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

African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV) that imposes a substantial economic burden on the global pig industry each year. When screening for the regulation of virus replication genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the 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 and TNF- , which are regulated by the NF- B signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKK and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and I B competitively inhibits the binding of the E3 ligase -TrCP to I B , thereby inhibiting the ubiquitination-dependent degradation of I B . Remarkably, despite encoding other inhibitors of NF- B, the 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 IL-1 and TNF- during the early stages of infection in 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.