ASFV pDP238L negatively regulates type I interferon production via inhibiting the methylation of TBK1

African swine fever (ASF) is an acute, severe, and hemorrhagic infectious disease of pigs caused by the African swine fever virus (ASFV), with a mortality rate of up to 100%. Type I interferons (IFN-I) play an important role in regulating innate and adaptive immune responses and viral proliferation. In this study, we demonstrated that ASFV pDP238L negatively regulated the production of IFN-I. The ectopic expression of DP238L significantly inhibited the methylation and phosphorylation of TANK binding kinase 1 (TBK1). Mechanistically, we demonstrated that protein arginine methyltransferase 5 (PRMT5) was involved in the methylation process of TBK1 in the cGAS-STING signaling pathway. We found that pDP238L interacted with PRMT5, thereby disrupting the interaction between TBK1 and PRMT5, and subsequently interfering with TBK1 methylation induced by PRMT5. Importantly, we found that amino acids E185, E186, and D191 in pDP238L work together to play a critical role in inhibiting type I interferon production by pDP238L. Our findings indicate that DP238L might play an important role in ASFV pathogenesis.