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.

猪繁殖与呼吸综合症病毒（PRRSV）对全世界的养猪业构成了重大威胁，开发有效且可持续的措施来控制PRRSV传播仍然是紧迫的问题。PRRSV非结构蛋白12（Nsp12）的功能，可能在病毒复制和生产中起重要作用，目前尚不清楚。在这项研究中，我们确定了一种新的宿主限制性因子，猪的RING指蛋白114（RNF114），可通过其降解病毒Nsp12抑制PRRSV复制。Western印迹，定量实时聚合酶链反应和病毒滴度测定表明RNF114过表达抑制PRRSV复制，而RNF114组合式增加病毒滴度和核衣壳蛋白水平。此外，我们观察到在猪的肺泡巨噬细胞和MARC-145细胞中，PPRSV感染在感染的中晚期均导致RNF114水平升高。此外，对PRRSV Nsps的筛选显示RNF114与病毒Nsp12相互作用，并且RNF114特异性抗PRRSV的作用与其泛素连接酶活性有关，这涉及K27连接的多泛素化和Nsp12通过蛋白酶体依赖性途径的降解。这些发现确定RNF114是PRRSV复制的关键调节器，并提供了Nsp12在PRRSV发病机理中的作用的见解。