Coxsackievirus A24 Variant Uses Sialic Acid-Containing O-Linked Glycoconjugates as Cellular Receptors on Human Ocular Cells

doi:10.1128/JVI.05597-11

J Virol. 2011 November; 85(21): 11283–11290.

PMCID: PMC3194947

Coxsackievirus A24 Variant Uses Sialic Acid-Containing O-Linked Glycoconjugates as Cellular Receptors on Human Ocular Cells

Nitesh Mistry,¹^* Hirotoshi Inoue,¹ Fariba Jamshidi,¹ Rickard J. Storm,¹ M. Steven Oberste,³ and Niklas Arnberg^1,²

¹Division of Virology, Department of Clinical Microbiology

²Laboratory for Molecular Infection Medicine in Sweden (MIMS), Umeå University, SE-90185 Umeå, Sweden

³Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

^*Corresponding author. Mailing address: Division of Virology, Department of Clinical Microbiology, Umeå University, SE-90185 Umeå, Sweden. Phone: 46 90 7851790. Fax: 46 90 129905. E-mail: nitesh.mistry/at/climi.umu.se.

Received July 5, 2011; Accepted August 17, 2011.

This article has been cited by other articles in PMC.

Abstract

Coxsackievirus A24 variant (CVA24v) is a main causative agent of acute hemorrhagic conjunctivitis (AHC), which is a highly contagious eye infection. Previously it has been suggested that CVA24v uses sialic acid-containing glycoconjugates as attachment receptors on corneal cells, but the nature of these receptors is poorly described. Here, we set out to characterize and identify the cellular components serving as receptors for CVA24v. Binding and infection experiments using corneal cells treated with deglycosylating enzymes or metabolic inhibitors of de novo glycosylation suggested that the receptor(s) used by CVA24v are constituted by sialylated O-linked glycans that are linked to one or more cell surface proteins but not to lipids. CVA24v bound better to mouse L929 cells overexpressing human P-selectin glycoprotein ligand-1 (PSGL-1) than to mock-transfected cells, suggesting that PSGL-1 is a candidate receptor for CVA24v. Finally, binding competition experiments using a library of mono- and oligosaccharides mimicking known PSGL-1 glycans suggested that CVA24v binds to Neu5Acα2,3Gal disaccharides (Neu5Ac is N-acetylneuraminic acid). These results provide further insights into the early steps of the CVA24v life cycle.

INTRODUCTION

Acute hemorrhagic conjunctivitis (AHC) is a highly contagious disease predominantly caused by two members of the Picornaviridae family, coxsackievirus A24 variant (CVA24v; human enterovirus species C) and enterovirus 70 (EV70; human enterovirus species D). AHC begins with sudden onset of ocular pain and foreign body sensation and is followed by swollen eyelids and subconjunctival hemorrhages. These viruses are also associated with symptoms in the upper respiratory tract, and neurological manifestations such as acute flaccid paralysis have also been noted (7, 24, 37, 76). Typically, AHC occurs in densely populated, humid regions across tropical and subtropical areas (32, 79). Three pandemics and numerous outbreaks have been described (6, 24, 35, 37, 46, 47, 54). Although EV70 was the first virus to be associated with AHC (during an outbreak in 1969 [13]), CVA24v, described for the first time in the Singapore outbreak of 1970 (41), has since the middle of the 1980s been reported as the main causative agent of AHC (9–12, 15, 24, 37, 48, 56, 70, 79). Neither vaccines nor antiviral drugs are available for prevention or treatment of AHC.

The cellular receptors for several members of the Picornaviridae family have been described, including decay-accelerating factor (DAF), intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDL-R), sialic acid, coxsackie and adenovirus receptor (CAR), various integrins, and heparan sulfate (25, 26, 34, 44, 49, 50, 53, 57, 61, 62, 75). CVA24v and EV70 replicate in both conjunctival and corneal cells in vivo and in vitro (50, 76). Whereas EV70 uses either DAF or α2,3-linked sialic acid, depending on the cell type, CVA24v does not bind to DAF and does not exhibit any obvious preference for α2,3- or α2,6-linked sialic acid. The usage of α2,3- and/or α2,6-linked sialic acid by other viruses appears to correlate with virus tropism and receptor distribution. EV70, adenovirus 37 (Ad37), and subtype 7 avian influenza A virus are ocular viruses that bind to α2,3-linked sialic acid, which is abundant on ocular tissue. Human influenza A virus, on the other hand, is a respiratory virus that binds to α2,6-linked sialic acid, which is found on cells in the upper respiratory tract (53, 55). CVA24v is associated with both ocular and respiratory disease, which correlates with its binding to both α2,3- and α2,6-linked sialic acid (50). Here, we investigated the nature of the sialic acid-containing receptors in binding and infection experiments using human ocular epithelial cells.

