| African swine fever virus(ASFV)is a highly pathogenic large DNA virus that causes African swine fever(ASF)in domestic pigs and European wild boars with mortality rate up to 100%.In August 2018,China reported the first case of ASF,and within a year the ASF quickly spread to all provinces in mainland China,bringing a fatal blow to the pig industry.Due to the complex immune escape mechanism and gene structure of ASFV,there is currently no effective commercial vaccine.The innate immune response plays an important role in the host's antiviral infection.ASFV can use its own encoded protein to antagonize the host's natural immune response and evade the host's immune surveillance.Type I interferon-mediated antiviral response is an important part of host defense against viral infection.It has been reported that part of the protein encoded by the ASFV Multi-gene Family is able to antagonize the production of type I interferon,hindering its antiviral effect mediated.12 L is a member of the ASFV Multi-gene Family 360.This study focuses on the molecular mechanism of ASFV MGF360-12 L that antagonizes the production of type I interferon and inhibits the interaction of host proteins and nuclear transporters.To investigate the effect of ASFV MGF360-12 L protein on type I interferon,analysis by Quantitative Real-time PCR and luciferase assay showed that MGF360-12 L can inhibit poly(I:C)induced IFN-? expression.In order to explore the molecular pathway of MGF360-12 L inhibiting the expression of IFN-?,the effect of MGF360-12 L on the transcription level of key cytokines in the type I interferon signaling pathway and the effect on the transcription of the transcriptional enhancer gene of IFN-? was analyzed by using Quantitative Real-time PCR and luciferase assay.The results show that MGF360-12 L can inhibit the transcription of key cytokines ISG54,ISG56,TBK1,STING and other genes in the type I interferon signaling pathway,and can also inhibit the IFN-? transcriptional enhancer NF-?B,IRF3 and AP-1 transcription,and this inhibition appears dose-dependent.To further verify that MGF360-12 L inhibits the production of NF-?B,explore whether MGF360-12 L will inhibit TNF?-induced NF-?B,using fluorescent quantitative PCR and dual luciferase detection system,the results show that MGF360-12 L can inhibit TNF? Induced NF-?B production,and showed dose-dependent inhibition,consistent with previous results.In order to further clarify the effect of MGF360-12 L on the NF-?B signaling pathway,indirect immunofluorescence,Western blotting,Co-IP and other technologies were used to deeply study the nuclear of classical nuclear localization signal(NLS),NF-?B subunits p50 and p65 Localization,nuclear transport protein expression,and interaction with MGF360-12 L.The results show that MGF360-12 L can inhibit the nuclear localization of NF-?B subunits p50 and p65 through classical nuclear localization signal(NLS),but MGF360-12 L does not Affecting the protein expression level of nuclear transporters,further research found that MGF360-12 L interacted with KPNA2,KPNA3,KPNA4,inhibited the interaction of p65 with KPNA2,KPNA3,KPNA4,thereby competitively inhibiting the nuclear positioning of NF-?B.Fluorescence quantitative PCR results showed that after overexpressing KPNA2,KPNA3,and KPNA4,the degree of MGF360-12 L inhibiting the transcription of IFN-? m RNA was alleviated.In order to investigate whether MGF360-12 L will affect the protein expression levels of TBK1,p TBK1,IRF3,and p IRF3,and whether it will interfere with the nuclear transport of p IRF3,Western blotting and nuclear protein analysis tests were used.The results showed that MGF360-12 L reduced the protein expression levels of TBK1,p TBK1,IRF3,and p IRF3,and inhibited the nuclear transport of p IRF3.These findings suggested that MGF360-12 L could inhibit the IFN-I production by blocking the interrelation of importin? and NF-?B signaling pathway,which might reveal a novel strategy for ASFV to escape the host innate immune response. |
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