Two distinct α2β1 integrin glycan interactions mediate cell entry of a picornavirus

Equine rhinitis A virus (ERAV), a picornavirus closely related to foot-and-mouth disease virus, causes respiratory infections in horses. We performed a HEK293T CRISPRi screen and identified integrin α2β1 as a protein receptor for ERAV. Using knock-out cells and pretreating cells with an antibody or soluble receptor, we confirmed the importance of this integrin for infection of multiple cell types, including equine fibroblasts. Cryo-EM and glycoproteomics analyses revealed that ERAV binds integrin α2β1 in its closed conformation by attaching to the β-propeller of the α2 chain, independent of divalent cations, as well as to a high-mannose glycan on α2 N112 and a sialylated glycan on β1 N269. Using glycan knockout and knock-in cells and by reconstituting integrin α2β1-depleted cells with mutant receptors, we demonstrated the importance of these glycans for virus entry. Our results identify a hitherto unseen combination of protein and glycan interactions between a virus and its receptor.