African swine fever virus pNP419L inhibits type I interferon signaling by blocking ISGF3-mediated transcriptional activation

African swine fever virus (ASFV) causes devastating hemorrhagic diseases characterized by significant mortality rates, thereby presenting substantial challenges to the global pork industry. ASFV employs multiple mechanisms to interfere with type I interferon (IFN-I) signaling, enabling it to evade host immune defenses. This study identifies pNP419L, one of the smallest DNA ligases encoded by ASFV, as a novel antagonist of IFN-I responses. pNP419L suppresses IFN-α-induced promoter activation of interferon-sensitive response elements (ISRE) and subsequent production of interferon-stimulated genes (ISGs), promoting viral replication. Mechanistically, pNP419L directly interacts with ISRE via its DNA-binding domain (DBD), a strategy that is evolutionarily linked to its viral DNA ligase activity and effectively disrupts the STAT1/STAT2/IRF9 ternary complex (ISGF3)-DNA interactions. Functional analyses reveal selective targeting of the IRF9 transcriptional activation module, as pNP419L inhibits IRF9/VP16(TA)-mediated ISRE activity but does not affect GAL4/STAT2(TA)- driven transcription. Truncation of the DBD abolishes ISG suppression, suggesting that this modified form could be a potential candidate for live-attenuated vaccine development. These findings elucidate an immune evasion mechanism employed by ASFV, enhance our understanding of viral IFN antagonism, and provide insights into vaccination and therapeutic strategies against African swine fever.