PEDV NSP5 promotes immune evasion by recruiting TRIM28 to degrade DDX3X

Porcine epidemic diarrhea virus (PEDV) has emerged as the predominant swine enteric coronavirus, causing vomiting, diarrhea, dehydration, and high mortality in piglets. Members of the DEAD-box family play critical roles in viral infection, yet their roles in PEDV infection remain undefined. Furthermore, it is unknown whether PEDV and its proteins regulate the expression of these DEAD-box proteins. In this study, reverse transcription quantitative PCR was used to examine the gene expression changes of 15 DEAD-box family members in the intestinal tissues of one-month-old PEDV-infected piglets. We found that the expression of DEAD-box helicase 3 X-Linked (DDX3X) was upregulated following PEDV infection, a result that was similarly observed in cellular experiments. Overexpression and knockdown of DDX3X significantly inhibited or promoted PEDV replication, respectively. Moreover, the use of specific DDX3X inhibitors demonstrated that DDX3X primarily restricts PEDV RNA accumulation. Our study also showed that DDX3X enhances RIG-I-like receptor (RLR) signaling by promoting the phosphorylation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), signal transducer and activator of transcription 1 (STAT1), and Janus kinase 1 (JAK1) proteins. Co-immunoprecipitation and immunofluorescence assays further demonstrated the association and colocalization of DDX3X with TBK1 and IRF3, potentially contributing to its antiviral function. We constructed all PEDV proteins and identified that NSP5 reduces DDX3X levels via ubiquitination. Additionally, we discovered that tripartite motif-containing protein 28 (TRIM28) interacts with DDX3X and mediates its K48-linked ubiquitination. Knockdown of TRIM28 significantly reduced NSP5-mediated DDX3X ubiquitination. In conclusion, this study clarified the molecular mechanism by which NSP5 antagonizes DDX3X-mediated antiviral immune responses via the ubiquitination pathway, providing new insights into PEDV-host interactions and potential targets for antiviral intervention.