African swine fever virus pE301R negatively regulates cGAS-STING signaling pathway by inhibiting the nuclear translocation of IRF3

African swine fever (ASF) is a highly contagious and lethal infectious disease of domestic pigs and wild boars by the African swine fever virus (ASFV). ASFV infects domestic pigs with the mortality rate approaching 100 % at acute stage of infection. The cGAS-STING-mediated antiviral responses are wildly accepted that cGAS acts as DNA sensor for sensing of viral DNA during DNA virus infection. However, the molecular mechanisms underlying negatively regulation of cGAS-STING signaling and type I IFN (IFN-I) production by ASFV proteins are not fully understood. In this study, we demonstrated that ASFV pE301R antagonize the activities of IFN-β-, NF-κB-, ISRE-luciferase (Luc) reporters-induced by cGAS-STING in a dose dependent manner. Consistent with these results, the mRNA levels of Ifnb1, Isg15, Isg56 are attenuated by ASFV pE301R. Furthermore, ASFV pE301R executes its inhibitory function at the downstream of IFN-regulatory factor 3 (IRF3) phosphorylation. Mechanistically, pE301R interacts with IRF3 via its amino acid (aa) 1–200 region, resulting in inhibition of the nuclear translocation of IRF3 induced by cGAMP and poly(dA:dT). Overall, our findings reveal that pE301R acts as a negatively regulator to inhibit IFN-I production and to subvert host antiviral innate immunity during ASFV infection.