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Structural basis for human coronavirus attachment to sialic acid receptors

Nature Structural & Molecular Biology volume 26, pages 481–489 (2019)Cite this article

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Abstract

Coronaviruses cause respiratory tract infections in humans and outbreaks of deadly pneumonia worldwide. Infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host receptors and fuses the viral and cellular membranes. To understand the molecular basis of coronavirus attachment to oligosaccharide receptors, we determined cryo-EM structures of coronavirus OC43 S glycoprotein trimer in isolation and in complex with a 9-O-acetylated sialic acid. We show that the ligand binds with fast kinetics to a surface-exposed groove and that interactions at the identified site are essential for S-mediated viral entry into host cells, but free monosaccharide does not trigger fusogenic conformational changes. The receptor-interacting site is conserved in all coronavirus S glycoproteins that engage 9-O-acetyl-sialogycans, with an architecture similar to those of the ligand-binding pockets of coronavirus hemagglutinin esterases and influenza virus C/D hemagglutinin-esterase fusion glycoproteins. Our results demonstrate these viruses evolved similar strategies to engage sialoglycans at the surface of target cells.

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Fig. 1: Cryo-EM structure of the apo-HCoV-OC43 S glycoprotein.
Fig. 2: Identification of a sialoglycan-binding site in the holo-HCoV-OC43 S glycoprotein structure.
Fig. 3: The identified HCoV-OC43 S interactions with sialosides are characterized by fast kinetics and are required for viral entry.
Fig. 4: Conservation of the receptor-binding groove among all 9-O-Ac-sialoglycan-recognizing coronaviruses.
Fig. 5: Conservation of the receptor-binding site architecture across coronavirus S, coronavirus HE and influenza virus HEF glycoproteins.

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Data availability

The cryo-EM maps and atomic models have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank with accession codes EMD-0557 and PDB ID 6NZK (holo-HCoV-OC43 S) and EMD-20070 and PDB ID 6OHW (apo-HCoV-OC43 S).

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Acknowledgements

Research reported in this publication was supported by the National Institute of General Medical Sciences (R01GM120553 to D.V.), a Pew Biomedical Scholars Award (D.V.) and an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund (D.V.), the Zoonoses Anticipation and Preparedness Initiative (IMI115760, F.A.R. and B.-J.B.), the Pasteur Institute (M.A.T. and F.A.R.), the Centre National de la Recherche Scientifique (F.A.R.), the LabEx Integrative Biology of Emerging Infectious Diseases (F.A.R.), the Netherlands Organization for Scientific Research (NWO TOP-PUNT 718.015.003, G.-J.B.) and CSC grant 2014-03250042 (Y.L.). This work was also partly supported by the Arnold and Mabel Beckman cryo-EM center and the Proteomics Resource (UWPR95794) at the University of Washington, and the Electron Imaging Center for NanoMachines supported by the NIH (1U24GM116792, 1S10RR23057 and 1S10OD018111), NSF (DBI-1338135) and CNSI at UCLA. We thank Y. Matsuura (Osaka University, Japan) for provision of the VSV-G pseudotyped VSVΔG/Fluc plasmids.

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Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, WA, USA

    M. Alejandra Tortorici, Alexandra C. Walls & David Veesler

  2. Institut Pasteur, Unité de Virologie Structurale, Paris, France

    M. Alejandra Tortorici & Félix A. Rey

  3. CNRS UMR 3569, Unité de Virologie Structurale, Paris, France

    M. Alejandra Tortorici & Félix A. Rey

  4. Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands

    Yifei Lang, Chunyan Wang, Danielle Koerhuis, Berend-Jan Bosch & Raoul J. de Groot

  5. Department of Chemical Biology and Drug Discovery, Utrecht University, Utrecht, the Netherlands

    Zeshi Li & Geert-Jan Boons

  6. Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, the Netherlands

    Zeshi Li & Geert-Jan Boons

  7. Department of Chemistry, University of Georgia, Athens, GA, USA

    Geert-Jan Boons

  8. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA

    Geert-Jan Boons

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  1. M. Alejandra Tortorici
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Contributions