MATERIALS AND METHODS

Cells and viruses.

L-PSGL-1.1, L-bsd, L-SCARB2, L-Empty, HCE (human corneal epithelial), and NHC (normal human conjunctival) cells were cultured as described previously (50, 52, 78). The CVA24v strains (110386, 110387, 110388, 110389, 110390, 110391, and 110392) used in this study originate from an outbreak in Malaysia (24). ³⁵S-Labeled CVA24v and unlabeled virions were generated as described previously (50).

Reagents and antibodies.

In this study, we used a mouse monoclonal antibody against enterovirus VP1 (DakoCytomation, Glostrup, Denmark), fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse immunoglobulin G (DakoCytomation), FITC-labeled streptavidin (DakoCytomation), mouse monoclonal anti-human P-selectin glycoprotein ligand-1 (PSGL-1; R&D systems, Minneapolis, MN), tunicamycin (Sigma-Aldrich, St. Louis, MO), benzyl N-acetyl-α-d-galactosaminide (benzyl-α-GalNAc; Sigma-Aldrich), peptide N-glycosidase F (PNGase F; Sigma-Aldrich), FITC-conjugated cholera toxin subunit B (CT-B; Invitrogen), neuraminidase from Vibrio cholerae (Sigma-Aldrich), FITC-conjugated Phaseolus vulgaris erythroagglutinating lectin (E-PHA; Vectorlabs, Burlingame, CA), 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP; Merck KGaA, Darmstadt, Germany), P4[(1R,2R)-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol], inactive enantiomer of P4 (1S,2S; kindly provided by Roland L. Schnaar), 3′-sialyl Lewis X (sLe^X; Carbosynth, Berkshire, United Kindom), 3′-sialyl-N-acetyllactosamine (3′SLN; Prozyme, Hayward, CA), 3′-sialyl-Thomsen-Friedenreich antigen (3′-sialyl-TF; Lectinity, Moscow, Russia), N-acetylneuraminic acid (sialic acid; Dextra, Reading, United Kindom), and d(+)galactose (Gal), N-acetyl-d-galactosamine (GalNac), l(−)fucose (Fuc), and N-acetyl-d-glucosamine (GlcNAc) (all from Sigma-Aldrich).

Binding experiments.

³⁵S-Labeled CVA24v (strain 110390) virions were used as previously described (4, 50). Briefly, adherent cells were detached with phosphate-buffered saline containing 0.05% EDTA (phosphate-buffered saline [PBS]–0.05% EDTA, Merck) and recovered in growth medium at 37°C. After 1 h, 2 × 10⁵ cells/sample were washed and diluted in 100 μl binding buffer (BB) containing Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich), HEPES (pH 7.4; EuroClone, Milan, Italy), and 1% bovine serum albumin (BSA; Roche, Stockholm, Sweden) before the addition of 5,000 ³⁵S-labeled CVA24v virions/cell. After 1 h of incubation at 4°C, cells were washed to remove nonbound virions before the radioactivity of the cells was measured using a Wallac 1409 scintillation counter (Perkin-Elmer, Waltham, MA).

CVA24v infection experiments.

(i) Immunofluorescence microscopy. This method was used to assess the impact of cell surface sialic acid during infection of multiple CVA24v strains and was performed as previously described (4, 50). Briefly, various dilutions of unlabeled CVA24v virions were allowed to attach to 2 × 10⁵ adherent HCE cells in 24-well plates at 4°C for 1 h. Cells were washed to remove nonbound virions and incubated in growth medium at 37°C for 16 to 18 h. After fixation with 99.5% ice-cold methanol, mouse monoclonal antibodies against enterovirus VP1 were diluted 1:200 in PBS and 300 μl was added per well. After incubation for 1 h at room temperature, the cells were washed again and incubated with 300 μl FITC-labeled rabbit anti-mouse immunoglobulin G (diluted 1:100 in PBS) per well at room temperature. One hour later, the cells were washed again, and the numbers of infected cells were quantified using a fluorescence microscope (Axioskop2; Zeiss, Jena, Germany) and ImageJ software (http://rsbweb.nih.gov/ij/).

