Integrin alphavbeta1 is a receptor for foot-and-mouth disease virus

Abstract

Infection by field strains of Foot-and-mouth disease virus (FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including alphavbeta3 and the epithelial integrin alphavbeta6. In this report we show that the integrin alphavbeta1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by alphavbeta1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric alpha5/alphav integrin subunits, in association with the beta1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of alphavbeta1, not alpha5beta1, thus providing further evidence for the receptor role of alphavbeta1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of alphavbeta1 and alphavbeta6.

FIG. 1.

Flow cytometric analysis of FMDV binding to CHOB2 and integrin-transfected CHOB2 cell lines. FMDV strain O1K-cad² (20 μg/ml) was bound to CHOB2 (A and B), αv/α5(F1-G232)/β1 (C and D), CHOB2-αvβ1 (E and F), α5/αv(F1-G223)/β1 (G and H), and CHOB2-αvβ6 (I and J) in the presence of Ca and Mg alone (A, C, E, G, and I) or in the presence of Ca, Mg, and Mn (B, D, F, H, and J). Virus binding (hatched histogram) was determined by using the anti-FMDV MAb C9 and a goat anti-mouse IgG2a-specific R-phycoerythrin conjugate. Background fluorescence (black histogram) was determined as described in Materials and Methods. Virus binding in the presence of the activating anti-β1-MAb 9EG7 (D, F, and H) is shown as the open histogram. One experiment representative of three is shown.

FIG. 2.

Anti-integrin MAbs inhibit binding of FMDV to CHOB2 cell lines expressing αvβ1 or αvβ6. CHOB2-αvβ1 (A) or CHOB2-αvβ6 (B) cells were pretreated with the anti-αv MAb, L230 (A), the anti-αvβ6 MAb, 10D5 (B), or the anti-αvβ5 MAb, P1F6 (A and B), at 100 μg/ml for 0.5 h prior to the addition of virus (O1Kcad²; 20 μg/ml). Virus binding to cells expressing αvβ1 was carried out in the presence of manganese. Virus binding was detected by flow cytometry as described in Materials and Methods and is expressed as the percentage of virus bound to cells pretreated with assay buffer alone (control). The means from two independent experiments are shown, and in each case the range of observations was within 5% of the mean.

FIG. 3.

RGD peptides differentially inhibit FMDV binding to CHOB2 cell lines expressing αvβ1 or αvβ6. CHOB2-αvβ6 (A) or CHOB2-αvβ1 (B) cells were pretreated with RGD or control RGE peptides at the indicated concentrations for 0.5 h prior to the addition of virus (O1Kcad²; 20 μg/ml). FMDV-RGD (VPNLRGDLQVLA) has its sequence derived from the FMDV RGD site. Virus binding to cells expressing αvβ1 was carried out in the presence of manganese. Virus binding was detected by flow cytometry as described in Materials and Methods and is expressed as the percentage of virus bound to cells pretreated with assay buffer alone (control). The means from two independent experiments are shown, and in each case the range of observations was within 10% of the mean.

FIG. 4.

Infection of integrin-transfected CHOB2 cells is inhibited by anti-integrin antibodies. Duplicate aliquots of CHOB2-αvβ1 (A) or CHO-αvβ6 (B) cells were pretreated with the anti-αv MAb (L230) (A), the anti-αvβ6 MAb (10D5) (B), or the anti-αvβ5 MAb (P1F6) (A and B) at 50 μg/ml for 0.5 h prior to the addition of cold virus (O1Kcad²) at a MOI of 1 PFU/cell for a further 0.5 h. The cells were washed to remove unbound virus, and infection was initiated by incubation at 37°C for 0.5 h. Virus that remained on the outsides of the cells was acid inactivated, and the infected cells were used in an infectious center assay. The infection of cells expressing αvβ1 was carried out in the presence of manganese. Control samples were incubated with assay buffer alone (control) before the addition of virus. The means from two independent experiments are shown, and in each case the range of observations was within 5% of the mean.

FIG. 5.

Infection of integrin-transfected CHOB2 cells is inhibited by RGD peptides. Duplicate cell aliquots of CHOB2-αvβ1 (A) or CHO-αvβ6 (B) were pretreated with RGD peptides at the indicated concentrations for 0.5 h prior to the addition of cold virus (O1Kcad²) at a MOI of 1 PFU/cell for a further 0.5 h. The cells were then treated as described for Fig. 4. Infection of cells expressing αvβ1 was carried out in the presence of manganese. Control samples were incubated with assay buffer alone (control) before the addition of virus. The means from two independent experiments are shown, and in each case the range of observations was within 10% of the mean.