Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins

Abstract

At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus adsorption. Virus incubated with soluble alphaVbeta6 had a lower sedimentation rate than native virus on sucrose density gradients, but the particles retained all of their structural proteins and still contained bound integrin and, therefore, were not exhibiting characteristics of a picornavirus A particle.

FIG. 1.

Analysis of soluble integrins secreted from transfected cells. COS-1 cells were transfected with soluble integrin subunits, as denoted in the figure, and labeled with [³⁵S]methionine-cysteine between 24 and 72 h after transfection as described in Materials and Methods. The medium was collected, and the presence of soluble integrins or integrin subunits was determined by RIP, using the antibodies denoted in the figure followed by nonreducing SDS-PAGE and autoradiography. NT, nontransfected cells. The specificities of the antibodies were as follows: rabbit polyclonal anti-α_Vβ₃ antibody (anti-α_Vβ₃); monoclonal anti-α_Vβ₆ antibody (10D5); monoclonal anti-β₆ antibody (CSβ6); monoclonal anti-α_Vβ₃ antibodies (LM609 and 23C6).

FIG. 2.

Analysis of binding of soluble α_Vβ₃ and α_Vβ₆ to FMDV. (a and b) Virus (1 μg) was captured on antibody-coated plastic wells as described in Materials and Methods. Culture medium from nontransfected HEK 293A cells (grey bars) or from transfected cells containing either soluble α_Vβ₆ (a) or α_Vβ₃ (b) (0.5 μg/well; black bars) was incubated with the virus, and binding of integrin was determined using either rabbit anti-α_Vβ₃ (a) or MAb CSβ6 (b) as described in Materials and Methods. (c) Wells were coated with either medium from nontransfected cells (grey bars) or from transfected cells containing soluble α_Vβ₃ (1 μg/ml; black bars) as described in Materials and Methods. Virus (1 μg) was incubated with the integrin, and the binding of virus was determined using an antiviral MAb.

FIG. 3.

Effect of soluble integrins on the adsorption of FMDV to cells. FMDV types A₁₂ and O₁C3056H, labeled with [³⁵S]methionine and purified as described in Materials and Methods, were incubated either with soluble α_Vβ₃ or α_Vβ₆ at the concentrations indicated in the figure or with medium from nontransfected HEK 293A cells. Binding assays were performed in BHK3α_Vβ₆ cells at 4°C, using a virus concentration of 3,000 particles/cell, as described in Materials and Methods. (a and b) Soluble α_Vβ₆ incubated with either type A₁₂ (a) or O₁C3056H (b). (c and d) Soluble α_Vβ₃ incubated with either type A₁₂ (c) or O₁C3056H (d).

FIG. 4.

Analysis of the virus-soluble α_Vβ₆ complex. The samples used for these binding assays were centrifuged in an SW41 rotor through a 10-to-50% (wt/vol) sucrose gradient in TBS+ at 17,000 rpm for 18 h at 4°C. The gradients were fractionated into 0.4-ml fractions, and 0.1-ml samples were counted. (a) [³⁵S]methionine-labeled type A₁₂ incubated with either medium from nontransfected cells (•) or soluble α_Vβ₆ (6 μg/ml) (○). (b) [³⁵S]methionine-labeled type O₁C3056H incubated with either medium from nontransfected cells (•) or soluble α_Vβ₆ (6 μg/ml) (○). Samples were run on parallel gradients but are represented on the same graph for clarity.