(ii) Flow cytometry. The effects of metabolic inhibitors of de novo glycosylation on CVA24v infection in corneal cells were measured by flow cytometry. Briefly, 2.5 × 10⁵ adherent HCE or NHC cells in 24-well plates were infected with ~70 nonlabeled CVA24v virions (strain 110390)/cell in 250 μl growth medium and incubated at 4°C with gentle agitation. After 1 h, nonbound virions were removed by washing with growth medium and the cells were incubated with 500 μl fresh growth medium at 37°C. After incubation for 16 to 18 h, the cells were detached with trypsin (Invitrogen), resuspended in BB to inactivate trypsin, and washed once with PBS prior to fixation with ice-cold 99.5% methanol. After being washed, the cells were incubated for 1 h at room temperature with 300 μl monoclonal mouse antibodies against enterovirus VP1 (diluted 1:200 in PBS) per well. Subsequently, the cells were washed with PBS and incubated with 300 μl FITC-labeled rabbit anti-mouse immunoglobulin G (diluted 1:100 in PBS) per well at room temperature. After 1 h, the cells were washed with PBS, resuspended in 2% formaldehyde, and analyzed using FACScan flow cytometry (Becton Dickinson).

Inhibition of glycosylceramide synthase.

To inhibit de novo synthesis of ganglioside biosynthesis, the cells were grown in the presence (or absence) of 25 μM PDMP, 2.5 μM P4 (1R,2R), or 25 μM inactive enantiomer of P4 (1S,2S) and incubated for 3 days at 37°C. Thereafter, the cells were washed and binding assays were carried out as described above. To demonstrate the effect of P4, 2 × 10⁵ P4-pretreated (or nontreated) cells were detached with PBS–0.05% EDTA, washed, and resuspended in PBS–0.1% BSA with prior incubation with 10 μg/ml ganglioside G_M1-binding FITC-conjugated CT-B diluted in PBS–0.1% BSA at 4°C. After 30 min, the cells were washed twice with PBS–0.1% BSA to remove nonbound CT-B, resuspended in PBS, and analyzed using FACScan flow cytometry.

Inhibition of N- and O-glycosylation synthesis.

Adherent NHC or HCE cells were grown in the presence or absence of either 0.3 μg/ml tunicamycin for 24 h or 3 mM benzyl-α-GalNAc for 48 h at 37°C (3). Prior to binding experiments and flow cytometry experiments, ³⁵S-labeled CVA24v (strain 110390) and nonlabeled CVA24v virions, respectively, were added to the cells. Cells were stained with trypan blue after tunicamycin and benzyl-α-GalNAc treatment to verify cell viability. To determine the effect of tunicamycin, cells treated with or without tunicamycin were first washed twice with buffer B (10 mM HEPES, pH 7.5, plus 0.15 NaCl) prior to incubation at 4°C with E-PHA (2 μg/ml for HCE cells and 3 μg/ml for NHC cells) diluted in 100 μl buffer B. After 1 h, the cells were washed twice in PBS, resuspended in 200 μl 2% paraformaldehyde, and analyzed using FACScan flow cytometry.

Binding inhibition with soluble glycans.

³⁵S-Labeled CVA24v (strain 110390) virions were incubated on ice with or without different concentrations of mono- and oligosaccharides diluted in 50 μl BB. After 1 h, virion-glycan mixtures were incubated with HCE cells prior to quantification of CVA24v binding as described above.

Enzymatic removal of cell surface glycans.

Two enzymes were used to cleave off cell surface glycans, as follows. (i) Amounts of 100 U/ml PNGase F in 100 μl BB were added to 2 × 10⁵ cells in suspension and incubated for 1 h at 37°C to remove N-linked glycans from cell surface proteins. The effect of PNGase F treatment on CVA24v binding was quantified as described above, as well as the overall efficiency of PNGase treatment (using E-PHA as described above). (ii) Sialic acid monosaccharides were enzymatically removed by incubating 2 × 10⁵ cells with 10 mU V. Cholerae neuraminidase diluted in 100 μl BB for 1 h at 37°C. After being washed, the cells were incubated with ³⁵S-labeled CVA24v (strain 110390) virions, and virion binding was quantified as described above.

Quantification of sLe^X and sialic acid levels on cellular surface.

