Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein

To identify cellular factors, other than DPP4, potentially involved in MERS-CoV cell attachment, we performed classical hemagglutination assays commonly used to detect virus–Sia interactions. Unlike SARS-CoV but similar to influenza A virus (IAV), MERS-CoV virions caused agglutination of human erythrocytes (Fig. 1). Because the spike glycoprotein is the only surface projection of clade C beta-CoVs, including MERS-CoV, we attempted to validate the observation in a hemagglutination assay using the recombinantly expressed S1 subunit of the MERS-CoV S protein that was transformed into a dimer by C-terminal fusion with the Fc part of human IgG. However, no hemagglutination was seen for MERS-CoV S1-Fc protein, not even when tested at concentrations of up to 500 μg/mL. We therefore considered the option that the observed hemagglutination by MERS-CoV was mediated through simultaneous low-affinity binding of multiple spike proteins on the virus particles with multiple receptors on the surface of human erythrocytes. Such low-affinity interactions may be augmented through multivalency-driven, high-avidity binding (34–36). Therefore, we opted to design a well-defined, self-assembling nanoparticle for arrayed presentation of S1 and S1 subdomains.

We selected the 154-residue-long lumazine synthase (LS) protein of the hyperthermophile Aquifex aeolicus bacterium that self-assembles into 15-nm-wide 60-meric particles with icosahedral symmetry (37). To allow multivalent presentation of Fc-tagged MERS-CoV S1, the LS protein was extended N-terminally with the 59-residue-long domain B of protein A (pA) of Staphylococcus aureus (Fig. 2A), known for its capacity to bind immunoglobulins via their Fc region (38). The pA-LS protein was provided with an N-terminal signal sequence to allow secretion from mammalian cells, and a C-terminal streptavidin tag was appended for affinity purification (SI Appendix, Fig. S1). The pA-LS protein was affinity-purified from cell culture supernatant of transiently transfected HEK-293T cells and migrated according to its calculated molecular mass of 26 kDa (SI Appendix, Figs. S2 and S3). Analysis of purified pA-LS by electron microscopy revealed spherical particles of ±15 nm in diameter (Fig. 2B), and size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) analysis of these pA-LS particles demonstrated that the mass of the main elution peak was close to that of the predicted 60-meric state of the nanoparticle (SI Appendix, Fig. S3). Binding of Fc-tagged S1 subunits (Fig. 2C) to the pA domain on pA-LS nanoparticles was confirmed by ELISA (SI Appendix, Fig. S4), and electron microscopy (Fig. 2B, Right).

Nanoparticle-displayed MERS-CoV S1 exhibited hemagglutination activity (2.5 μg of S1-Fc combined with 0.5 μg of pA-LS; HA titer = 128) (Fig. 3A). Since no hemagglutination activity was observed for the (dimeric) MERS-CoV S1-Fc, multivalent presentation of S1-Fc appeared critical for the hemagglutination phenotype. No hemagglutination was observed with human erythrocytes desialylated by pretreatment with bacterial neuraminidase (NA; Arthrobacter ureafaciens), indicating that the observed MERS-CoV S1–erythrocyte interaction is strictly Sia-dependent (Fig. 3A).

The pA-LS/S1-Fc ratio showing the highest hemagglutination titer was determined to be 1 μg per 2.5 μg (molar ratio = 1:0.6), indicating that ∼36 MERS-CoV S1 receptor-binding subunits, presented as 18 S1-Fc dimers, can be accommodated on the 60-meric pA-LS nanoparticle providing optimal hemagglutination efficiency (Fig. 3B and SI Appendix, Table S1). Aside from S1-Fc proteins of MERS-CoV and several previously documented Sia-interacting CoVs [TGEV, porcine epidemic diarrhea virus (PEDV), BCoV, HCoV-OC43, and infectious bronchitis virus (IBV) (39–43)], none of the tested S1 subunits, including those of the MERS-CoV–related Betacoronavirus clade C bat CoVs HKU4 and HKU5, displayed hemagglutination activity in the nanoparticle-based assay (SI Appendix, Table S2).

