Article | Published:

Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy

Nature Structural & Molecular Biology volume 23, pages 899905 (2016) | Download Citation

Abstract

The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.

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Protein Data Bank

Proteomics Identifications Database

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NCBI Reference Sequence

Protein Data Bank

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Acknowledgements

Research reported in this publication was supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under award number 1R01GM120553-01 (D.V.) and T32GM008268 (A.C.W.). J.S. acknowledges support from the Netherlands Organization for Scientific Research (NWO, Rubicon 019.2015.2.310.006) and the European Molecular Biology Organisation (EMBO, ALTF 933-2015). M.A.T. and F.A.R. acknowledge support from the Institute Pasteur and the CNRS. The authors acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by the NIH (1S10RR23057 and 1S10OD018111), NSF (DBI-1338135) and CNSI at UCLA. The authors are grateful to H. Choe (The Scripps Research Institute) for providing the HCoV-NL63 S gene, J. Labonte (Johns Hopkins School of Medicine) for providing the Rosetta code handling glycans and P.J.M. Rottier (Utrecht University) for scientific advice. This work was partly supported by the University of Washington's Proteomics Resource (UWPR95794), and the authors thank P.D. von Haller and J.K. Eng for their assistance and expertise. SPR experiments were performed with the help of J. Sumida at the University of Washington School of Pharmacy Analytical Biopharmacy Core. Part of this research was facilitated by the Hyak supercomputer system at the University of Washington.

Author information

Affiliations

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

    • Alexandra C Walls
    • , Brandon Frenz
    • , Joost Snijder
    • , Frank DiMaio
    •  & David Veesler
  2. Institut Pasteur, Unité de Virologie Structurale, Paris, France.

    • M Alejandra Tortorici
    •  & Félix A Rey
  3. CNRS UMR 3569 Virologie, 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.

    • Wentao Li
    •  & Berend-Jan Bosch

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Contributions

B.-J.B. designed and cloned the protein construct. M.A.T. carried out protein expression and purification under the supervision of F.A.R. A.C.W. performed cryo-EM sample preparation, data collection and processing under the supervision of D.V. A.C.W., B.F., F.D. and D.V. built the atomic model. A.C.W. and J.S. performed the MS experiments. W.L. performed the hemagglutination assays and ELISAs under the supervision of B.-J.B. A.C.W. performed the SPR experiments under the supervision of D.V. All authors analyzed the data. A.C.W., J.S., B.-J.B. and D.V. prepared the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Berend-Jan Bosch or David Veesler.

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    Supplementary Table 1

    Characterization of the HCoV-NL63 S glycans by using mass spectrometry and cryo-EM

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https://doi.org/10.1038/nsmb.3293

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