Briefly, the cells were detached with PBS–0.05% EDTA and recovered in growth medium at 37°C. After 1 h, the cells were washed and resuspended in 5 ml PFN buffer (PBS containing 1% fetal calf serum [FCS; Sigma-Aldrich]) and then transferred to a 96-well plate with 3 × 10⁵ cells/well. Cells were then incubated for 1 h on ice with 10 μg/ml of biotinylated wheat germ agglutinin lectin (WGA; binds sialic acid and GlcNAc) in 100 μl PFN buffer and sodium azide (OneMed Lab, Gothenburg, Sweden) or with 2 μg/ml PSGL-1 antibody (sLe^X-bearing core 2 O-glycan) in 100 μl PBS. WGA-incubated cells were then washed twice with PFN and incubated for 1 h in the dark with 100 μl of 5 μg/ml FITC-labeled streptavidin diluted in PFN, whereas antibody-treated cells were washed twice and incubated with 100 μl of 1 μg/ml FITC-labeled rabbit anti-mouse antibody diluted in PFN. In either case, the cells were then washed twice and resuspended in 200 μl PFN and analyzed with FACScan flow cytometry (Becton Dickinson).

Glycan array.

Purified CVA24v virions (strain 110390) were labeled with Alexa Fluor 488 (AF-488) carboxyl acid, succinimidyl ester (Invitrogen Life Technologies, Carlsbad CA) as previously described (71). Briefly, purified CVA24v virions, as described previously, were diluted to 3 × 10¹¹ virions/ml in 0.1 M sodium bicarbonate buffer, pH 8.4, and 2 ml virions was added to 100 μl dimethyl sulfoxide (DMSO) containing 1 mg AF-488. The mixture was vortexed for 30 s and then incubated for 1 h at room temperature with agitation in the dark. After incubation, the mixture was transferred into a 10-kDa Slide-A-Lyzer cassette (Thermo Scientific, Rockford, IL) and dialyzed overnight at 4°C against three changes of Tris-buffered saline, pH 7.4. A concentration of 10% glycerol was added to the dialyzed AF-488-labeled CVA24v virions, which were then aliquoted and stored at −20°C. The virion concentration was determined (70 μg/ml) and a glycan array (version 4.2) with AF-488-labeled CVA24v was performed by the Consortium for Functional Glycomics (CFG, core H). Full assay procedures are also available on the CFG website (http://www.functionalglycomics.org/; CFG accession number cfg_rRequest_1629). To confirm the sialic acid-binding function of AF-488-labeled CVA24v virions, 2 × 10⁵ adherent HCE cells in a 24-well plate were treated with or without 20 mU V. Cholerae neuraminidase in 200 μl BB for 1 h at 37°C. After being washed, 2 × 10⁹ AF-488-labeled CVA24v virions were diluted in 200 μl BB and incubated with the cells for 1 h on ice with gentle agitation. After being washed, the cells were fixed in 2% paraformaldehyde and analyzed in a fluorescence microscope.

Statistical analysis.

A two-tailed, paired Student's t test was used to determine the statistical significance of data wherever indicated, and P values of less than 0.05 were considered significant.

RESULTS

Multiple CVA24v clinical strains require sialic acid for infection of corneal cells.

We have previously shown that one strain (110390) of CVA24v infects corneal cells through binding to cell surface sialic acid (50). To determine whether this mechanism is shared by other clinical CVA24v strains, corneal (HCE) cells were treated with Vibrio cholerae neuraminidase before infection with seven different low-passage-number CVA24v strains isolated from the 2002 to 2003 AHC outbreak in Malaysia (24). In each case, in comparison to nontreated cells, the number of CVA24v-infected cells was 7 to 12% after neuraminidase treatment (Fig. 1). This demonstrated that sialic acid-containing molecules are required for infection of corneal cells by multiple CVA24v clinical strains, suggesting that it may be a general property of CVA24v.

Fig. 1.

Multiple CVA24v strains require sialic acid for infection of human corneal epithelial cells. HCE cells were not treated or treated with V. cholerae neuraminidase prior to infection with different strains of CVA24v. The bars indicate the number of CVA24v-infected (more ...)

CVA24v binding to corneal cells does not require gangliosides.

