Porcine USP18 inhibits Japanese encephalitis virus replication by regulating the type I interferon signaling pathway

Ubiquitin-specific peptidase 18 (USP18) possesses both deubiquitinating and deISGylating activities and plays a crucial role in regulating innate immune responses and antiviral defense. However, the function of USP18 in Japanese encephalitis virus (JEV) replication remains unclear. In this study, the expression pattern of the porcine USP18 gene following JEV infection was examined by quantitative real-time PCR (qRT-PCR) and Western blot analysis. The effects of USP18 overexpression and siRNA-mediated knockdown on JEV replication and the expression of type I interferon (IFN-I) signaling pathway genes were examined. Protein-protein interactions between USP18 and IFN-I pathway components were predicted by AlphaFold3 and validated by co-immunoprecipitation (Co-IP) and confocal immunofluorescence colocalization assays. The results showed that JEV infection significantly upregulated USP18 expression. Overexpression of USP18 significantly inhibited JEV replication, whereas USP18 knockdown enhanced viral replication and suppressed IFN-I signaling-related gene expression. Moreover, USP18 was found to interact with the key IFN-I signaling proteins IRF3 and MAVS, and USP18 promoted IRF3 phosphorylation, suggesting that USP18 suppresses JEV replication by positively regulating IFN-I-mediated antiviral responses. These findings provide new insights into the antiviral mechanism of porcine USP18 and offer potential molecular targets for disease-resistant breeding and antiviral therapy in pigs.