The S273R protein of African swine fever virus antagonizes the canonical NF- <i>κ</i> B signaling pathway by I <i>κ</i> B <i>α</i>

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-<i>κ</i>B 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-<i>κ</i>B signaling pathway independently of its protease activity. The ectopically expressed pS273R markedly inhibited the tumor necrosis factor-alpha or interleukin-1 beta-triggered NF-<i>κ</i>B 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-<i>κ</i>B complex and interrupted the translocation of I<i>κ</i>B<i>α</i> into the proteasome, resulting in the increased stability of I<i>κ</i>B<i>α</i> 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-<i>κ</i>B 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-<i>κ</i>B 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.