Previous attempts to characterize CVA24v receptors by protease treatment of target cells indicated that the CVA24v receptor contains at least one sialylated protein component (50). To exclude the possibility that gangliosides (sialylated glycolipids) could function as CVA24v receptors, we treated HCE cells with the glycosylceramide synthase inhibitors PDMP and P4 (1R,2R) and, as a control, the inactive form of P4 (1S,2S). PDMP interferes in the first step of glycosphingolipid glycosylation but may also have an inhibitory and cytotoxic effect on cells due to increasing intracellular ceramide levels (1, 40). P4 (1R,2R), on the other hand, is a more potent inhibitor of glycosylceramide synthase with no inhibition of cell growth or elevation of intracellular ceramides. Pretreatment of HCE cells with these compounds did not reduce CVA24v binding to HCE cells (Fig. 2A), suggesting that gangliosides are not important for binding of CVA24v to corneal cells. The effect of P4 on HCE cell ganglioside biosynthesis was demonstrated using pentameric, FITC-labeled, ganglioside G_M1-binding cholera toxin subunit B (CT-B) (39). As expected, pretreatment of HCE cells with P4 1R,2R (but not 1S,2S) efficiently reduced (93%) CT-B binding, clearly showing that P4 1R,2R was functional (Fig. 2B).

Fig. 2.

Gangliosides are not involved in CVA24v binding to corneal cells. HCE cells were preincubated with PDMP, P4 (1R,2R), or inactive P4 (1S,2S) to inhibit glycosylceramide synthase. (A) The bars indicate the percentages of cell-bound ³⁵S-labeled CVA24v (strain (more ...)

CVA24v binding to and infection of corneal cells require O-linked glycans.

Recently, it was shown that sialic acid was required for efficient CVA24v binding to and infection of corneal but not conjunctival cells (50). To further characterize the receptor used by CVA24v on these cells, we treated HCE and conjunctival (NHC) epithelial cells with PNGase F to remove N-linked glycans from cell surface glycoproteins before quantification of CVA24v binding. CVA24v binding was 73% in comparison to the results for the control in NHC cells (Fig. 3A), and it was 83% in HCE cells (Fig. 3B), but these reductions were not significant (P = 0.077 and 0.18, respectively). Next, N- and O-linked de novo glycosylation was inhibited with tunicamycin and benzyl-α-GalNAc, respectively. As expected, no or little effect of either inhibitor was observed for CVA24v binding to NHC cells (Fig. 3A), and tunicamycin treatment of HCE cells had no effect on CVA24v binding (Fig. 3B). However, CVA24v binding to HCE cells was efficiently reduced (30% of the binding of the control; P = 0.0037) when inhibiting the de novo synthesis of O-linked glycans with benzyl-α-GalNAc. Next, we tested whether O-linked and/or N-linked glycans were essential for CVA24v infection. Treatment of HCE cells with benzyl-α-GalNAc efficiently inhibited CVA24v infection (28% infected cells in comparison to the control; P = 0.0093), and a minor effect was observed after tunicamycin treatment (73% infected cells in comparison to the control; P = 0.0461) (Fig. 3D). However, only minor, nonsignificant effects were observed after tunicamycin (77% infected cells in comparison to the control; P = 0.0529) and benzyl-α-GalNAc (120% infected cells in comparison to the control; P = 0.1796) treatment of NHC cells (Fig. 3C). These results suggested that CVA24v used glycans that are O-linked (via Ser or Thr) to membrane proteins for binding to and infection of corneal cells but not conjunctival cells. The efficiency of tunicamycin and PNGase F treatment was demonstrated by quantification of bound Phaseolus vulgaris erythroagglutinating lectin (E-PHA), which is specific for N-linked biantennary carbohydrate chains (36). Removal of N-linked glycans with PNGase F reduced the binding of FITC-conjugated E-PHA lectin by 60% in NHC cells and 32% in HCE cells (data not shown), and tunicamycin inhibited 50% of E-PHA binding to HCE cells and 40% to NHC cells (data not shown).

Fig. 3.

CVA24v binding to and infection of corneal cells depend on O-linked glycans. Bars indicate percentages of CVA24v binding to (A, B) or infection of (C, D) NHC (A, C) or HCE (B, D) cells pretreated with PNGase F, benzyl-α-GalNAc, or tunicamycin. (more ...)

CVA24v virions bind better to cells overexpressing glycoprotein PSGL-1.

