Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

Abstract Influenza A virus (IAV) hijacks host cellular machinery, but many virus–IAV interactions and contacting protein sites remain uncharacterised, particularly those dependent on intact cellular architecture, such as membrane-associated or phase-separated compartments. Here, we applied in-cell cross-linking mass spectrometry (XL-MS), integrated with AlphaFold-based structural modelling and functional assays, to map protein-protein contact sites in IAV-infected human cells. This approach revealed previously unrecognised virus–host interactions linked to spatially organised processes, including the maturation pathway of HA through the membrane-bound ER– Golgi system, the novel interaction of M2 with the membrane-embedded LAT1 amino acid transporter, and the progressive disassembly of paraspeckles–phase-separated compartments in the nucleus. We validate M2-LAT1 interaction and paraspeckle disassembly in human primary lung epithelial cells and show that the paraspeckle disassembly constitutes a new and unique infection mechanism through which IAV releases RNA-binding proteins that support viral RNA replication. These findings advance the understanding of IAV manipulation of host cellular processes and illustrate how the integrative in-cell structural system biology approach captures native host-pathogen interactomes, infection pathways, and host cell perturbations.