Proteasomal degradation of nonstructural protein 12 by RNF114 suppresses porcine reproductive and respiratory syndrome virus replication.

• RNF114 exhibits significant anti-PRRSV activity. • RNF114 knockdown facilitates PRRSV replication. • PRRSV infection leads to endogenous RNF114 upregulation. • The E3 ligase RNF114 interacts with Nsp12 and promotes its proteasomal degradation. • RNF114 catalyzes K27-linked polyubiquitination of PRRSV Nsp12. Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the swine industry worldwide, and the development of effective and sustainable measures to control PRRSV transmission remains a pressing problem. The function of PRRSV nonstructural protein 12 (Nsp12), which might play essential roles in viral replication and production, remains unknown. In this study, we identified a new host-restricted factor, porcine RING finger protein 114 (RNF114), as an inhibitor of PRRSV replication through its degradation of viral Nsp12. Western blot, quantitative real-time polymerase chain reaction, and viral titer assays indicated that RNF114 overexpression suppressed PRRSV replication, whereas RNF114 knockdown increased viral titer and nucleocapsid protein levels. Additionally, we observed that PPRSV infection led to increased RNF114 levels during the middle and late phases of infection in both porcine alveolar macrophages and MARC-145 cells. Moreover, screening of PRRSV Nsps showed that RNF114 interacted with viral Nsp12, and that RNF114-specific anti-PRRSV effects were associated with its ubiquitin ligase activity, which involves K27-linked polyubiquitination and degradation of Nsp12 through a proteasome-dependent pathway. These findings identified RNF114 as a critical regulator of PRRSV replication and offer insights into the roles of Nsp12 in PRRSV pathogenesis. [ABSTRACT FROM AUTHOR]