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

Influenza A virus (IAV) exploits the host cellular machinery to facilitate its replication, yet many host-IAV interactions remain unexplored in the cellular context. Detailed information about interacting domains is also largely lacking. To address this, we employed in-cell cross-linking mass spectrometry (XL-MS) to capture a vast network of protein-protein contact sites within IAV-infected human cells. This approach uncovered previously unrecognised host factors, delineated steps of the hemagglutinin maturation pathway, and revealed that IAV progressively disrupts paraspeckles. We further found that paraspeckle disruption is mediated by NP and NS1 interactions, PA-X activity, and inhibition of RNA polymerase II, thereby liberating RNA-binding proteins that enhance viral replication. Our work advances the understanding of IAV manipulation of host cellular processes and demonstrates the utility of in-cell XL-MS for comprehensive mapping of host-pathogen interactomes and infection pathways.