Recently, two novel receptors for enterovirus 71 (EV71) and coxsackievirus A16 were discovered: human P-selectin glycoprotein-1 (PSGL-1) and scavenger receptor class B, member 2 (SCARB2) (52, 78). PSGL-1 is a mucinlike glycoprotein involved in binding and rolling of leukocytes on the endothelium, and SCARB2 is a membrane protein associated with endocytosis of high-density lipoproteins (20, 45). To test whether PSGL-1 and SCARB2 cells could serve as attachment receptors for CVA24v, we quantified the binding of ³⁵S-labeled CVA24v virions to L929 cells (a mouse fibroblastlike cell line) transfected with cDNAs encoding PSGL-1 or SCARB2. CVA24v did not bind more efficiently to SCARB2-expressing cells than to control (L-Empty) cells (Fig. 4) but did bind to PSGL-1-expressing cells (L-PSGL-1.1) 3-fold better than to mock-transfected (L-bsd) cells (Fig. 4B). Moreover, we demonstrated that both L-PSGL-1.1 and L-bsd cells express the same level of sialic acid (Fig. 5A), and CVA24v binding to L-PSGL-1.1 and L-bsd cells was only 0.1% and 1.2%, respectively, after neuraminidase treatment (Fig. 4B). These results suggested that the overall level of CVA24v binding to PSGL-1-transfected cells depended entirely on sialic acid-containing glycans and that the increased binding was due to an increase of specific sialic acid-containing carbohydrates. These results also suggested that CVA24v did not bind directly to the PSGL-1 protein but, rather, bound only to the sialylated form of PSGL-1.

Fig. 4.

CVA24v binding is enhanced in cells overexpressing PSGL-1. Bars represent binding of CVA24v to cells transfected with SCARB2 (A) or PSGL-1 (B) cDNAs or to corresponding mock-transfected cells (L-Empty and L-bsd, respectively). (B) Black bars represent (more ...)

Fig. 5.

Sialic acid and sLe^X expression on target cells. (A) Bars represent WGA binding to L-PSGL1.1 and control L-bsd cells. (B) Bars represent binding of anti-sLe^X-bearing core 2 O-glycan antibody to L-PSGL1.1, L-bsd, HCE, and NHC cells. Data in the figures (more ...)

Sialic acid-containing glycans inhibit CVA24v binding to corneal cells.

PSGL-1 is covered by O-linked glycans and contains the tetrasaccharide sialyl Lewis X [sLe^X; Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc] glycan (Fig. 6C) (2, 72), and we therefore set out to verify whether this glycan may also be found on target cells. By using an antibody recognizing sLe^X-bearing core 2 O-glycan structures, we could demonstrate that PSGL-1-transfected cells express a higher level of sLe^X than mock-transfected (L-bsd) cells, and we also found that the corneal cells expressed more sLe^X than NHC cells and twice as much sLe^X as the PSGL-1-transfected cells (Fig. 5B). Combined with the results showing that CVA24v binds better to L-PSGL-1.1 cells than to L-bsd cells, despite the fact that these cells express similar levels of sialic acid, this opened up the possibility that CVA24v binds to sLe^X. Consequently, we next tested whether sLe^X and other soluble, PSGL-1-containing glycans could interfere with CVA24v binding to HCE cells. As expected, sialic acid alone inhibited CVA24v binding (50% inhibitory concentration [IC₅₀], 20 mM) (Fig. 6A), and the other monosaccharides had no or minor effect. Since PSGL-1 is covered by complex glycans that are not available for purchase, we also tested whether smaller tri- and tetrasaccharides matching parts of the more complex glycans could inhibit CVA24v binding. sLe^X and the trisaccharides 3′SLN (Neu5Acα2-3Galβ1-4GlcNAc) and 3′-sialyl-TF (Neu5Acα2-3Galβ1-3GalNAc) (Fig. 6C) inhibited CVA24v with IC₅₀s of 8, 5, and 3 mM, respectively (Fig. 6B). These results suggested that the combination and linkage of underlying saccharides of sialic acid are important and that the galactose linked to sialic acid via α2,3-glycosidic bonds (Neu5Acα2,3Gal) may also interact with the CVA24v capsid. The fucose did not appear to contribute to sLe^X binding.

Fig. 6.

CVA24v binding to corneal cells is inhibited by sialic acid-containing glycans. CVA24v (strain 110390) was incubated with different concentrations of monosaccharides (A) or with tri- or tetrasaccharides (B) prior to incubation with corneal cells. Each (more ...)

CVA24v did not bind to any of the 511 glycans in a glycan array.

