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

Kotova, I.; Muehlberg, L.; Gilep, K.; Yu, D.; Ziemianowicz, D.; Stanelle-Bertram, S.; Beck, S.; Baeg, K.; Duss, O.; Gabriel, G.; Liu, F.; Bogdanow, B.; Kosinski, J.

doi:10.1101/2025.03.09.642134

bioRxiv · 10.1101/2025.03.09.642134

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

Kotova, I.·Muehlberg, L.·Gilep, K.·Yu, D.·Ziemianowicz, D.·Stanelle-Bertram, S.·Beck, S.·Baeg, K.·Duss, O.·Gabriel, G.·Liu, F.·Bogdanow, B.·Kosinski, J.

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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.

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Kotova, I., Muehlberg, L., Gilep, K., Yu, D., Ziemianowicz, D., Stanelle-Bertram, S., Beck, S., Baeg, K., Duss, O., Gabriel, G., Liu, F., Bogdanow, B., Kosinski, J.. 2025-03-11. Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection. https://doi.org/10.1101/2025.03.09.642134

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