablated in the Fc-MFSD6(L3) produced in ΔGALE/ΔGALK2 cells, resulting in a substantial mass loss and simplification of the intact protein mass spectra, due to reduction of multiple overlapping ion signals from the various glycoforms. (c) Western blot of Fc-MFSD6(L3) produced in wild-type and O-linked glycosylation deficient cells. The proteins were incubated with StcE, a protease that specifically cleaves mucin-domain glycoproteins. (d) Tandem MS analysis by HCD confirming the presence of core fucosylated N-glycan structure (Man3GlcNAc2Fuc1) located on N207 (representative glycopeptide LnVSDTVTLPTAPNMNSEPTLQPQTGEITNR) and resolved by the cryo-EM map shown in Fig. 4b. All b and y fragment ions are represented by the cyan and pink fragment labels, respectively. (e) Representative tandem MS spectra showing the diversity of the various N-glycan structures (Man3GlcNAc5Fuc1, Man3GlcNAc6Fuc1, and Man3GlcNAc4Gal1NeuAc1Fuc1) characterized by HCD at N207 and represented in Fig. 4c. All b and y fragment ions are represented by the cyan and pink fragment labels, respectively. All spectral assignments are within 2 ppm of total mass error.
Extended Data Fig. 7 Cryo-EM data processing workflow.
Workflow for cryo-EM data processing for (a) EV-D68 apo and (b) EV-D68 + Fc-MFSD6(L3). Data was processed in Relion until 3D classification, after which particle stacks were imported into cryoSPARC. Icosahedral symmetry was applied only in cryoSPARC during Ab Initio and Homogeneous Refinement. Following refinement in cryoSPARC, the unsharpened maps were input into Phenix for auto-sharpening.
Extended Data Fig. 8 Q-score model validation for cryo-EM models.
Representative images of proteins, waters, ions, or glycans and corresponding Q-score plots for the EV-D68 apo map and model, depicting (a) VP1 (chain A, blue), (b) VP2 (chain B, green), (c) VP3 (chain C, red), (d) VP4 (chain D, brown), and (e) waters (each dot denotes a single water); and for the EV-D68 + Fc-MFSD6(L3) map and model, depicting (f) VP1 (chain A), (g) VP2 (chain B), (h) VP3 (chain C), (i), VP4 (chain D), (j) waters and ions (each dot denotes a single water or ion), and (k) Fc-MFSD6(L3) (chains E-F), with separate plots for the protein backbone (chain E) and glycan chain (chain F). Maps are shown in grey with transparency. Models are coloured by chain or by atomic colouring. For Fc-MFSD6(L3), protein Q-scores are plotted in black, and glycan Q-scores are plotted with colours matching the monosaccharide identity (GlcNAc, denoted NAG in blue, mannose, denoted BMA or MAN, in green, and fucose, denoted FUC, in red). Q-scores for protein residues, water, and glycans are plotted with filled circles. Q-scores for ions (3 points in (j)) are plotted with open circles. Dashed lines indicate the most commonly observed Q-score (black, Q_Peak) for maps in the EMDB at similar resolutions in addition to upper bounds (blue, Q_High 95%) and lower bounds (red, Q_Low 95%) that enclose 95% of observed maps for that resolution.
Extended Data Fig. 9 EV-D68 residues interacting with Fc-MFSD6(L3).
(a) Interactions within 4 Å distance from the Fc-MFSD6(L3) (chain E). The model for EV-D68 in complex with Fc-MFSD6(L3) is coloured, with VP1 (purple) and VP3 (red). The model for apo EV-D68 is grey. Residues between L1208 and T1216 are part of the VP1 GH loop. Fc-MFSD6(L3) is yellow, with start and end residues labelled. (b) Hydrogen bonding and salt bridge interactions and distances from PISA. *Indicates interaction not found by PISA but measured in Chimera. The atom ID starts with the atom (H (hydrogen), O (oxygen), N (nitrogen)). Protein atom IDs with one letter belong to the main chain; others belong to the side chain. Glycan atom IDs are relative to carbon numbering. NAG represents GlcNAc. (c-d) Selected interactions (also shown in Supplementary Video 1) show residues interacting with (c) R205 with H-bonds and (d) two GlcNAcs with H-bonds. The model is in atomic colouring (carbon (grey), hydrogen (white), oxygen (red), nitrogen (blue)). The map is transparent grey. Hydrogen bonds are shown with dashed lines. (e) EV-D68 RIVEM roadmap plot showing interacting residues. The large triangle marks the boundary of the projected asymmetric unit. The icosahedral symmetry axes are indicated by an oval, triangle, and pentagon. Residues interacting with Fc-MFSD6(L3) (chains E-F) within 4 Å distance are coloured by capsid protein with VP1 (purple), VP2 (green), and VP3 (red). A bright red outline marks capsid residues with H-bonds or a salt bridge with Fc-MFSD6(L3) (also in (b)). (f) EV-D68 RIVEM roadmap coloured by radial distance from 135 Å (blue) to 160 Å (red). A bright yellow outline marks capsid residues interacting with Fc-MFSD6(L3) within 4 Å distance and encloses part of the canyon, a depressed region (blue).
Extended Data Fig. 10 Comparison of EV-D68 receptor binding with other enterovirus ligands.
Asymmetric units of ligand-bound enteroviruses for structural comparison. A legend shows colourings for enterovirus capsid proteins, with VP1 in light purple, VP2 in light green, and VP3 in light red. Residues within a 4 Å interaction radius are shown in bright purple, green, and red surfaces, respectively. VP4 (behind) is not visible in this view. Ligand-bound enteroviruses include (a) RV-B14 and ICAM-1 (PDB ID: 7BG7); (b) EV-D68 and Fc-MFSD6(L3), in which the interaction involves Fc-MFSD6(L3) chains E-F but chain F is removed for clarity; (c) EV-D68 and sialic acid (PDB ID: 5BNO); (d) EV-D68 and 8F12 Fab (PDB ID: 7EC5); (e) EV-D68 and 2H12 Fab (state S1) (PDB ID: 7EBZ); and (f) EV-D68 and EV68-228 Fab (PDB ID: 6WDT). Ligands are shown in yellow (b-c) or transparent orange (a, d-f).
Supplementary information
Supplementary Table 1
Dataset of A549 EV-D68 CRISPR screens. Genome-scale CRISPR screen datasets were analysed using the MAGeCK algorithm. P values are calculated using MAGeCK. P values are one-sided. FDR-adjusted P values were multiple-hypothesis corrected using the Benjamini–Hochberg procedure
Supplementary Table 2
Dataset of U87-MG EV-D68 CRISPR screens. Genome-scale CRISPR screen datasets were analysed using the MAGeCK algorithm. P values are calculated using MAGeCK. P values are one-sided. FDR-adjusted P values were multiple-hypothesis corrected using the Benjamini–Hochberg procedure.
Supplementary Video 1
Video of interactions between EV-D68 and Fc–MFSD6(L3) resolved by cryo-EM. Video showing the cryo-EM structure of EV-D68 in complex with Fc–MFSD6(L3) and visualizing the interaction interface
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Varanese, L., Xu, L., Peters, C.E. et al. MFSD6 is an entry receptor for enterovirus D68. Nature 641, 1268–1275 (2025). https://doi.org/10.1038/s41586-025-08908-0
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DOI: https://doi.org/10.1038/s41586-025-08908-0
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