Structural basis for human coronavirus attachment to sialic acid receptors

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.Structural and functional analyses reveal how 9-O-acetyl sialic acid is recognized by the human coronavirus OC43 S glycoprotein and how this interaction promotes viral entry.

[1]  Ning Wang,et al.  Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus , 2017, PLoS pathogens.

[2]  E. Huizinga,et al.  Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity , 2017, Cell Host & Microbe.

[3]  P. Lemey,et al.  Complete Genomic Sequence of Human Coronavirus OC43: Molecular Clock Analysis Suggests a Relatively Recent Zoonotic Coronavirus Transmission Event , 2005, Journal of Virology.

[4]  H. Klenk,et al.  The receptor‐destroying enzyme of influenza C virus is neuraminate‐O‐acetylesterase. , 1985, The EMBO journal.

[5]  H. Takagi,et al.  Unique Directional Motility of Influenza C Virus Controlled by Its Filamentous Morphology and Short-Range Motions , 2017, Journal of Virology.

[6]  Conrad C. Huang,et al.  UCSF ChimeraX: Meeting modern challenges in visualization and analysis , 2018, Protein science : a publication of the Protein Society.

[7]  Fang Li,et al.  Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor , 2011, Proceedings of the National Academy of Sciences.

[8]  Lisa E. Gralinski,et al.  A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence , 2015, Nature Medicine.

[9]  Fang Li,et al.  Crystal Structure of Bovine Coronavirus Spike Protein Lectin Domain* , 2012, The Journal of Biological Chemistry.

[10]  Erik Lindahl,et al.  New tools for automated high-resolution cryo-EM structure determination in RELION-3 , 2018, eLife.

[11]  R. Henderson,et al.  High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy☆ , 2013, Ultramicroscopy.

[12]  S. Sawasdikosol,et al.  The Structure of HPK1 Kinase Domain: To Boldly Go Where No Immuno-Oncology Drugs Have Gone Before. , 2019, Structure.

[13]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[14]  E. Huizinga,et al.  Coronavirus receptor switch explained from the stereochemistry of protein–carbohydrate interactions and a single mutation , 2016, Proceedings of the National Academy of Sciences.

[15]  Keith S Wilson,et al.  Privateer: software for the conformational validation of carbohydrate structures , 2015, Nature Structural &Molecular Biology.

[16]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[17]  Matthias J. Brunner,et al.  Atomic accuracy models from 4.5 Å cryo-electron microscopy data with density-guided iterative local refinement , 2015, Nature Methods.

[18]  Lisa E. Gralinski,et al.  SARS-like WIV1-CoV poised for human emergence , 2016, Proceedings of the National Academy of Sciences.

[19]  S. Perlman,et al.  Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism , 2016, Proceedings of the National Academy of Sciences.

[20]  S. Barondes,et al.  X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution. , 1998, The Journal of biological chemistry.

[21]  L. Pelkmans,et al.  Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner , 2014, PLoS pathogens.

[22]  Fang Li,et al.  Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State , 2017, Journal of Virology.

[23]  Fang Li,et al.  Cryo-EM structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins , 2018, PLoS pathogens.

[24]  John Doyle,et al.  Rules of engagement , 2007, Nature.

[25]  T. Stehle,et al.  Viruses and sialic acids: rules of engagement , 2011, Current Opinion in Structural Biology.

[26]  Gary R. Whittaker,et al.  Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis , 2014, Virus Research.

[27]  L. Mitnaul,et al.  Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species. , 1997, Virology.

[28]  T. Pierson,et al.  Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses , 2014, Journal of Virology.

[29]  R. Groot Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses , 2006, Glycoconjugate Journal.

[30]  Yoshihiro Kawaoka,et al.  Receptor binding by a ferret-transmissible H5 avian influenza virus , 2013, Nature.

[31]  L. Enjuanes,et al.  Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity , 1996, Journal of virology.

[32]  B. Bosch,et al.  The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex , 2003, Journal of Virology.

[33]  A. Walls,et al.  Crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo‐electron microscopy , 2016, Protein science : a publication of the Protein Society.

[34]  F. Dimaio,et al.  Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer , 2016, Nature.

[35]  T. Stehle,et al.  The sweet spot: defining virus–sialic acid interactions , 2014, Nature Reviews Microbiology.

[36]  Barney S. Graham,et al.  Pre-fusion structure of a human coronavirus spike protein , 2016, Nature.

[37]  B. Bosch,et al.  Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A , 2019, Proceedings of the National Academy of Sciences.

[38]  Y. Matsuura,et al.  Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64 , 2010, Journal of Virology.

[39]  A. Cheng,et al.  2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy , 2015, eLife.

[40]  Jasenko Zivanov,et al.  A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis , 2018, bioRxiv.

[41]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[42]  Haixia Zhou,et al.  Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding , 2016, Cell Research.

[43]  G. Gao,et al.  Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses , 2016, Trends in Microbiology.

[44]  Conrad C. Huang,et al.  Visualizing density maps with UCSF Chimera. , 2007, Journal of structural biology.

[45]  Xi Chen,et al.  Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform , 2015, Biomolecules.

[46]  C. D. de Haan,et al.  Binding of Avian Coronavirus Spike Proteins to Host Factors Reflects Virus Tropism and Pathogenicity , 2011, Journal of Virology.

[47]  E. Huizinga,et al.  Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution , 2008, Proceedings of the National Academy of Sciences.

