The S273R protein of African swine fever virus antagonizes the canonical NF-κB signaling pathway by IκBα.

African swine fever virus (ASFV) is a large double-stranded DNA virus, which is the causative agent of African swine fever (ASF), a devastating disease of suids epidemic in many countries. The virus has developed multiple strategies to evade surveillance from the host immune system. Inflammatory responses, especially the NF-κB signaling pathway, play central roles in ASFV pathogenesis and immunoevasion. In this study, we identified the ASFV S273R protein (pS273R) as an antagonist of the canonical NF-κB signaling pathway independently of its protease activity. The ectopically expressed pS273R markedly inhibited the tumor necrosis factor-alpha or interleukin-1 beta-triggered NF-κB signaling pathway in HEK293T and PK-15 cells. Silencing pS273R by RNA interference led to elevated expression levels of proinflammatory cytokines in the ASFV-infected primary porcine alveolar macrophages. Mechanistically, pS273R functioned independently of its protease activity. pS273R was associated with the NF-κB complex and interrupted the translocation of IκBα into the proteasome, resulting in the increased stability of IκBα and subsequently impaired nuclear translocation of p65. Furthermore, the core domain (amino acids 83-273) of pS273R was essential for the pS273R-mediated inhibition of the NF-κB signaling pathway. These findings demonstrate the immunosuppressive role of pS273R and provide novel insights into ASFV biological characteristics.IMPORTANCEAfrican swine fever (ASF) is a hemorrhagic disease of suids caused by African swine fever virus (ASFV), with morbidity and mortality rates of up to 100%. The disease has led to significant economic losses to the global swine industry. In this study, we identify the ASFV S273R protein (pS273R) as an antagonist of the canonical NF-κB signaling pathway. Our findings demonstrate the immunosuppressive role of pS273R, which will contribute to a better understanding of the pathogenesis of ASFV and may contribute to the development of antiviral therapies against ASF.