M.A.T., A.C.W., Y.L., R.J.d.G. and D.V. designed the experiments. C.W. and B.-J.B. designed and cloned the protein constructs. M.A.T. and C.W. carried out protein expression and purification. Z.L. and G.-J.B. provided key reagents; M.A.T. and A.C.W. performed cryo-EM sample preparation and data collection. M.A.T., A.C.W. and D.V. processed the cryo-EM data. M.A.T. and D.V. built and refined the atomic models. Y.L. and D.K. carried out the binding and pseudovirus assays. M.A.T., A.C.W., Y.L., R.J.d.G., F.A.R. and D.V. analyzed the data. M.A.T., A.C.W. and D.V. prepared the manuscript with input from all authors.

Corresponding author

Correspondence to David Veesler.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 CryoEM analysis of apo and holo HCoV-OC43 S glycoprotein.

a, c, Representative electron micrographs (defocus: 1.4 μm and 1.2 µm) and class averages of apo (a) and holo (c) HCoV-OC43 S trimer embedded in vitreous ice. Scale bars: 100 nm (micrograph) and 200 nm Å (class averages). b, d Gold-standard (blue) and model/map (red) Fourier shell correlation curves. The resolution was determined to 2.9 Å and 2.8 Å for the apo and holo HCoV-OC43 S glycoprotein maps, respectively. The 0.143 and 0.5 cut-off values are indicated by horizontal dashed lines.

Supplementary Figure 2 Carbohydrate ligands are recognized by distinct regions of the HCoV-OC43 S, human galectin-3 or rotavirus VP8* β-sandwich.

(a-c) Ribbon diagrams in two orthogonal orientations showing 9-O-Ac-Me-Sia bound to the HCoV-OC43 S domain A (a), galactose bound to human galectin-3 (b, PDB 1A3K) and sialic acid bound to rotavirus VP8* (c, PDB 1KQR). Each domain is shown in the same two orientations for comparison.

Supplementary Figure 3 Zoomed-in view of the sialoglycan-binding site in the holo-HCoV-OC43 S glycoprotein structure.

The A domain is rendered as a ribbon diagram with the side chains of key ligand-interacting residues shown as sticks and the corresponding cryoEM density shown as a blue mesh. The ligand is rendered as sticks with atoms colored by elements (carbon: grey, nitrogen: blue, oxygen: red).

Supplementary Figure 4 Free 9-O-Ac-Me-Sia receptor does not trigger HCoV-OC43 S fusogenic conformational changes.

(a) SDS-PAGE. (b) Purified wild-type HCoV-OC43 S ectodomain trimer in the prefusion conformation. (c-f) The wild-type HCoV-OC43 S ectodomain trimer remained in the prefusion conformation after cleavage with 28 µg.mL−1 trypsin (c), incubation with 100 mM 9-O-Ac-Me-Sia of pre-cleaved trimer (d) or trypsin cleavage of 9-O-Ac-Me-Sia-bound trimer (e), cleavage with 28 µg.mL−1 trypsin and incubation at pH 4.5 for 1 hour at room temperature (f), as visualized by single-particle electron microscopy of negatively stained samples. The wild-type HCoV-OC43 S ectodomain trimer was cleaved with 28 µg.mL−1 trypsin and heated for 20 minutes at 50 °C in the absence (g) or presence (h) of 10% isopropanol (used to dissolve the trypsin inhibitor added to stop the proteolytic reaction). Only the latter condition led to the formation of postfusion rosettes. Scale bars: 200 nm.

Supplementary Figure 5 Coronavirus S galectin-like A domains have a conserved architecture.

(a–d) Ribbon diagrams of the HCoV-OC43 (a), BCoV (b, PDB 4H14), PHEV (c, PDB 6QFY) and HCoV-HKU1 (d, PDB 5I08) A domains.

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Tortorici, M.A., Walls, A.C., Lang, Y. et al. Structural basis for human coronavirus attachment to sialic acid receptors. Nat Struct Mol Biol 26, 481–489 (2019). https://doi.org/10.1038/s41594-019-0233-y

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