[48]  Chengsheng Zhang,et al.  The Acetyl-Esterase Activity of the Hemagglutinin-Esterase Protein of Human Coronavirus OC43 Strongly Enhances the Production of Infectious Virus , 2013, Journal of Virology.

[49]  Frank DiMaio,et al.  Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta , 2016, bioRxiv.

[50]  Fang Li,et al.  Crystal Structure of Bovine Coronavirus Spike Protein Lectin , 2012 .

[51]  David Baker,et al.  Modeling Symmetric Macromolecular Structures in Rosetta3 , 2011, PloS one.

[52]  G. Gao,et al.  An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism , 2016, PLoS pathogens.

[53]  D. Hamilton Outbreak Investigation , 2014 .

[54]  Barney S. Graham,et al.  Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen , 2017, Proceedings of the National Academy of Sciences.

[55]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[56]  E. Huizinga,et al.  The Murine Coronavirus Hemagglutinin-esterase Receptor-binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture , 2012, PLoS pathogens.

[57]  Christian Drosten,et al.  Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC , 2013, Nature.

[58]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[59]  D. Isaacs,et al.  Epidemiology of coronavirus respiratory infections. , 1983, Archives of disease in childhood.

[60]  Shibo Jiang,et al.  Receptor Usage and Cell Entry of Porcine Epidemic Diarrhea Coronavirus , 2015, Journal of Virology.

[61]  Daniel Wrapp,et al.  Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis , 2018, Scientific Reports.

[62]  Therése L. Eriksson,et al.  The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis , 2011, Nature Medicine.

[63]  Johannis P Kamerling,et al.  Exploration of the Sialic Acid World , 2018, Advances in Carbohydrate Chemistry and Biochemistry.

[64]  Frank DiMaio,et al.  Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy , 2016, Nature Structural &Molecular Biology.

[65]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[66]  Marion P G Koopmans,et al.  Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation , 2013, The Lancet Infectious Diseases.

[67]  G. Herrler,et al.  Structural and Functional Analysis of the Surface Protein of Human Coronavirus OC43 , 1993, Virology.

[68]  A. Walls,et al.  Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion , 2017, Proceedings of the National Academy of Sciences.

[69]  Yi Shi,et al.  Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains , 2017, Nature Communications.

[70]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[71]  Yi Shi,et al.  Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26 , 2013, Nature.

[72]  S. Barondes,et al.  X-ray Crystal Structure of the Human Galectin-3 Carbohydrate Recognition Domain at 2.1-Å Resolution* , 1998, The Journal of Biological Chemistry.

[73]  T. Sakai,et al.  Influenza A virus hemagglutinin and neuraminidase act as novel motile machinery , 2017, Scientific Reports.

[74]  Jared Adolf-Bryfogle,et al.  Automatically Fixing Errors in Glycoprotein Structures with Rosetta , 2018, Structure.

[75]  A. R. Collins HLA class I antigen serves as a receptor for human coronavirus OC43. , 1993, Immunological investigations.

[76]  J. Paulson,et al.  Kinetic analysis of the influenza A virus HA/NA balance reveals contribution of NA to virus-receptor binding and NA-dependent rolling on receptor-containing surfaces , 2018, PLoS pathogens.

[77]  Glycan Shield and Fusion Activation of a Deltacoronavirus Spike Glycoprotein Fine-Tuned for Enteric Infections , 2017, Journal of Virology.

[78]  J. Skehel,et al.  Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus , 1998, Nature.

[79]  M. Tortorici,et al.  Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein , 2017, Proceedings of the National Academy of Sciences.

[80]  Gerhard Wagner,et al.  The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site , 2002, The EMBO journal.

[81]  A. Walls,et al.  Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion , 2019, Cell.

[82]  P. Palese,et al.  Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[83]  G. Whittaker,et al.  Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites , 2009, Proceedings of the National Academy of Sciences.

[84]  R. Baric,et al.  Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme , 2015, Journal of Virology.

[85]  Meitian Wang,et al.  Crystal structure of the receptor binding domain of the spike glycoprotein of human betacoronavirus HKU1 , 2017, Nature Communications.

[86]  S. Harrison,et al.  Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor , 2005, Science.

[87]  J. Epstein,et al.  Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor , 2013, Nature.

[88]  Ajit Varki,et al.  Complexity and Diversity of the Mammalian Sialome Revealed by Nidovirus Virolectins , 2015, Cell Reports.

[89]  Nathan A. Baker,et al.  PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations , 2004, Nucleic Acids Res..

[90]  Early events during human coronavirus OC43 entry to the cell , 2018, Scientific Reports.

[91]  Dimitry Tegunov,et al.  Real-time cryo–EM data pre-processing with Warp , 2018, Nature Methods.

[92]  P. Woo,et al.  Molecular Epidemiology of Human Coronavirus OC43 Reveals Evolution of Different Genotypes over Time and Recent Emergence of a Novel Genotype due to Natural Recombination , 2011, Journal of Virology.

[93]  R. D. de Groot Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses , 2006, Glycoconjugate Journal.

[94]  T. Stehle,et al.  Crystal Structure of Reovirus Attachment Protein σ1 in Complex with Sialylated Oligosaccharides , 2011, PLoS pathogens.

[95]  Xinquan Wang,et al.  Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2 , 2018, PLoS pathogens.

[96]  G. Whittaker,et al.  Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein , 2014, Proceedings of the National Academy of Sciences.