Low-affinity binding in to P2YR mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β integrins. Previous studies have implicated a cognate interaction between the bent conformation β/β integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2YR. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αβ integrins and (RGD)P2YR expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2YR removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2YR on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αβ 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 protein from binding to the cytoplasmic domain of the β integrin prevents outside-in signaling and infection. We propose that the interaction with P2YR 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.