The D129L protein of African swine fever virus interferes with the binding of transcriptional coactivator p300 and IRF3 to prevent beta interferon induction

African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease, against which there is no vaccine available except in Vietnam. ASFV has developed multiple strategies to evade the antiviral innate immune responses. Here, we revealed that the D129L protein (pD129L), a nonstructural protein of ASFV, counteracted the type I interferon (IFN) responses and IFN-stimulated genes (ISGs) expression. Furthermore, we demonstrated that knockdown of D129L by specific small interfering RNAs enhanced the transcription of IFN-beta mRNA in the ASFV-infected primary porcine alveolar macrophages (PAMs). Mechanistically, pD129L did not affect the expression of adaptors in the cGAS-STING signaling pathway or the nuclear translocation of IFN regulatory factor 3 (IRF3). Importantly, pD129L specifically bound to the transcriptional coactivators CBP/p300 in the nucleus and inhibited the interaction of IRF3 with CBP/p300, which in turn suppressed the activation of the IFN-beta promoter to antagonize the IFN-beta induction. More specifically,specifically, the HrcA domain of pD129L and the IRF3-binding domain of p300 were mapped crucial for their interaction. Taken together, our findings demonstrate a novel immunoevasion mechanism evolved by ASFV to escape the host antiviral response, which provides a new target for the development of antiviral strategies. IMPORTANCE African swine fever (ASF) is an acute, hemorrhagic, and severe porcine infectious disease caused by African swine fever virus (ASFV). ASF outbreaks severely threaten the global pig industries and result in serious economic losses. No safe and efficacious commercial vaccine is currently available except in Vietnam. To date, large gaps in the knowledge concerning viral biological characteristics and immunoevasion strategies have hindered the ASF vaccine design. In this study, we demonstrate that pD129L negatively regulates the type I interferon (IFN) signaling pathway by interfering with the interaction of the transcriptional coactivator p300 and IRF3, thereby inhibiting the induction of type I IFNs. This study reveals a novel immunoevasion strategy employed by ASFV, shedding new light on the intricate mechanisms for ASFV to evade the host immune responses.