To identify the S1 domain of the MERS-CoV spike protein involved in Sia binding, we expressed (SI Appendix, Fig. S2) and assessed the hemagglutination activity of Fc-tagged MERS-CoV S1^A and S1^B domains, as these are the two domains known to facilitate receptor interactions for Betacoronavirus. In contrast to the MERS-CoV S1^B domain, nanoparticle-displayed MERS-CoV S1^A-Fc was capable of mediating hemagglutination (Fig. 4). These data demonstrate that the Sia-binding capacity of the MERS-CoV S1 subunit resides in its domain A.

Sialoglycans occur in an extraordinary structural variety, which arises from the composition and complexity of the glycan chain, differences in the glycosidic linkage through which the Sia is joined to the adjacent sugar residue, and differential modification of Sia at carbon 5 [either N-acetyl moiety [N-acetylneuraminic acid (Neu5Ac)] or N-glycolyl moiety [N-glycolylneuraminic (Neu5Gc)] in combination with modifications of any of the hydroxyl groups at C4, C7, C8, and C9, most often by acetate esters. To assess the specificity of MERS-CoV, glycan array analysis was performed with Fc-tagged MERS-S1^A-loaded pA-LS nanoparticles against a library of 135 glycans. We used NA-treated MERS-CoV S1^A as desialylation increased the Sia-binding potential of the MERS-CoV S1 subunit (SI Appendix, Table S3), similar to what has been reported for the Alphacoronavirus TGEV and Gammacoronavirus IBV (43, 44) for which Sia interaction has been shown to be biologically relevant (31, 45), consequently enhancing glycan array sensitivity. A recombinant Fc-tagged HA ectodomain of a human H1N1 IAV (A/California/04/2009) with a binding preference for α2,6-linked sialylated glycans (46) was taken along as a positive control. Unloaded nanoparticles, included as a negative control, displayed no binding on the glycan array (Fig. 5A). Indeed, nanoparticles loaded with dimeric HA-Fc displayed specific binding to α2,6-linked sialoglycoconjugates, as was previously reported the for trimeric HA ectodomain (46) (Fig. 5 and SI Appendix, Table S4). MERS-CoV spike protein domain A predominantly bound to short, sulfated, α2,3-linked mono-sialotrisaccharides (glycan nos. 11, 12, and 15), as well as to long, branched, α2,3-linked di-Sia and tri-Sia glycans with a minimum extension of 3 Galβ1–4GlcNAcβ1–3 (LacNAc) tandem repeats (Fig. 5 and Dataset S1). Generally, no or low binding was observed to α2,6-linked sialosides. Strikingly, MERS-CoV S1^A did not bind Neu5Gc containing α2,3-linked glycans in contrast to the α2,3-linked Neu5Ac glycan counterparts (Fig. 5B). In accordance, nanoparticle-displayed MERS-CoV S1^A did not agglutinate equine erythrocytes that exclusively contain Neu5Gc sialoglycans (47), irrespective of the presence of potentially interfering O-acetyl moieties on the C4 and C9 positions of erythrocyte sialoglycans, which were enzymatically removed using CoV hemagglutinin-esterase proteins (SI Appendix, Table S4). In contrast, the PEDV S1 protein that can bind to Neu5Ac and Neu5Gc (42) did agglutinate erythrocytes of horse origin.

The MERS-CoV spike protein appears to bind to nonmodified Neu5Ac sialosides, which suggests that, other than for beta 1 CoVs (40, 41, 48), Sia O-acetylation is not a prerequisite for binding. We wondered what effect Sia 9-O-acetylation would have on MERS-CoV spike–Sia interaction, and hence used a bovine submaxillary mucin (BSM)-based ELISA, as BSM is a well-characterized substrate rich in 9-O-acetylated Sias with only a minor population of unmodified Neu5Ac (49). While MERS-CoV S1^A was unable to interact with mock-treated mucin of bovine origin (Fig. 6A), pretreatment of BSM with sialate–9-O-acetylesterase [i.e., BCoV hemagglutinin-esterase (HE)] enabled its interaction. Similarly, pretreatment of BSM with sialate–9-O-acetylesterase clearly enhanced binding of IAV HA to bovine mucin (Fig. 6A), for which it is known that the acetyl moiety at the Sia C9 position prevents recognition of the saccharide by HA (50). No binding of MERS-CoV S1^A and IAV HA was observed upon pretreatment of BSM with NA, confirming that the spike–mucin interaction was Sia-dependent. Consistent with these observations, treatment of rat erythrocytes, which are rich in 9-O-acetylated sialo-glycoconjugates, with sialate–9-O-acetylesterase revealed receptors compatible with MERS-CoV S1^A–mediated hemagglutination (SI Appendix, Fig. S5). We conclude that O-acetylation of the Sia glycerol side chain inhibits MERS-CoV S1^A binding. Conclusively, we offer that MERS-CoV S1^A comprises a Sia binding preference for α2,3-coupled, 5-N-acetylated neuraminic acid in which both 5-N-glycolylation and 9-O-acetylation are not tolerated.

