[PubMed] [CrossRef] [Google Scholar] 37. using the stimulator of interferon genes (STING) proteins formulated with a 2,3-cGAMP-binding theme and stage mutations in the Y76S and N78A proteins of EP364R that impaired relationship with 2, 3-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease Methylene Blue in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2,3-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2,3-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines. in the family (1, 2). The genomic size of ASFV is approximately 170 to 193 kbp, and the genome encodes 150 to 167 proteins that play roles in virus structure formation, viral replication, and immune evasion. However, many viral proteins have unknown functions (3,C5). ASFV replicates mainly in the cytoplasm of monocyte- and macrophage-lineage cells (6), where replication predominates in the perinuclear cytoplasmic region, called the viral factory (7). African swine fever has caused headlines with a surge in cases worldwide. This highly contagious hemorrhagic viral disease in pigs has a mortality rate of nearly 100% and threatens the global pork supply and food security (8, 9). However, no effective drugs or vaccines are commercially available for this deadly disease (7). When a DNA virus infects a permissive cell, viral DNA is released into the Methylene Blue cell cytoplasm before viral protein synthesis. Although various cytosolic HESX1 DNA sensors have been identified, mainly cytosolic viral DNA is recognized by cyclic GMP-AMP (cGAMP) synthase (cGAS), which allows the rearrangement of the cGAS catalytic pocket for the subsequent binding of ATP and GTP as cGAS substrates for the synthesis of 2,3 cyclic GMP-AMP (2,3-cGAMP) (10, 11). The synthesis of 2,3-cGAMP is a crucial first step in initiating cGAS-mediated downstream signaling (12, 13). Synthesized 2,3-cGAMP acts as a second messenger that can bind to the endoplasmic reticulum (ER) membrane adaptor-stimulator of interferon gene (STING) (also called MITA, ERIS, and MPYS) and induces conformational changes, activating STING (12). Activated STING then migrates from the ER to the ER-Golgi intermediate compartment (ERGIC), and upon reaching ERGIC and Golgi compartments, STING recruits TANK-binding kinase 1 (TBK1), which phosphorylates the interferon regulatory factor 3 (IRF3). Phosphorylated IRF3 dimerizes and enters the nucleus, leading to the induction of type I interferons (IFNs) and other antiviral genes (14, 15). In contrast, STING activates the inhibitor of nuclear factor-B (IB) kinase to release NF-B, which translocates to the cell nucleus and activates the transcription of proinflammatory cytokine-related genes (16, 17). The type I IFN response is the first-line defense mechanism against invading viruses, including ASFV (18). Therefore, viruses have evolved diverse antagonistic strategies to evade the type I IFN response and facilitate Methylene Blue rapid replication in host cells (19). The virulent ASFV strain Armenia/07 has been shown to inhibit IFN- production via the cGAS-STING pathway (20), and ASFV has been shown to be sensitive to type I and II IFNs (21). Thus, ASFV also employs various immune evasion mechanisms that regulate various steps in the type I IFN signaling pathways. For instance, ASFV MGF505-7R degrades STING through the autophagy lysosomal pathway (22) and inhibits IRF3 nuclear translocation via IRF3 interaction (23). ASFV DP96R also interferes with the cGAS-STING-TBK1 axis (24). E120R was shown to interact with IRF3 to inhibit the interaction between TBK1 and IRF3 (25). In contrast, ASFV I215L (E2 ubiquitin-conjugating enzyme) impairs IFN- via the K63-linked ubiquitination of TBK1 and NF-B signaling (26, 27). Consequently, various ASFV proteins are involved in immune-suppressive mechanisms along with diverse target molecules in type I IFN and NF-B signaling (28,C32). However, the exact mechanism by which the ASFV protein targets cGAMP in type I.