African swine fever virus protein R298L opposes the NF-κB signaling pathway and interferon-mediated pathway

African swine fever virus (ASFV) is a large DNA virus that is highly pathogenic to domestic pigs and severely threatens the global pig industry. ASFV has evolved a range of strategies to evade or suppress the innate antiviral immunity of the host in order to promote its own infection and proliferation. However, the molecular mechanisms underlying the inhibition of innate antiviral immunity by ASFV-encoded proteins remain unclear. Here, we report the ASFV-encoded protein R298L (p298L) inhibits inflammation and interferon (IFN)-mediated signaling pathways. To verify the function of pR298L in vivo, a recombinant ASFV with deletion of the R298L gene (ASFV_ΔR298L) was constructed and assessed. The results showed that ASFV_ΔR298L infection induced higher levels of inflammatory cytokines and IFN-stimulated gene factors compared with its parental ASFV CN/GS/2018 strain. Mechanistically, as a bridge protein, pR298L prevented p65 from phosphorylation and activation by blocking the interaction between IKKβ and p65 in the NF-κB pathway. The expression of pR298L prevents the important kinases, JAK1 and JAK2, in the interferon pathway from interacting with their substrates, which blocks the activation of the IFN-triggered signaling pathway. These findings offer new insights into the functions of the ASFV-encoded R298L gene and its role in the viral infection-induced immune response, which may aid in the development of antiviral agents and live attenuated vaccines to treat African swine fever. IMPORTANCE: African swine fever virus (ASFV) causes a highly fatal swine disease that is prevalent in numerous countries and has a significant impact on the pig industry. The lack of clarity regarding the mechanisms of ASFV infection and immune escape is a major impediment to vaccine development. The ASFV genome is large and encodes more than 200 proteins whose functions are unknown. To gain a more in depth understanding of how ASFV regulates the host immune response, we conducted a comprehensive screening and functional validation of viral proteins that regulate the interferon pathway and inflammatory response. This study focused on investigating the production and regulation of antiviral and inflammatory factors in the natural antiviral immune response of the host. This study reveals the significant contribution of ASFV-encoded protein R298L (p298L) in both the NF-κB signaling pathway and IFN-mediated pathway. This study offers novel perspectives on the immune evasion mechanisms of ASFV, thereby contributing to the development of effective vaccines and therapeutics.