Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential

Abstract

Binding to target cell receptors is a critical step in the virus life cycle. Coxsackievirus A24 variant (CVA24v) has pandemic potential and is a major cause of acute hemorrhagic conjunctivitis, but its cellular receptor has hitherto been unknown. Here we show that CVA24v fails to bind to and infect CHO cells defective in sialic acid expression. Binding of CVA24v to and infection of corneal epithelial cells are efficiently inhibited by treating cells with a sialic acid-cleaving enzyme or sialic acid-binding lectins and by treatment of the virus with soluble, multivalent sialic acid. Protease treatment of cells efficiently inhibited virus binding, suggesting that the receptor is a sialylated glycoprotein. Like enterovirus type 70 and influenza A virus, CVA24v can cause pandemics. Remarkably, all three viruses use the same receptor. Since several unrelated viruses with tropism for the eye use this receptor, sialic acid-based antiviral drugs that prevent virus entry may be useful for topical treatment of such infections.

FIG. 1.

CVA24v requires cell surface sialic acid for efficient binding to and infection of CHO cells. (A) CHO cells in suspension were first pretreated at 37°C for one hour with or without V. cholerae neuraminidase, incubated with 5,000 ³⁵S-labeled CVA24v virions per cell at 4°C for one hour, washed to remove unbound virions, and then analyzed in a scintillation counter for cell-associated radioactivity. CPM, counts per minute. (B) Adherent CHO cells were infected with 20,000 CVA24v virions per cell, fixed in methanol, and stained as described in Materials and Methods. CVA24v antigen-positive cells were then quantified in a fluorescence microscope. The results are presented as means ± standard deviations of duplicate samples from at least three independent experiments (MOCK, CHO-MOCK [mock with respect to CAR]; CAR, CHO-CAR; CD55, CHO-CD55; ICAM1, CHO-ICAM-1; CD46, CHO-CD46; 2241, CHO-2241; Lec2; and Pro-5 [parental cell line to Lec2]).

FIG. 2.

CVA24v virions efficiently infect and bind to human ocular epithelial cells. (A) Adherent cells (2 × 10⁵) were incubated with 5,000 CVA24v virions per cell at 4°C for one hour, washed to remove unbound virions, and incubated for 16 to 18 h at 37°C. The cells were fixed with methanol, stained as described in Materials and Methods, and quantified in a fluorescence microscope. (B) Binding experiments were carried out exactly as described for Fig. 1A, but without neuraminidase and with different cells. The results are presented as means ± standard deviations of duplicate samples from at least three independent experiments. CPM, counts per minute.

FIG. 3.

Flow cytometry analysis of candidate receptors on epithelial cells. Epithelial cells (5 × 10⁵) in suspension were incubated with biotinylated WGA to detect sialic acid, followed by incubation with streptavidin-FITC (A) or with monoclonal antibodies to human CAR (B), human CD55 (C), human CD46 (FITC conjugated) (D), or human ICAM-1 (FITC conjugated) (E), followed by FITC-conjugated anti-antibodies (against CAR or CD55), and analyzed by flow cytometry as described in Materials and Methods. Data represent the geometric means ± standard deviations of the fluorescence measured in three experiments in duplicate.

FIG. 4.

Cell surface sialic acid is required for efficient binding of CVA24v to and infection of human ocular cells. Binding was carried out as described for Fig. 1. (A) Cells (2 × 10⁵) in suspension were pretreated with or without neuraminidase from V. cholerae prior to incubation of the cells with 5,000 ³⁵S-labeled CVA24v virions per cell. (B) Cells (2 × 10⁵) in suspension were preincubated with or without WGA prior to incubation with 5,000 ³⁵S-labeled CVA24v virions per cell. (C) ³⁵S-labeled CVA24v virions (5,000 virions/cell) were preincubated with or without 13-valent sialic acid linked to albumin (SA-HSA) prior to virion-cell incubation (2 × 10⁵ cells/sample; concentrations of SA-HSA are in mM and with respect to sialic acid monosacharrides). (D to F) Adherent HCE (D), NHC (E), or Hep2 (F) epithelial human cell lines were pretreated with or without neuraminidase from V. cholerae or preincubated with or without WGA prior to incubation with virus (500 virions per cell) at 4°C. After one hour, the cells were washed to remove unbound virions and then incubated for 16 to 18 h at 37°C. They were fixed with methanol, stained as described in Materials and Methods, and quantified in a fluorescence microscope. The results are presented as means ± standard deviations of duplicate samples from at least three independent experiments. CPM, counts per minute.

FIG. 5.

Roles of SAα2,3Gal and SAα2,6Gal in binding of CVA24v to human ocular cells. (A) Flow cytometric analysis of expression of SAα2,3Gal (MAA II binding) and SAα2,6Gal (SNA binding) on NHC and HCE cells. Cells (5 × 10⁵) in suspension were first incubated with biotinylated lectins, followed by streptavidin-FITC, prior to flow cytometric analysis. Data represent the geometric means ± standard deviations of the fluorescence measured in three experiments in duplicate. (B) Effect of SAα2,3Gal-binding MAA II and SAα2,3Gal-binding SNA on CVA24v virion binding to NHC and HCE cells. Cells (2 × 10⁵) in suspension were preincubated with lectins prior to incubation with 5,000 ³⁵S-labeled CVA24v virions/cell. Cell-associated radioactivity (CPM [counts per minute] bound) was determined by using a scintillation counter. The results are presented as means ± standard deviations of duplicate samples from at least three independent experiments.

FIG. 6.

Protease treatment of ocular cells inhibits binding of CVA24v virions to NHC and HCE cells. Cells (2 × 10⁵) in suspension were pretreated with bromelain and ficin proteases (which cleave at cysteine residues) or V8 protease (which cleaves at glutamic acid or aspartic acid residues), prior to incubation with 5,000 ³⁵S-labeled CVA24v virions/cell. Cell-associated radioactivity (CPM [counts per minute] bound) was determined by using a scintillation counter. Cell viability was assessed with the trypan blue method immediately after proteolytic digestion and no cytotoxic effects mediated by the protease treatment could be seen, compared to untreated control cells. The results are presented as means ± standard deviations of duplicate samples from at least three independent experiments.