The African swine fever virus MGF300-4L protein is associated with viral pathogenicity by promoting the autophagic degradation of IKK and increasing the stability of IB

African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV), which imposes a substantial economic burden on the global pig industry. When screening for the virus replication-regulating genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the MGF300-4L gene. However, the role of MGF300-4L in ASFV infection remains unexplored. In this study, we found that MGF300-4L could effectively inhibit the production of proinflammatory cytokines IL-1 beta and TNF-alpha, which are regulated by the NF-kappa B signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKK beta and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and I kappa B alpha competitively inhibits the binding of the E3 ligase beta-TrCP to I kappa B alpha, thereby inhibiting the ubiquitination-dependent degradation of I kappa B alpha. Remarkably, although ASFV encodes other inhibitors of NF-kappa B, the MGF300-4L gene-deleted ASFV (Del4L) showed reduced virulence in pigs, indicating that MGF300-4L plays a critical role in ASFV pathogenicity. Importantly, the attenuation of Del4L was associated with a significant increase in the production of IL-1 beta and TNF-alpha early in the infection of pigs. Our findings provide insights into the functions of MGF300-4L in ASFV pathogenicity, suggesting that MGF300-4L could be a promising target for developing novel strategies and live attenuated vaccines against ASF.