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. 2019 Jun;26(6):481-489.
doi: 10.1038/s41594-019-0233-y. Epub 2019 Jun 3.

Structural basis for human coronavirus attachment to sialic acid receptors

M Alejandra Tortorici  1   2   3 , Alexandra C Walls  1 , Yifei Lang  4 , Chunyan Wang  4 , Zeshi Li  5   6 , Danielle Koerhuis  4 , Geert-Jan Boons  5   6   7   8 , Berend-Jan Bosch  4 , Félix A Rey  2   3 , Raoul J de Groot  4 , David Veesler  9
Affiliations

Structural basis for human coronavirus attachment to sialic acid receptors

M Alejandra Tortorici et al. Nat Struct Mol Biol. 2019 Jun.

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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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Cryo-EM structure of the apo-HCoV-OC43 S glycoprotein.
a, Ribbon diagrams of the HCoV-OC43 S ectodomain trimer in two orthogonal orientations. The individual protomers are each in a different color, and the glycans are rendered as dark blue spheres. b, Ribbon diagrams of the superimposed HCoV-OC43 (light pink) and HCoV-HKU1 (dark gray) B domains in two orthogonal orientations. The N and C termini are labeled.
Fig. 2
Fig. 2. Identification of a sialoglycan-binding site in the holo-HCoV-OC43 S glycoprotein structure.
a, Molecular surface representation of the holo-HCoV-OC43 S ectodomain trimer structure with the bound ligand shown as sticks. Protomers are individually colored. b, Surface representation of the ligand-binding site colored by electrostatic potential from −12 to +12 kBT/ec. c, Two orthogonal views of the 9-O-Ac-Me-Sia binding site. The A domain is rendered as a ribbon diagram with the side chains of surrounding residues shown as sticks. The cryo-EM density is shown as a blue mesh. In a−c, the ligand is rendered as sticks with atoms colored by element (carbon, gray; nitrogen, blue; oxygen, red). Dashed lines show a salt bridge and hydrogen bonds formed between the ligand and domain A.
Fig. 3
Fig. 3. The identified HCoV-OC43 S interactions with sialosides are characterized by fast kinetics and are required for viral entry.
a, Biolayer interferometry showing binding of wild-type or W90A monomeric HCoV-OC43 domain A to immobilized 6-sialyl-5-N-acetyl,9-O-acetyl-lactosamine (9-O-Ac-6SLN), 6-sialyl-5-N-acetyl-lactosamine (6SLN) or HE-pre-treated 6-sialyl-5-N-acetyl,9-O-acetyl-lactosamine before binding (9-O-Ac-6SLN, pre-HE) or after a successful association/dissociation event (9-O-Ac-6SLN, post-HE). b, Binding of different concentrations of wild-type monomeric A domain to immobilized 9-O-Ac-6SLN. c, Steady-state affinity determination using the curves shown in b. HCoV-OC43 A engages 9-O-Ac-6SLN with a KD = 49.7 ± 10.7 µM. d, Asn27, a key 9-O-Sia-interacting residue visualized in the holo-HCoV-OC43 S glycoprotein structure was substituted with alanine, and binding was assessed using a solid-phase lectin binding assay. Data points are averages from three independent technical triplicates. The data are normalized relative to the wild type. e, Sialoside binding to the identified site is necessary for HCoV-OC43 S-mediated entry of pseudotyped VSV-ΔG particles into host cells. n = 3 pseudovirus experiments (technical replicates). Data are normalized relative to wild type and shown as mean and s.d. of technical triplicates. f, Western-blot analysis of VSV-ΔG pseudotyped with wild-type or mutant HCoV-OC43 S. VSV-N was used as a quantitative control for the amount of virions analyzed.
Fig. 4
Fig. 4. Conservation of the receptor-binding groove among all 9-O-Ac-sialoglycan-recognizing coronaviruses.
a–d, Zoomed-in view of the binding sites rendered as ribbon diagrams with surrounding residues shown as sticks for HCoV-OC43 (a), BCoV (b), PHEV (c), HCoV-HKU1 (d). Residues are colored by conservation, based on the analysis of all the S glycoprotein sequences available for each virus. In a, the 9-O-Ac-Me-Sia ligand is rendered as sticks with atoms colored by elements (carbon, gray; nitrogen, blue; oxygen, red). HCoV-OC43, 192 sequences; BCoV, 150 sequences; PHEV, 12 sequences; HCoV-HKU1, 28 sequences.
Fig. 5
Fig. 5. Conservation of the receptor-binding site architecture across coronavirus S, coronavirus HE and influenza virus HEF glycoproteins.
a, HCoV-OC43 S bound to 9-O-Ac-Me-Sia. b, BCoV HE bound to 5-N-acetyl-4,9-di-O-acetyl-neuraminic acid α-methylglycoside (PDB 3CL5). c, Influenza virus C HEF in complex with 9-N-Ac-Sia. In all panels, the glycoprotein is rendered as a gray surface with the bound ligand shown as sticks. The hydrogen bond formed with the carbonyl of the 9-O/N-acetyl group is shown by dashed lines.
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