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Abstract 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 and transmit IAV entry signals. However, how IAV engages and activates EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV attachment factors and its functional receptor at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AF and EGFR are organized in partially overlapping submicrometer clusters in the apical plasma membrane of A549 cells. Within AF domains, which are distinct from microvilli, the local AF concentration, a parameter that directly influences virus-cell binding, 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 membrane, revealing that the distinct mobility pattern of IAVs is dominated by the local AF concentration, consistent with live cell single-virus tracking data. In contrast to AF, EGFR resides in clusters of rather low molecular density. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, instead relying on 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, and thus we relate 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. It is a complex and dynamic organelle that needs to protect as a barrier but also process subtle signals into and out of the cell. For IAV, an enveloped virus, it represents a major obstacle that it needs to overcome during infection as well as the site for the assembly of progeny virus particles. However, the organisation of the plasma membrane in particular the sites of virus interaction at the scale of an infecting particle (length scales < 100 nm) remains largely unknown. Sialic acids 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 still remains speculative. Here we use super resolution microscopy to study the lateral organization as well as the functional relationship of plasma membrane-bound molecules involved in IAV infection. We find that molecules are organized in submicrometer nanodomains and, in combination with virus diffusion simulations, present a mechanistic view for how IAV first engages with AFs in the plasma membrane to then engage and trigger entry-associated membrane receptors.

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