The Molecular Mechanism Underlying Of ASFV PI215L Inhibiting The Type ? Interferon Production

African swine fever(ASF)is a severe animal infectious disease caused by ASF virus(ASFV),and the morbidity and mortality of pigs associated with the virulent ASFV isolates infection are as high as 100%.Previous studies showed that the ability of ASFV to antagonize interferon(IFN)production is closely related to its pathogenicity.(1)We used ASFV HLJ/18 strain to infect porcine alveolar macrophages(PAMS)as a model.By q RT-PCR and ELISA,we found that ASFV HLJ/18 strain hardly induced the production of IFN-I(such as IFN-?and IFN-?)in porcine alveolar macrophages(PAMS).In addition,ASFV HLJ/18 strain can significantly inhibit the production of IFN-I activated by c GAMP,suggesting that the ASFV-encoded protein may inhibit the production of IFN-I.(2)Dual-Luciferase Reporter Assay System were used to screen the inhibitory effects of 102 ASFV on the activity of IFN-?promoter mediated by cGAS-STING.We found that the E2 ubiquitin binding enzyme(pI215L)had the most significant inhibitory effect.In addition,knockdown of pI215L expression significantly inhibited the replication of ASFV and enhanced the production of IFN-?and ISG56.It proved that pI215L is essential for viral replication and interferon production.(3)In order to determine the key target proteins of ASFV PI215L in the cGAS-STING signaling pathway,Dual-Luciferase Reporter Assay System and real-time quantitative PCR(q RT-PCR)showed that ASFV pI215L significantly inhibited the IFN-?promoter activity and IFN-?m RNA levels mediated by STING and TBK1.However,pI215L did not inhibit IFN-?promoter activity and IFN-?m RNA level mediated by IRF3-5D,suggesting that pI215L may targets TBK1.Co-immunoprecipitation assay and confocal microscopy analysis showed that pI215L significantly inhibited K63-linked ubiquitination of TBK1.Using pI215L-C85A mutant without its E2 enzyme activity we found that the ASFV pI215L protein inhibits type I IFN production is independent on its E2 enzyme activity.(4)In order to elucidate the molecular mechanism by which ASFV pI215L inhibits the K63-liked ubiquitination of TBK1,we then found that the E3 ubiquitin ligase RNF138 was a novel pI215L binding partner by IP-MS screening.Using knockout cell line HEK293T-RNF138^-/-,we found that pI215L inhibitory effect on the type I IFN production was dependent on the presence of RNF138,and pI215L recruited RNF138 to enhance the inhibition of the K63-linked ubiquitination of TBK1.Further study showed that pI215L and RNF138 interacted with E3 ubiquitin ligase RNF128,which enhanced the K63-linked ubiquitin modification of TBK1,and promoted RNF128 degradation.Mechanistically,pI215L promotes RNF128 degradation by recruiting RNF138,which resulted in reduced K63-linked polyubiquitination of TBK1 and IFN-I production,and thereby inhibiting the type I IFN signaling pathway.Taken together,our findings elucidates a novel immune escape mechanism of ASFV,which provides a clue to design and develop attenuated live vaccine.