A dual-receptor checkpoint primes enterovirus D68 for respiratory cell entry

Figures

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  Fig. 1. Structure of EV-D68 apo and in complex with MFSD6-L3. a The colored surface from left to right represents cryo-EM maps of the EV-D68 strain Fermon mature virion, procapsid, mature virion in complex with MFSD6-L3, and B-particle in complex with MFSD6-L3. The densities were displayed as a three-dimensional reconstruction, colored by radius, ranging from blue through cyan, green, yellow, and orange to red, from the lowest to the highest radius. The 5-fold, 3-fold, and 2-fold icosahedral axes are indicated by a pentagon, triangles, and an oval, respectively. The i5 capsid, i3 capsid and i2 capsid are indicated by the labels “i5”, “i3” and “i2” respectively. b Comparison of the structure and SPH analysis of the pocket factor in mature virion with the crystal structure of fatty acid in 4WM8. c Cryo-EM maps of the MFSD6-L3 binding region in mature virion alone (left panel) and mature virion in complex with MFSD6-L3 (right panel), displayed as two asymmetric units extracted from the reconstructed 5-fold. d Details for the EV-D68-mature:MFSD6-L3 interaction. The interacting region is presented in enlarged views. All polypeptides are shown as cartoon diagrams. Interacting residues are…

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  Fig. 2. Glycoproteomics analysis of MFSD6-L3 and mapping of the binding segment. a–d Comparative site-specific glycosylation profiling of MFSD6-L3-Fc and MFSD6-L3-N207A-Fc. a Global identification statistics. Comparison of the total numbers of identified MS/MS spectra, intact glycopeptides, mapped glycosylation sites, and glycoform distributions classified by sialylation status (sialylated versus non-sialylated glycans) between MFSD6-L3-Fc and MFSD6-L3-N207A-Fc. b Sialylated glycan occupancy at individual glycosylation sites. For each glycosylation site, numbers inside the circles indicate the absolute MS/MS spectral counts corresponding to sialylated intact glycosites. Percentages denote the proportion of sialylated glycan spectra relative to the total glycopeptide spectra identified at that site, representing site-specific occupancy of sialylated glycans. c Numbers of intact O-GalNAc glycosylation sites carrying specific O-glycans. The left and right panels represent glycosite distributions identified using MFSD6-L3-Fc and MFSD6-L3-N207A-Fc, respectively. Bars indicate the number of unique intact O-GalNAc glycosylation sites modified by the indicated O-glycan types. d For each glycan…

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  Fig. 3. The Neu5Ac in MFSD6 is important for EV-D68 infection. a Cryo-EM density maps of EV-D68 Fermon bound to MFSD6- R196-L226. Two asymmetric units extracted from the reconstructed five-fold axis are shown. The color scheme for VP1/VP2/VP3 domains and MFSD6-L3 is as follows: VP1, pale blue; VP2, pale green; VP3, pale cyan; MFSD6-L3, red. b Details for the virus-Neu5Ac interaction in EV-D68:MFSD6-R196-L226. The color scheme is the same as that used in Fig. 3a. c Cryo-EM density maps of EV-D68 Fermon with MFSD6-R196-L226-NA-treated. Two asymmetric units extracted from the reconstructed five-fold axis are shown. The color scheme for VP1/VP2/VP3 domains and MFSD6-L3 is as follows: VP1, pale blue; VP2, pale green; VP3, pale cyan; MFSD6-L3, red. The model of MFSD6-R196-L226-NA-treated and pocket factor are shown with the protein backbone in red (right panel). d, e Viral attachment assay (left panel). A549 cells, and A549-NA-treated cells were washed with cold PBS before infection, followed by incubation with EV-D68 at an MOI of 10.0 (Fermon, d, MO, e) at 4 °C for 2 h to facilitate virus attachment. Viral entry assay (right panel): A549 cells and A549-NA-treated cells were washed with…

