Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by IAV and transmit cell entry signals. However, how IAV's binding to sialic acid leads to engagement and activation of EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV's AFs and its functional receptor EGFR at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AFs and EGFR are organized in partially overlapping submicrometer clusters in the plasma membrane of A549 cells. Within AF domains, the local AF concentration reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a flat membrane. The results predict that the local AF concentration strongly influences the distinct mobility pattern of IAVs, in a manner consistent with live-cell single-virus tracking data. In contrast to AFs, EGFR resides in smaller clusters. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, indicating the activation of pre-formed clusters, which we show are long-lived. Taken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, thus linking their spatial organization to their functional role during virus-cell binding and receptor activation. Author summary The plasma membrane is the major interface between a cell and its environment. This complex and dynamic organelle needs to protect, as a barrier, but also transmit subtle signals into and out of the cell. For the enveloped virus IAV, the plasma membrane represents both a major obstacle to overcome during infection, and the site for the assembly of progeny virus particles. However, the organisation of the plasma membrane-a key to understanding how viral entry works-at the scale of an infecting particle (length scales < 100 nm) remains largely unknown. Sialylated glycans serve as IAV attachment factors but are not able to transmit signals across the plasma membrane. Receptor tyrosine kinases were identified to be activated upon virus binding and serve as functional receptor. How IAV engages and activates its functional receptors while initially binding glycans still remains speculative. Here, we use super resolution microscopy to study the lateral organization of plasma membrane-bound molecules involved in IAV infection, as well as their functional relationship. We find that molecules are organized in submicrometer nanodomains and, in combination with virus diffusion simulations, present a mechanistic model for how IAV first engages with AFs in the plasma membrane to subsequently engage and trigger entry-associated membrane receptors.