Single influenza A viruses induce nanoscale cellular reprogramming at the virus-cell interface

Viruses, as nanoscale entities with limited proteomes, must efficiently infect target cells using their available resources. During infection, individual virions induce specific cellular signaling within the virus-cell interface, a nanoscale patch of the plasma membrane in contact with the virus. However, virus-induced receptor recruitment and cellular activation are transient processes that occur within minutes and at the nanoscale level. Hence, the temporal and spatial kinetics of such early events often remain poorly understood due to technical limitations. To address this challenge, we developed a novel protocol to covalently immobilize unmodified influenza A viruses on glass surfaces before exposing them to live epithelial cells. This approach extends the observation time for virus-plasma membrane interaction while preserving the viruses' native state for uncompromised cell interaction. Using single-molecule super-resolution microscopy, we investigated virus-receptor interaction showing that viral receptors are not immobilized by the virus but rather slowed down, which leads to a specific local receptor accumulation and turnover. We further followed the dynamics of clathrin-mediated endocytosis at the single-virus level and demonstrate the recruitment of adaptor protein 2 (AP-2), previously thought to be uninvolved in influenza A virus infection. Finally, we examined the nanoscale organization of the actin cytoskeleton at the virus-binding site, showing a local and dynamic response of the cellular actin cortex to the infecting virus. Our findings provide novel insights into the fundamental process of virus-cell interaction and demonstrate the versatility and potential impact of our approach on virus-cell biology.