Influenza a Viruses use Multivalent Sialic Acid Clusters for Cell Binding and Receptor Activation

Abstract

Binding of influenza A virus to the host cell is mediated by the major viral surface protein hemagglutinin (HA). HA recognises sialic acid (SA), a plasma membrane glycan that functions as the specific primary virus attachment factor. Since, sialic acid alone can not fulfil a signalling function, the virus needs to activate downstream factors in order to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase (RTK) family, was shown to be involved and transmit virus entry signals. However, how influenza viruses engage with and activate EGFR is largely unknown. Using quantitative super-resolution microscopy (STORM and PALM), we show that SA and EGFR are organized in submicrometer clusters in the apical plasma membrane of different permissive cell lines. We found that the local SA concentration, a parameter that directly influences virus-cell binding, strongly increases towards the cluster center, thereby representing a multivalent virus-binding platform. By using 2-color super-resolution microscopy, we show that SA and EGFR clusters co-localize, eventually allowing SA-mediated virus-EGFR interaction. Finally, we show that virus binding activates EGFR and that this process occurs without a major EGFR redistribution, suggesting the activation of preformed clusters. Further, our quantitative characterization of SA distribution on the host cell membrane, enabled us to simulate 2D virus trajectories, which could be confirmed by live cell single-virus tracking. Taken together, our results provide a quantitative functional model of the initial events of influenza virus infection and provide insights into how the virus finds and activates EGFR.