Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to the swine industry worldwide. The viral matrix (M) protein is essential for virion assembly and infectivity. In this study, we found that the M protein is degraded through the ubiquitin-proteasome system. Immunoprecipitation and mass spectrometry identified the host deubiquitinase OTUB1 as an M-binding partner. Overexpression of OTUB1 increased M protein stability and expression levels, while OTUB1 knockdown promoted M degradation. OTUB1 interacted with the C-terminal ectodomain of the M protein and specifically removed K48-linked ubiquitin chains that target proteins for proteasomal degradation. Mechanistically, a catalytically inactive OTUB1 mutant (C91A) retained the ability to stabilize and deubiquitinate M, while a mutant defective in the non-canonical pathway (D88A) lost this function, indicating that OTUB1 acts primarily by sequestering E2 ubiquitin-conjugating enzymes. Furthermore, we identified UBE2D2 as the specific E2 ubiquitin-conjugating enzyme sequestered by OTUB1 to prevent M protein degradation. Depletion of OTUB1 significantly attenuated PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages. OTUB1 specifically targeted the M protein without stabilizing other PRRSV proteins. Importantly, OTUB1-mediated stabilization was conserved across M proteins from diverse PRRSV strains, including PRRSV-1, PRRSV-2, and emerging NADC30-like and NADC34-like variants. Collectively, our findings revealed a host mechanism exploited by PRRSV to enhance replication and identified OTUB1 as a potential target for antiviral development.