The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin ␣v6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD ؉1 and RGD ؉4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to ␣v6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin ␣v6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.Foot-and-mouth disease virus (FMDV) is the type species of the genus Aphthovirus within the family Picornaviridae and the etiological agent of foot-and-mouth disease, a severe vesicular condition affecting a large number of artiodactyls, including domesticated ruminants and pigs (1, 34). Presently, the virus is endemic in many parts of the world, including South America, Africa, and Asia (19). Foot-and-mouth disease is highly contagious and difficult to control as FMDV has a wide host range (see above) and a rapid replication cycle, small amounts of virus can initiate infection, and infected animals excrete high levels of virus. In addition, multiple modes of transmission have been recognized, including airborne spread, sometimes over long distances, including overseas (1,10,12,31).Field isolates of FMDV use integrins to initiate infection (14, 15, 29). The integrin family of cell adhesion receptors are a conserved series of ␣ heterodimers, which bind in a divalent cation-dependent manner to ligands through recognition of short motifs that usually include one of the acidic residues glutamate (E) or aspartate (D) (13). Examples of such motifs include arginine-glycine-aspartate (RGD) or leucine-aspartate-valine (LDV), and short peptides containing these motifs can interact similarly with integrins (13). Recognition of RGDcontaining proteins can proceed in a stepwise manner where the initial RGD binding is enhanced by a second stabilizing interaction involving so-called synergy sites on the ligand (2, 21, 23). The concept of a synergy site was first described for binding of ␣51 to fibronectin (Fn). Thus, high-affinity binding of Fn to ␣51 requires the RGD motif located on the 10th type III domain of Fn and a second synergy site in the 9th type III domain (23). Similarly, the large extracellular matrix protein ...