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  Fig. 4. The N-glycan at N207 is important for EV-D68 infection. a Cryo-EM maps and structure of EV-D68 with MFSD6-L3-N207A. The color scheme is the same as that used in Fig. 3. The model of MFSD6-L3-N207A and pocket factor are shown with the protein backbone in red. The map density of MFSD6-L3-N207A and pocket factor is transparent red, and EV-D68 is transparent grey. b, c Viral attachment assay (left panel). Vero cells-MFSD6-WT cells, and Vero-MFSD6-N207A cells were washed with cold PBS before infection followed by incubation with EV-D68 at an MOI of 10.0 (Fermon, c, MO, d) at 4 °C for 2 h to facilitate virus attachment. Viral entry assay (right panel): cells were washed with prewarmed PBS before infection and then incubated with EV-D68 at an MOI of 10.0 (Fermon, b, MO, c). N = 3 (b, c) biological replicates. Data are represented as mean ± SD. Two-tailed t-test (b, c) was used to assess statistical significance. Source data are provided as a Source Data file.

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  Fig. 5. Cell-based assays for the impacts of MFSD6-L3 mutants on EV-D68 infection. a Detection of EV-D68-induced CPEs. Images are representative of three independent experiments. Scale bars equal 20 µm. b At 48 h post-infection at an MOI of 2.0, cell lysates were subjected to immunoblot analysis. Representative of three biologically independent experiments. c EV-D68 MO viruses were pre-incubated with control microbodies (control-Fc) or the indicated MFSD6 microbodies (MFSD6-Fc) for 30 minutes prior to infection of A549 cells. The inhibitory effects of different MFSD6-Fc fusion proteins on progeny virus titers were measured. The error bars denote the SD. ns indicates no statistical significance, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. d EV-D68 Beijing-R0132 viruses were pre-incubated with control microbodies (control-Fc) or the indicated MFSD6 microbodies (MFSD6-Fc) for 30 minutes prior to infection of human bronchial epithelial cells (HBECs). The inhibitory effects of different MFSD6-Fc fusion proteins on progeny virus titers were measured. The error bars denote the SD. ns indicates no statistical significance, ** indicates P < 0.01, *** indicates P < 0.001.…

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  Fig. 6. Conserved pocket factor occupancy and MFSD6 engagement across EV-D68 strains. a Cryo-EM analysis of EV-D68 in the apo state. The top row shows the cryo-EM density maps of EV-D68 Fermon, MO, and KY (left to right), displayed as two asymmetric units extracted from the reconstructed virion. The bottom row shows the corresponding pocket factor densities and fitted atomic models, visualized in COOT, highlighting the occupancy of the pocket factor in the VP1 hydrophobic pocket. The pocket factor density is clearly resolved in all three strains. Relevant residues lining the pocket are indicated (I217 for Fermon and MO; I216 for KY). b Cryo-EM analysis of EV-D68 in complex with MFSD6-L3. The top row shows the cryo-EM density maps of EV-D68 Fermon, MO, and KY bound to MFSD6-L3 (left to right), again displayed as two asymmetric units and one MFSD6-L3, with MFSD6-L3 shown in red. The bottom row shows the corresponding fitted atomic models, demonstrating displacement of the pocket factor upon MFSD6 engagement. In all three strains, MFSD6 binding correlates with loss of pocket factor density, indicating a conserved receptor-induced pocket factor egress mechanism. VP1, VP2, VP3, and MFSD6-L3…

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  Fig. 7. Comparison of EV-D68:MFSD6-L3-Fc binding mode with other Picornaviruses. Asymmetric units of virus-receptor for structural comparison. The color scheme for VP1/VP2/VP3/receptor domains and receptor is as follows: VP1, pale blue; VP2, pale green; VP3, pale cyan; receptor, red. VP4 (behind) is not visible in this view. Virus-receptor structures include (a) EV-D68:sialic acid (PDB: 5BNP)11; b EV71:SCARB2 (PDB: 6I2K)27; c HRV 14:ICAM-1 (PDB: 7BG7)26; d Coxsackievirus B1:CAR (PDB: 7DQ1)28; e EV-D68:MFSD6-L3-Fc.