African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

African swine fever virus (ASFV) infection causes a severe hemorrhagic disease in pigs, characterized by excessive inflammatory responses and tissue damage, posing substantial threats to the pig industry worldwide. Given the lack of vaccines and effective antiviral treatments, as well as the largely unknown functions of most ASFV-encoded proteins, it's urgent to study the proteins that are crucial in triggering inflammatory responses and how they do so. This study demonstrated that ASFV exploited the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome to induce pyroptosis and inflammatory responses, effectively replacing the non-functional porcine AIM2 pseudogene. Screening over 150 proteins encoded by the ASFV genome, EP364R was identified as the viral factor responsible for driving NLRP3-mediated pyroptosis and high-level cytokine production. Ectopic expression of EP364R in mice elicited significant upregulation of serum pro-inflammatory cytokines and splenomegaly, while its expression in bone marrow-derived macrophages (BMDMs) from NLRP3-knockout mice abrogated pyroptosis and related effects. Mechanistic investigation revealed that the helicase DDX3X acted as a molecular bridge, enabling EP364R to interact with NLRP3 to promote the aggregation and activation of inflammasomes. Depletion of DDX3X abolished EP364R's ability to induce NLRP3-dependent pyroptosis and pro-inflammatory cytokine production. We found that the NACHT domain of porcine NLRP3 interacted with DDX3X, and EP364R established a connection with the NACHT and LRR domains of NLRP3 through DDX3X. However, EP364R bound to all the domains of DDX3X. Molecular docking analysis revealed that DDX3X interacted with EP364R through a spatially defined interface, thereby exerting its function. Furthermore, a natural compound library was employed to screen functional compounds targeting EP364R, and HAMNO was identified as an inhibitor that bound to E256, K259, and D260 of EP364R, consequently suppressing ASFV replication. Our findings explain how ASFV triggers pyroptosis and excessive cytokine release, and identify a potent small-molecule inhibitor of ASFV, aiding the development of vaccines and therapies to prevent and control African swine fever (ASF) caused by ASFV infection.