To further identify specific glycans to which CVA24v could bind, we analyzed the glycan binding of AF-488-labeled CVA24v virions in the glycan array (version 4.2) provided by the Consortium for Functional Glycomics (CFG) (data not shown). The glycan array consisted of 511 structurally defined oligosaccharides, of which 122 oligosaccharides contained at least one sialic acid residue. Of these sialic acid-containing oligosaccharides, 27 contained a terminal GalNAc, which is often the monosaccharide in O-linked glycans that binds Ser/Thr on mucin-type glycoproteins (30). However, no CVA24v binding was observed to any of the glycans in the array, despite the presence of sLe^X and other glycans found in PSGL-1. We therefore assumed that the glycans in the array did not accurately present the sialylated oligosaccharides specific for CVA24v binding. For example, if the sialic acid-binding site is localized in a canyon or pocket of the CVA24 capsid, it is possible that the length and flexibility of the glycans in the array did not permit binding to the virions. The functionality of AF-488-labeled CVA24v virions was, however, demonstrated by treating or not treating HCE cells with neuraminidase prior to analysis of bound virions with fluorescence microscopy. The binding of CVA24v to neuraminidase-treated HCE was reduced (data not shown), suggesting that the AF-488-labeled virions were functional and that the Alexa Fluor dye did not interfere with CVA24v binding to its sialylated attachment receptor.

DISCUSSION

Multiple viruses use sialic acid-containing molecules as cellular receptors, including epidemic keratoconjunctivitis (EKC)-causing Ad37, Newcastle disease virus (NDV), AHC-causing picornaviruses, and avian and human influenza A virus (3–5, 38, 50, 55, 67). Gangliosides are used by Sendai virus, bovine adeno-associated virus (BAAV), and human parainfluenza virus types 1 and 2 (21, 60, 66), but bovine parvovirus (BPV), influenza A virus, BK virus, JC virus, EV70, human norovirus, rotavirus, and adeno-associated virus serotypes 4 and 5 (AAV4 and AAV5) use sialic acid-containing glycoproteins as receptors (8, 16, 18, 19, 33, 42, 53, 59, 74). In many of these cases, the underlying glycan-carrying molecule is poorly understood, and this is also the case for AHC-causing CVA24v. In this work, we set out to further characterize the sialic acid-containing receptor(s) used by CVA24v for infection of ocular cells. Previous studies using proteases suggested that the CVA24v receptor(s) include one or more protein component(s). However, this did not exclude gangliosides from being involved in virus binding. Our results from experiments using glycosylceramide synthase inhibitors P4 and PDMP clearly demonstrated that the sialic acid-containing component used by CVA24v for binding to corneal cells is not a ganglioside. PNGase F cleaves between GlcNAc and the asparagine residues of N-linked proteins, except in glycans containing α1,3-linked fucose (68). This treatment may leave some N-linked sialic acid-containing saccharides left on the cell surface that CVA24v may bind to. However, since we obtained similar results from experiments where cells were treated with tunicamycin, we conclude that N-linked glycans do not contribute to CVA24v binding to ocular cells to any large extent. In contrast, treatment of corneal cells with benzyl-α-GalNAc resulted in efficient reduction in both binding and infection, thus suggesting that CVA24v binding to and infection of corneal cells requires one or more glycoproteins that contain O-linked glycans. In agreement with previous studies suggesting that CVA24v binding to conjunctival cells does not involve sialic acid, we did not observe any substantial effect of tunicamycin, benzyl-α-GalNAc, or PNGase F on CVA24v binding to these cells, and thus, we concluded that CVA24v binding to conjunctival cells does not require cell surface glycans, in contrast to CVA24v binding to corneal cells.

