biostudies-literatureZhang DNational Natural Science Fund for Excellent Young Scientists Fund ProgramFundamental Research Funds for the Provincial Universities of ZhejiangThe Start-up funding from Life Sciences Institute, Zhejiang UniversityMOST | National Natural Science Foundation of China (NSFC)National Natural Science Fund for Excellent Young Scientists Fund Program (Oversea)MOST | National Natural Science Foundation of Chinae0175824https://www.ebi.ac.uk/biostudies/studies/S-EPMC11784292biostudies-literatureUnknown99(1)Enteroviral 3C protease (3Cpro) is an essential enzyme for viral replication and is responsible for combating the host anti-viral immune response by targeting cellular proteins for cleavage. The identification and characterization of 3Cpro substrates will contribute to our understanding of viral pathogenesis. In this study, we performed a motif search for 3Cpro substrates in the human protein database using FIMO, which refers to a common cleavage sequence of 3Cpro. We identified and characterized NEDD4-binding protein 1 (N4BP1), a key negative regulator of the NF-κB pathway, as a novel 3Cpro substrate. N4BP1 is cleaved at residue Q816 by 3Cpro from several human enteroviruses, resulting in the loss of its ability to regulate tumor necrosis factor alpha-activated NF-κB signaling. In additioJournal of virologyEnteroviral 3C protease cleaves N4BP1 to impair the host inflammatory response.PMC11784292K2024014520230QZJH6082372249Fan WZhang DXie YCao JHuang LfalseEnteroviral 3C protease cleaves N4BP1 to impair the host inflammatory response.Enteroviral 3C protease (3Cpro) is an essential enzyme for viral replication and is responsible for combating the host anti-viral immune response by targeting cellular proteins for cleavage. The identification and characterization of 3Cpro substrates will contribute to our understanding of viral pathogenesis. In this study, we performed a motif search for 3Cpro substrates in the human protein database using FIMO, which refers to a common cleavage sequence of 3Cpro. We identified and characterized NEDD4-binding protein 1 (N4BP1), a key negative regulator of the NF-κB pathway, as a novel 3Cpro substrate. N4BP1 is cleaved at residue Q816 by 3Cpro from several human enteroviruses, resulting in the loss of its ability to regulate tumor necrosis factor alpha-activated NF-κB signaling. In additio2025-01-01T00:00:00Z2025 Jan2026-06-01T21:58:44.943Z2025-04-05T23:36:20.153ZS-EPMC117842923965595710.1128/jvi.01758-24