HERPUD1 suppresses porcine epidemic diarrhea virus replication by recruiting HRD1 to degrade viral ORF3 protein

The open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to virus pathogenicity. A previous study found that PEDV ORF3 protein localizes in the endoplasmic reticulum (ER) to trigger the unfolded protein response (UPR). Nevertheless, the precise interactions and underlying mechanisms between ORF3 and UPR pathway-associated proteins remain largely unexplored. In this study, we demonstrate that ORF3 protein is capable of inducing ER stress through activating the transcription factor-6 (ATF6) pathway and PKR-like ER kinase (PERK) pathway. Through yeast two-hybrid screening, we identified that HERPUD1 interacts with ORF3 protein. Further investigations revealed that HERPUD1 recruits the E3 ubiquitin ligase HRD1 to facilitate ORF3 ubiquitination and subsequent degradation via ER-associated degradation (ERAD). This process in turn alleviates ORF3-induced ER stress and suppresses PEDV replication. Subsequently, it was found that lysine 61 of ORF3 is a crucial site for ORF3 degradation. Then, a recombinant PEDV with ORF3 mutation (rPEDV-ORF3-K61R) was constructed. The rPEDV-ORF3-K61R presented a higher level of ORF3 expression, induced a greater degree of ER stress and more effectively inhibited interferon-β (IFN-β), accompanied by an approximately 0.5 log increase in viral titer compared with the rPEDV. Overall, this study indicates that HERPUD1 inhibits PEDV replication by negatively regulating the stability of PEDV ORF3, providing an updated understanding of the pathogenic mechanism of viral infection. IMPORTANCE: Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, and high mortality in newborn piglets. ORF3 is an important virulence factor of PEDV, which can induce endoplasmic reticulum stress and antagonize interferon production, facilitating virus replication. However, it is unclear whether the host can reversely regulate ORF3 protein and inhibit its functions. This study found that HERPUD1 interacts with PEDV ORF3 protein and mediates ORF3 degradation, thereby inhibiting PEDV replication. The lysine at position 61 of ORF3 protein is a key site for ubiquitination and degradation. Modifying this site can increase the virus titer. This study is the first to identify HERPUD1 as a novel host factor restricting PEDV. These findings provide new insights into the pathogenesis of PEDV and offer new approaches for modifying PEDV vaccine strains to increase virus titer.