Reportedly, 9-O-acetylated Sias are scarce in the respiratory tissue of humans (49), while glycolylated Sias are considered to be absent altogether (51, 52). Accordingly, we examined whether mucin derived from human respiratory epithelial cells could be bound by MERS-CoV S1^A. Nanoparticle-displayed MERS-CoV S1^A showed binding to human mucin in a concentration-dependent manner similar to IAV HA (Fig. 6B), whereas negative control pAPN did not. Pretreatment of human mucin with sialidase completely inhibited binding by MERS-CoV S1^A and IAV HA, demonstrating the Sia-dependent nature of the observed interactions (Fig. 6B). Consistent with previous binding assays, MERS-CoV S1^A binding to mucin was only detected when presented on nanoparticles, indicating that the multivalent lectin–carbohydrate interactions enhance binding affinity (SI Appendix, Fig. S6). In addition, MERS-CoV S1^A binding to human mucin was observed to be temperature-sensitive and displayed weaker binding at higher temperatures (SI Appendix, Fig. S7). These results contrast with the observations made for BSM and suggest that the MERS-CoV S1^A protein is particularly suited to bind human respiratory mucin.

With the preferential binding of MERS-CoV S1^A to α2,3-linked sialoglycans in mind, we next studied the distribution of these glycans in the upper (nose) and lower (lung) respiratory tissues of humans and the natural reservoir of MERS-CoV: dromedary camels. Lectin histochemistry on these tissues suggests that α2,3-linked sialoglycans are abundant in the camel nasal respiratory epithelium and the alveoli of the human lung (Fig. 6C), which coincides with DPP4 expression and the site of MERS-CoV replication in these mammals (53).

To study whether the MERS-CoV Sia-binding activity may aid virus cell entry, African green monkey kidney Vero cells and human airway epithelial Calu-3 cells were depleted for cell surface Sias by NA treatment or mock-treated. The cells were then inoculated with MERS-CoV and IAV [multiplicity of infection (MOI) = 0.1], and infection levels were assessed by immunostaining. As expected, NA treatment rendered Vero and Calu-3 cells almost completely resistant to the Sia-dependent IAV. In Vero cells, NA treatment did not affect MERS-CoV entry efficiency. However, Sia depletion of Calu-3 cells reduced MERS-CoV entry by more than 70% compared with control-treated cells (Fig. 7 A and B). The difference between Vero and Calu-3 cells may be explained by a difference in the levels of Sia cell surface expression. As shown by flow cytometry, both cell types display similar levels of the entry receptor DPP4 (Fig. 7C), but in Calu-3 cells, those of cell surface MERS-CoV S1^A glycotopes are fivefold higher (Fig. 7D). Our findings provide direct evidence that sialylated proteins or glycolipids on the surface of human airway epithelial cells can be used as an attachment receptor by MERS-CoV, and thereby increase infection efficiency.

