Identification and structural characterisation of a picornavirus integrin receptor with an essential role of two distinct glycans for virus infection

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 ⍺2β1 as a protein receptor for ERAV. Using knock-out cells and by 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 ⍺2β1 in its closed conformation by attaching to the β-propeller of the ⍺2 chain, independent of divalent cations, as well as to a high-mannose glycan on ⍺2 N112 and a sialylated glycan on β1 N269. Using glycan knockout and knock-in cells and by reconstituting integrin ⍺2β1-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.