Low-affinity binding incisto P2Y₂R mediates force-dependent integrin activation during hantavirus infection

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β 3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β 5 /β 3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y 2 R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant α IIb β 3 integrins and (RGD)P2Y 2 R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y 2 R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y 2 R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing α IIb β 3 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 Gα 13 protein from binding to the cytoplasmic domain of the β 3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y 2 R 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.