Genes encoding the 6,7-dimethyl-8-ribityllumazine synthase (LS; GenBank accession no. WP_010880027.1) of A. aeolicus and domain B of pA (UniProt accession no. P38507; amino acids 212–270) of S. aureus were synthesized using human-preferred codons obtained from GenScript USA, Inc. The cysteine at position 37 and asparagine at position 102 of LS were mutated to alanine and glutamine, respectively. A pA-LS expression vector was generated by ligating the pA-encoding sequence in-frame with the N terminus of the LS-encoding sequence via a Gly-Ser linker and subsequent cloning into the pCAGGS mammalian expression vector. In addition, LS ORFs in the LS and pA-LS expression vectors were provided with a sequence encoding an N-terminal CD5 signal peptide, and a streptomycin tag purification tag sequence (IBA) (SI Appendix, Fig. S1). To generate the MERS-CoV S1^A-LS and S1^B-LS expression vectors, the pA sequence in the pA-LS expression vector was replaced by a sequence encoding the MERS-CoV S1^A domain (residues 19–357) (protein sequences are provided in SI Appendix, Fig. S1). Plasmids encoding pA-LS were polyethylenimine (PEI)-transfected into 60% confluent HEK-293T cells for 6 h, after which transfections were removed and medium was replaced with 293 SFM II-based expression medium (Gibco Life Technologies) and incubated at 37 °C in 5% CO₂. Tissue culture supernatants were harvested 5–6 d posttransfection, and expressed proteins were purified using StrepTactin Sepharose beads (IBA) according to the manufacturer’s instruction. Purified proteins were stored at 4 °C until further use.

Expression of Fc-tagged S1 subunits of spike proteins of TGEV (GB: ABG89325.1), HCoV-229E (GB: NP_073551.1), HCoV-NL63 (GB: YP_003767.1), PEDV (GB: AOG30832.1), HCoV-HKU1 (GB: YP_173238.1), BCoV (GB: P15777.1), HCoV-OC43 (GB: AAR01015.1), SARS-CoV (GB: AAX16192.1), MERS-CoV (GB: YP_009047204.1), bat CoV HKU4.2 (GB: ABN10848.1), bat CoV HKU5 (GB: ABN10893.1), IBV (GB: AAW33786.1), and porcine delta coronavirus (PDCoV) (AML40790.1) was performed as described previously (26). Briefly, plasmids encoding the CoV-S1 subunit (or a domain thereof), C-terminally fused to the Fc part of human IgG1, were generated, and proteins were expressed in HEK-293T cells as described above. Similarly, an Fc-tagged version of the HA ectodomain of IAV [A/California/04/2009 (H1N1)] containing T200A and E227A substitutions in the Sia-binding site enhancing Sia affinity (46) and of the ectodomain of porcine APN (GB: XP_005653580.1) were generated. Fc-tagged proteins were purified from tissue culture supernatants 5–6 d posttransfection by pA affinity chromatography (17-0780-01; GE Healthcare), eluted using acid solution (0.1 M citric acid at pH 3.0), and neutralized immediately using Tris at pH 8.8 (0.2 M final concentration), with the exception of HA-Fc, which was purified via a C-terminal streptomycin tag and StrepTactin Sepharose beads and eluted using StrepTactin elution buffer (100 mM Tris⋅HCl, 150 mM NaCl, 1 mM EDTA, 2.5 mM biotin). Purified proteins were analyzed on a 12% SDS/PAGE gel under reducing conditions and stained with GelCodeBlue stain reagent (Thermo Scientific). Purified proteins were stored at 4 °C until further use.

Before the application of samples, 400-mesh copper grids with a pure carbon film were exposed to a glow discharge in air for 20 s to make them hydrophilic. Ten microliters of purified LS, pA-LS, or pA-LS + MERS-CoV S1-Fc (premixed at a 1:1.2 ratio) at 0.2–0.3 mg/mL was applied to the grids and incubated for 1 min. Excess sample was blotted with a filter paper, and for negative staining, 10 μL of 1% phosphotungstic acid at pH 6.8 was applied. After 1 min, excess stain was blotted and grids were left to dry. The specimens were examined in a JEOL JEM2100 transmission electron microscope at 200 kV, and images were taken at a magnification of 80,000× with a Gatan US4000 4K × 4K camera. At least 50 particles were measured to calculate the mean diameter and SD of the pA-LS particles.

