African Swine Fever Virus MGF360-13L Inhibited Type ? Interferon Production Through CGAS-STING Pathway

African swine fever virus(ASFV)is a complex double-stranded DNA virus that replicates primarily in the host cytoplasm,persists in a variety of environmental conditions,and spreads easily.ASFV causes a highly contagious hemorrhagic disease in pigs with morbidity and mortality rates as high as 100%.Since its discovery in Kenya in1921,ASFV has gradually spread to Europe and Asia,causing significant economic losses to the world of pig farming.Over the years,despite the ongoing exploration of ASFV vaccines,research on safe and efficient ASFV vaccines has not been a major success.Innate immunity is the host's first line of defense against invading pathogens,and ASFV adopts multiple strategies to antagonize the host's natural immunity to achieve efficient virus infection and replication.Several members of the MGF505 and MGF360 multigene families currently antagonize type I IFN production,but their mechanisms of action remain to be elucidated.In this study,we investigated the effect of MGF360-13 L on c GAS-STING pathway-mediated type I IFN production,revealed the mechanism of ASFV immune escape,and provided new insights into the host-virus interaction.Major findings:1.MGF360-13 L inhibited type ? IFN production and its mediated signal transductionTo investigate the inhibitory effect of ASFV on IFN-I signaling pathway,multiple viral genes from MGF505 and MGF360 multigene families were transfected into HEK293 T cells,and the m RNA expression level of IFN-? was detected by q PCR;STING or c GAS/STING were cotransfected into HEK 293 T cells,and a dual luciferase reporter system was used to study IFN-? promoter activity.The results showed that MGF505-3R and-A528 R,MGF360-12 L,-13 L,-14 L and-A276 R could significantly inhibit the expression of IFN-? induced by poly(I:C)and all could significantly inhibit the promoter activity of IFN-?.MGF360-13 L was transfected with 3D4/21 cells and treated with interferon-stimulated DNA(ISD)fragments or poly(I:C).q PCR was performed to detect the transcript levels of m RNAs of key molecules of type I IFN signaling pathway and JAK-STAT signaling pathway,and a dual luciferase reporter system was used to study the IFN-? promoter activity.The results showed that MGF360-13 L significantly attenuated ISD or poly(I:C)-mediated key genes of type I IFN signaling pathway(IFN-?,ISG15,ISG54,ISG56,STING,TBK1)and key genes of JAK-STAT signaling pathway(JAK1,STAT1,STAT2,STAT3 STAT6,IRF9)m RNA transcript levels and IFN-? promoter activity.2.MGF360-13 L inhibited c GAS-STING-mediated IFN-? activationTo investigate the effect of MGF360-13 L on c GAS/STING-induced signaling effects,MGF360-13 L was cotransfected with c GAS/STING or STING,respectively,in HEK293 T cells,and the promoter activities of IFN-?,IRF3 and NF-?B were detected by dual luciferase reporter system.The results showed that MGF360-13 L ectopic expression significantly inhibited the activation of IFN-?,IRF3 and NF-?B promoters,and MGF360-13 L reversed the activation of IFN-?,IRF3 and NF-?B induced by 2'3'-c GAMP stimulation of STING.To identify the target molecules of MGF360-13 L that inhibit the c GAS/STING pathway,MGF360-13 L was cotransfected with downstream bridging protein particles of STING in HEK 293 T cells,respectively,and the dual luciferase reporter system assayed the promoter activity of IFN-?.The results showed that MGF360-13 L could inhibit both TBK1,IKK?,p65 and IRF3/5D overexpression-mediated promoter activity of IFN-?.3.Molecular mechanism of MGF360-13 L inhibition of type I IFN productionTo investigate whether MGF360-13 L affects the interaction between STING,TBK1 and IRF3,MGF360-13 L was transfected with 3D4/21 cells and treated with ISD,and the interaction between STING,TBK1 and IRF3 was detected by co-immunoprecipitation technique.The results showed that MGF360-13 L could inhibit the interaction between STING and TBK1 or IRF3,but it was not clear whether MGF360-13 L inhibited the phosphorylation of TBK1 or IRF3.MGF360-13 L was transfected with 3D4/21 cells and treated with ISD for Western Blot assay.The results showed that MGF360-13 L inhibited ISD-induced phosphorylation of TBK1 and IRF3 in 3D4/21 cells.Meanwhile,the Native Page results showed that ISD-induced IRF3 dimerization was also significantly inhibited upon MGF360-13 L overexpression.Furthermore,MGF360-13 L was cotransfected with c GAS/STING in HEK 293 T,and the Western Blot results showed that MGF360-13 L inhibited c GAS/STING-induced phosphorylation of TBK1 and IRF3,and MGF360-13 L did not affect the levels of IRF3 and p IRF3 in the cytoplasm,but significantly decreased the expression of p IRF3 in the nucleus.Indirect immunofluorescence results showed that after MGF360-13 L transfection,IRF3 accumulated around the nucleus and showed a tight fit to the nuclear region,indicating that MGF360-13 L could inhibit the nuclear localization of IRF3.Based on the above results,the following conclusions were drawn.1.MGF360-13 L antagonized the production of type I IFN and inhibited its mediated signal transduction.2.MGF360-13 L inhibited c GAS-STING-mediated IFN-? activation and antagonized the recruitment of TBK1 and IRF3 to STING.3.MGF360-13 L inhibited phosphorylation,dimerization of TBK1 and IRF3 and nuclear translocation of IRF3.