Low-affinity binding in cis to P2Y₂R mediates force-dependent integrin activation during hantavirus infection

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β₃ integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β₅/β₃ integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y₂R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant α_IIbβ₃ integrins and (RGD)P2Y₂R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y₂R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y₂R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing α_IIbβ₃ integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα₁₃ protein from binding to the cytoplasmic domain of the β₃ integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y₂R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.