BPV, AAV4, Ad37, and EV70 also bind to O-linked glycans on membrane glycoproteins. The latter two viruses are known to cause eye diseases. In contrast, studies on NDV, another virus with ocular tropism in humans (28), suggested that gangliosides are important for binding and N-linked glycans are crucial for entry (23). Thus, O-linked glycans appear not to be universal receptors for ocular viruses. Nevertheless, the apical surface of the ocular epithelium is rich in mucins, which are high-molecular-weight glycoproteins containing multiple O-linked glycans and with primarily α2,3-linked sialic acids (29). Here, we show that binding and infection of CVA24v are reduced if the target cells express fewer O-linked glycans, allowing us to speculate that O-linked glycans present on membrane-bound mucins, such as PSGL-1, are involved in CVA24v infection in the eye. In our search for receptors, we analyzed the binding of ³⁵S-labeled CVA24v to PSGL-1- and SCARB2-transfected L-929 cells. CVA24v did not use SCARB2, which is in agreement with its high levels of N-linked but not O-linked glycans (14). In contrast, cell surface PSGL-1 efficiently promoted CVA24v binding, suggesting that PSGL-1 glycoproteins contain one or more components that mediate CVA24v binding to target cells. PSGL-1 is a heavily O-glycosylated, mucinlike dimeric glycoprotein that is rich in serine/threonine repeat regions, with no less than 70 potential sites for O-glycosylation and only 3 potential sites for N-glycosylation on each subunit (17, 63). The majority of the glycans on PSGL-1 are nonfucosylated, nonsialylated core 2 O-glycans, and thus, only a few of these structures contain sLe^X (2, 72). Here, we demonstrate that sLe^X is present mainly on corneal cells but also on conjunctival cells and that soluble sLe^X is a better inhibitor of CVA24v binding to corneal cells than sialic acid monosaccharides. The other glycans on PSGL-1, 3′SLN and 3′-sialyl-TF, reduced CVA24v binding to corneal cells even better than sLe^X, indicating that the fucose monosaccharide present in sLe^X is not important for binding. The tri- and tetrasaccharides all contained the Neu5Acα2,3Gal motif, suggesting that this motif is important for binding to corneal cells. However, this does not exclude the possibility that CVA24v may also bind to α2,6-linked sialic acids, which may be important for binding to upper respiratory tract tissues. In general, viruses that infect ocular tissues preferentially bind α2,3-linked sialic acids, which are abundant in ocular epithelial cells (5, 53), and α2,6-linked sialic acids are instead used as receptors by viruses that infect upper respiratory tract tissues (55). CVA24v appears to bind to both α2,3-linked and α2,6-linked sialic acids, and this may at least partially explain why CVA24v causes respiratory disease more frequently than EV70, which mainly binds α2,3-linked sialic acid and almost exclusively causes ocular disease (50, 53, 76). On PSGL-1, the 3′-sialyl-TF trisaccharide is linked to either 3′SLN or sLe^X, thus creating a disialic acid-containing glycan (2, 72), which may provide even better binding to CVA24v. In agreement with this suggestion, we demonstrated recently that branched, disialylated glycans are functional receptors for another human ocular pathogen, Ad37, and showed that this soluble glycan was 200-fold more efficient in inhibiting Ad37 binding to corneal cells (51).

In an attempt to determine the structure of the sialylated glycan chain that binds CVA24v, we analyzed the binding of FITC-labeled CVA24v virions in a glycan array that included 511 different glycan structures. Six of the glycan structures in the glycan array performed by CFG contained sLe^X motifs. Unfortunately, CVA24v did not bind to any of the glycans in the array, perhaps because the binding site is localized in the assumed canyons of CVA24v, which are present in other picornaviruses and harbor receptor binding pockets (31, 77). In this case, however, we assumed that these sites are not available for the glycans in the array, possibly for reasons of spacer length and glycan flexibility.

The ability to bind host cell receptors is thought to be of importance in virus tropism and pathogenesis (22). Multiple viruses use more than one receptor on the cellular surface. The attachment and entry of viruses such as herpes simplex virus (43, 64), HIV (80), and rotavirus (27), for example, require multiple interactions between viral proteins and target cell receptors. Studies of the Enterovirus genus (CVA, CVA21, and EV70) provide further support for multireceptor usage (34, 53, 58, 62, 69, 73). Our results suggest that CVA24v utilizes at least two different cell surface receptors. The conjunctival receptor remains to be identified but is assumed to be a membrane protein, and if glycosylated, these glycans play no major role in virus binding. The corneal receptor, on the other hand, appears to contain O-linked oligosaccharides attached to one or more cell surface glycoprotein(s). The O-glycans involved as receptors by CVA24v appear to be identical or highly similar to the group of glycans (sLe^X, 3′SLN, and 3′-Sialyl-TF) that are found on PSGL-1, which may be one candidate receptor for CVA24v. Our findings provide novel insights into the molecules that determine the early events of the CVA24v life cycle and may also provide a target interaction for the development of antiviral drugs, similar to what has been suggested for sialic acid-binding Ad37 (51, 65).

ACKNOWLEDGMENTS

We thank Roland L. Schnaar for supplying the glycosphingolipid biosynthesis inhibitor (1R,2R)-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (P4) and the inactive (1S,2S) enantiomer of P4, and we thank Satoshi Koike for L929 cells transfected with SCARB2 and L-Empty cells. We also thank Yorihiro Nishimura and Hiroyuki Shimizu for providing L-PSGL-1.1 and L-bsd cells.

This work was supported by a grant from the Swedish Research Council (grant no. 2007-3402), the Swedish Foundation for Strategic Research (grant no. F06-0011), and the Kempe foundation (to MIMS; grant no. SMK-2752).

The authors declare that there are no conflicts of interest.

Footnotes

Published ahead of print on 31 August 2011.

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