For SEC-MALS analysis, purified LS nanoparticles [150 μg in 20 mM Tris⋅HCl (pH 8.0), 150 mM NaCl] were loaded onto a Superose 6 10/300 GL column (0.4 mL⋅min⁻¹; GE Life Sciences) and passed through a Wyatt DAWN Heleos II EOS 18-angle laser photometer coupled to a Wyatt Optilab TrEX differential refractive index detector. Data were analyzed, and weight-averaged molecular masses and mass distributions (polydispersity) for each sample were calculated using Astra 6 software (Wyatt Technology Corp.).

Nunc Maxisorp plates were coated with 100 μL of D-PBS (PBS with Ca²⁺/Mg²⁺) containing 50 ng of MERS-CoV S1-Fc per well and incubated for 4 h at room temperature. Plates were washed three times with washing buffer (PBS + 0.05% Tween-20) and subsequently blocked overnight at 4 °C with 200 μL of blocking buffer (PBS + 0.1% Tween-20, containing 2% BSA). Plates were put at room temperature and washed three times with washing buffer before use. Streptomycin-tagged LS and pA-LS were threefold serially diluted in blocking buffer and incubated for 1 h at 37 °C. Plates were washed three times with washing buffer and once with PBS + 2% BSA, and then incubated for 1 h at 37 °C with 100 μL of a 1:10,000 StrepMAB-classic HRP (IBA) dilution in PBS + 2% BSA. Plates were washed three times with washing buffer. One hundred microliters of TMB Super Slow One Component HRP Microwell Substrate (SurModics) was added to the wells and developed for 4 min. Reaction was stopped with 100 μL of 12.5% sulfuric acid per well, and absorbance was measured at 450 nm using an ELx808 Absorbance Microplate Reader (Bio-Tek Instruments).

The classical hemagglutination assay was performed according to standard procedures. Briefly, twofold dilutions of the MERS-CoV (EMC strain), SARS-CoV (HKU39849 strain), and IAV (PR8 strain) [starting dilution of 10⁷ TCID₅₀ (tissue culture infectious dose at which 50% of cells display cytopathogenic effects) per milliliter] were incubated with 0.5% washed human erythrocytes (diluted in PBS) in V-bottom, 96-well plates (Greiner Bio-One) for 30 min at 4 °C. Plates were incubated for 2 h at 4 °C, after which hemagglutination titer was scored. For the nanoparticle-based hemagglutination assay, Fc-tagged chimeric proteins were complexed with pA-LS at a 0.6:1 molar ratio (unless stated otherwise) for 30 min at 4 °C in blocking buffer (PBS + 0.1% BSA) and subjected to hemagglutination assay as described above. In some experiments, human, rat, or equine erythrocytes were pretreated for 3 h at 37 °C with NA from A. ureafaciens (Roche; diluted to 20 mU/mL in PBS), Fc-tagged ectodomains of the bovine CoV-LUN hemagglutinin-esterase (20 μg/mL), or Fc-tagged ectodomains of MHV-S HE (4 μg/mL), the latter two of which were produced and purified as described by Zeng et al. (73). In some instances, MERS-S1^A-Fc protein was treated with 20 mU/mL NA in PBS for 3 h at 37 °C before complexation to pA-LS nanoparticles.

Glycans containing Neu5Gc were synthesized essentially as described for the corresponding glycans terminating in Neu5Ac (74), except that enzymatic addition of Sia in the last step was done with the donor substrate CMP-Neu5Gc instead of CMP-Neu5Ac. Microarrays were printed as described previously (75). Microarray slides were blocked with PBS + 0.1% Tween-20 and 5% Protifar skimmed milk. Empty pA-LS nanoparticles or nanoparticles complexed with A. ureafaciens NA-treated MERS-S1^A-Fc or IAV HA-Fc at a 0.6:1 molar ratio (as described above) were diluted in PBS + 1% BSA to a concentration of 500 μM (based on the pA-LS concentration) and incubated for 120 min at 4 °C on the microarray slides under a microscope cover glass in a humidified chamber at room temperature. Microarray slides were subsequently washed by successive rinses with PBS + 0.05% Tween-20 and overlaid with a Chromeo488-conjugated secondary StrepMAB antibody (IBA). Slides were repeatedly washed with PBS + 0.05% Tween-20, PBS, and deionized water, and immediately subjected to imaging as described elsewhere (75).

