NSMCE1 suppresses FMDV replication by promoting the ubiquitination and degradation of 3A

1. The E3 ubiquitin ligase NSMCE1 is a novel host factor that suppresses FMDV replication. 2. NSMCE1 interacts with the FMDV 3A and mediates its K33-linked ubiquitination at K16, leading to proteasomal degradation of 3A. 3. Mutation of 3A K16 enhances FMDV replication capacity both in vitro and in vivo, confirming the critical role of NSMCE1-mediated ubiquitination in restricting viral infection. Foot-and-mouth disease virus (FMDV) is a highly contagious picornavirus that causes severe economic losses in livestock worldwide. The nonstructural protein 3A of FMDV is essential for viral replication and virulence, and mutations in 3A are associated with altered host tropism, highlighting its role in mediating host-virus interactions. However, the molecular mechanisms underlying the interplay between 3A and host cellular factors remain poorly understood. Here, through systematic screening and functional analyses, we identify the E3 ubiquitin ligase NSMCE1 as a novel host-encoded negative regulator of FMDV replication. NSMCE1 interacts directly with the FMDV 3A protein and mediates its K33-linked ubiquitination at lysine 16 (K16). This modification promotes the proteasomal degradation of 3A, thereby suppressing FMDV replication. Consistent with this mechanism, recombinant virus with a mutation at lysine 16 of 3A enhances the replication capacity of FMDV both in vitro and in vivo, confirming the critical role of this regulatory event. Our findings reveal a previously unrecognized role for NSMCE1 in limiting FMDV infection through targeted regulation of viral protein 3A and uncover a regulatory role of the ubiquitin-proteasome system in picornavirus replication. These insights advance our understanding of host antiviral defense mechanisms and provide a potential foundation for the development of novel antiviral therapies targeting the ubiquitin pathway.