Proximity labelling of internalizing influenza A viruses reveals a role for neogenin in virus uptake

Influenza A virus (IAV) is a respiratory pathogen of global concern. Entry of most IAVs is mediated by binding of viral hemagglutinin to cellular sialic acid, facilitating virus attachment. A subsequent interaction with a surface receptor(s) triggers viral uptake. Although multiple host factors involved in viral entry are known, the identity of these receptors remains unclear. Here, we utilized proximity labelling to acquire the interactome of epsin 1, an adaptor protein utilized by IAV for clathrin-mediated endocytosis, during virus internalization to identify them. We uncover neogenin (Neo1), a member of the immunoglobulin superfamily expressed in primary human airway cultures, as a potential epsin 1 interactor and virus receptor candidate. Knockdown of Neo1 led to a reduction in replication of H1N1, H2N2 and H5N1 IAVs in primary and immortalized lung cells. Moreover, human recombinant Neo1 was found to bind IAV with a KD of 21 ± 14 nM by atomic force microscopy and Neo1 could co-localize with incoming IAV at early times post-infection, as well as affect viral entry. As Neo1 can interact with IAV and its depletion impairs IAV entry, this study reveals its potential as an IAV internalization receptor.