TurboID ‐Based Proximity Labeling Map Reveals the Protein Interaction Network and Anti‐Viral Mechanism Against Influenza A H1N1

Influenza A virus (IAV) remains a significant public health threat due to its high variability and pathogenicity. Systematic identification of viral-host interfaces is critical for developing targeted therapies. In the present study, TurboID proximity labeling was applied to map interactomes of 10 H1N1 viral proteins in human alveolar epithelial cells A549. Key interactions were validated via co-immunoprecipitation (Co-IP), confocal microscopy, dual-luciferase reporter assays, and functional studies. Results showed that Hemagglutinin (HA) and polymerase acidic (PA) directly bound mitochondrial antiviral-signaling protein (MAVS), significantly suppressing interferon-beta (IFN-β) promoter activity and impairing phosphorylation of TANK-binding kinase 1 (TBK1)/signal transducer and activator of transcription (STAT) signaling proteins. HA exploited integrin alpha-2 (ITGA2) as a novel entry cofactor, with their interaction confirmed by bidirectional Co-IP and co-localization. ITGA2 silencing markedly reduced viral titers. Tripartite motif-containing protein 56 (TRIM56) restricted H1N1 replication by binding viral nucleoprotein (NP) and inducing proteasomal degradation via K48-linked ubiquitination. As a conclusion, this study delineates H1N1's coordinated tactics to hijack host pathways, identifying MAVS, ITGA2, and TRIM56 as pivotal nodes for combination therapies. TurboID-driven interactomics provides a promising framework for system-level antiviral discovery.