The α2,3-Sia expression on human and dromedary camel nose and lung tissues was assessed by lectin histochemistry. Human and camel tissue samples used in this study were similar to those described in our previous study (53). The staining was performed as previously described using biotinylated Maackia amurensis lectin II (76). The use of human materials was approved by the local medical ethical committee (MEC approval: 2014-414). Human tissues were residual human biomaterials, which are collected, stored, and issued by the Erasmus MC Tissue Bank under ISO 15189:2007 standard operating procedures. Use of these materials for research purposes is regulated according to the Human Tissue and Medical Research code of conduct for responsible use (https://www.federa.org/sites/default/files/digital_version_first_part_code_of_conduct_in_uk_2011_12092012.pdf.). Experimental procedures using dromedaries were approved by the local Ethical Committee of the Autonomous University of Barcelona (no. 8003).

Maxisorp flat-bottom, 96-well plates were coated per well with 100 μL of PBS containing 1% (vol/vol) mucin from human airway epithelium (Epithelix) or with 100 μL of D-PBS containing 1 μg of BSM, and incubated for 4 h at room temperature. Plates were washed three times with washing buffer (PBS + 0.05% Tween-20) and subsequently blocked for 2 h at 37 °C with 200 μL of blocking buffer (PBS + 0.1% Tween-20, containing 5% Protifar skimmed milk). Plates were put at room temperature and washed three time with washing buffer before use. In some instances, mucin-coated plates were mock-treated (with PBS), treated with 20 μg/mL sialate–9-O-acetylesterase (BCoV-HE), or treated with 20 mU/mL NA (A. ureafaciens; Roche) (diluted in PBS, 100 μL per well) for 3 h at 37 °C. MERS-CoV S1^A-Fc, IAV HA-Fc [A/California/04/2009 (H1N1)] and pAPN-Fc were mock-treated or treated with 20 mU/mL NA (A. ureafaciens; Roche) in PBS for 3 h at 37 °C. Fc-tagged proteins were complexed with pA-LS (2:1 molar ratio) for 30 min at 4 °C, and twofold serial dilutions of mixtures were made using ice-cold blocking buffer. Complexed nanoparticles were incubated on mucin-coated plates for 2 h at 4 °C, after which bound LS nanoparticles were detected by ELISA using the HRP-conjugated anti-StrepMAB antibody that recognizes their C-terminally appended streptomycin tag.

Confluent layers of Vero or Calu-3 cells were mock-treated or treated with NA (A. ureafaciens) for 2 h at 37 °C, followed by inoculation with MERS-CoV (EMC isolate) or IAV (PR8 strain) (MOI = 0.1). Inoculum was removed after 1 h, and cells were washed once before addition of fresh culture medium. At 8 h postinfection, cells were fixed with formaldehyde, permeabilized with 70% ethanol, and stained using monoclonal antibody against MERS-CoV Nucleoprotein (Immunosource) (32) or a monoclonal antibody against influenza nucleoprotein (IgG2A, Clone Hb65; American Type Culture Collection) according to standard protocols using Alexa Fluor 488-conjugated goat α-rabbit or goat α-mouse antibodies as a second step (25). The percentage of infected cells was determined by immunostaining and counting.

To measure DPP4 expression, Vero and Calu-3 cells were incubated with 5 μg/mL polyclonal goat anti-hDPP4 antiserum (clone AF1180; R&D Systems), washed three times with FACS buffer (2 mM EDTA and 0.05% BSA in PBS), and then subsequently stained with rabbit anti-goat IgG antibodies conjugated with Alexa Fluor 488 in a 1:250 dilution (Life Technologies). To measure Sia expression, Vero and Calu-3 cells were incubated with 7.5 μg/mL S1^A-Fc–loaded pA-LS nanoparticles, followed by rabbit antistreptomycin antibodies (1:200), after which cells were washed three times with FACS buffer and stained with Alexa Fluor 633-conjugated secondary anti-rabbit IgG in a 1:250 dilution (Invitrogen). Cells were washed and resuspended for flow cytometric analysis using a BD FACS Canto II (BD Biosciences). Data were analyzed using FlowJo software.