The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKα and IKKβ through the recruitment of TOLLIP.

The multigene family genes (MGFs) in the left variable region (LVR) of the African swine fever virus (ASFV) genome have been reported to be involved in viral replication in primary porcine alveolar macrophages (PAMs) and virulence in pigs. However, the exact functions of key MGFs in the LVR that regulate the replication and virulence of ASFV remain unclear. In this study, we identified the MGF300-2R gene to be critical for viral replication in PAMs by deleting different sets of MGFs in the LVR from the highly virulent strain ASFV HLJ/18 (ASFV-WT). The ASFV mutant lacking the MGF300-2R gene (Del2R) showed a 1-log reduction in viral titer, and induced higher IL-1β and TNF-α production in PAMs than did ASFV-WT. Mechanistically, the MGF300-2R protein was found to interact with and degrade IKKα and IKKβ via the selective autophagy pathway. Furthermore, we showed that MGF300-2R promoted the K27-linked polyubiquitination of IKKα and IKKβ, which subsequently served as a recognition signal for the cargo receptor TOLLIP-mediated selective autophagic degradation. Importantly, Del2R exhibited a significant reduction in both replication and virulence compared with ASFV-WT in pigs, likely due to the increased IL-1β and TNF-α, indicating that MGF300-2R is a virulence determinant. These findings reveal that MGF300-2R suppresses host innate immune responses by mediating the degradation of IKKα and IKKβ, which provides clues to paving the way for the rational design of live attenuated vaccines to control ASF.