The African Swine Fever Virus gene MGF_360-4L inhibits interferon signaling by recruiting mitochondrial selective autophagy receptor SQSTM1 degrading MDA5 antagonizing innate immune responses

Multigene family (MGF) 360 genes, which are African swine fever virus (ASFV) virulence genes, primarily target key host immune molecules to suppress host interferon (IFN) production and interferon-stimulated gene (ISG) transcription, impairing host innate immune responses for efficient viral replication. However, the interactions between MGF 360 virulence genes and host molecules, as well as the mechanisms through which MGF 360 genes regulate host immune responses and interferon signaling, require further elucidation. In this study, we discovered that ASFV MGF_360-4L interacts with MDA5 and recruits the mitochondrial selective autophagy receptor SQSTM1 to degrade MDA5, thus impairing interferon signaling and compromising host innate immune responses. Furthermore, MGF_360-4L inhibits the interaction between MDA5 and MAVS, blocking ISG15-mediated ISGylation of MDA5. MGF_360-4L deficiencysignificantly attenuated virus-induced mitochondrial autophagy in vitro. Additionally, OAS1 ubiquitinates MGF_360-4L at residues K290, K295 and K327. Finally, a recombinant ASFV lacking the MGF_360-4L gene (ASFV-?MGF_360-4L) was generated using ASFV-CN/SC/2019 as the backbone, which demonstrated that the replication kinetics of ASFV-ΔMGF_360-4L in PAM cells were like those of the highly virulent parental ASFV-WT in vitro. Domestic pigs infected with ASFV-ΔMGF_360-4L exhibited milder symptoms than those infected with parental ASFV-WT, and ASFV-ΔMGF_360-4L-infected pigs presented with enhanced host innate antiviral immune response, confirming that the deletion of the MGF_360-4L gene from the ASFV genome highly attenuated virulence in pigs and provided effective protection against parental ASFV challenge. In conclusion, we identified a novel ASFV virulence gene, MGF_360-4L, further elucidating ASFV infection mechanisms and providing a new candidate for vaccine development.