Study On The Molecular Mechanism Of African Swine Fever Virus F334l Inhibiting Type ? Interferon Signaling Pathway

African swine fever(ASF)caused by African swine fever virus(ASFV)is a severe infectious disease which has resulted in immeasurable losses to the global swine industry.The huge genome structure and complex immune escape mechanism,there is still no effective commercial vaccine.In the process of virus invading the host,type ? interferon is an important part of the host defense against virus infection.However,in order to achieve the purpose of reproducing in the cells and infecting the host,the virus usually evolve different strategies to antagonize the host antiviral effect.It has been shown that ASFV has the ability to use its own encoded protein to antagonize the host type ? interferon production and signal transduction.F334 L is a non-structural protein encoded by ASFV,but its role in host antiviral innate immunity is still unclear.Therefore,this study takes ASFV F334 L as the research object,aiming to explain the molecular mechanism in inhibiting type ? interferon signal transduction pathway.To explore whether ASFV F334 L involved in inhibiting the type ? interferon signal transduction pathway,the eukaryotic expression plasmid of ASFV F334 L was transfected into HEK-293 T cells at different concentrations.The ISRE promoter activity and the transcription level of ISGs were detected and analyzed by using the dual luciferase reporter system and real-time quantitative PCR assay.The results showed that ASFV F334 L could inhibit ISRE promoter activity in a dose-dependent manner and expression of ISGs(ISG15,ISG54,ISG56 and OAS1),suggesting that ASFV F334 L has an inhibitory effect on type ? interferon signal transduction pathway.In order to further determine the specific target protein that F334 L antagonizes the type ? interferon signal transduction pathway,we detected the effect of F334 L on the m RNA levels of key molecules in this pathway by using real-time quantitative PCR assay.The results showed that ASFV F334 L had no effect on transcription levels of these proteins.Subsequently we tested whether overexpression of F334 L has an influence on the protein levels of these proteins in the pathway by Western blot.The analysis showed that IRF9 was degraded by ASFV F334 L in a dose-dependent manner.Taken together,ASFV F334 L inhibits type ? interferon signal transduction by down-regulating the protein level of IRF9 rather than the transcription level.In order to further investigate the inhibitory effect of ASFV F334 L on type ? interferon signaling pathway,the interaction between IRF9 and F334 L was studied by indirect immunofluorescence and Co-IP methods.The results showed that the over-expressed F334 L specifically interacted with IRF9 and the nuclear translocation of IRF9 was partly blocked.Meanwhile,the over-expressed F334 L was located in the cytoplasm of the host cells.Since IRF9 is a key component of ISGF3 in the type ? signaling pathway,we detected whether the other two important components(p-STAT1,p-STAT2)will undergo the nuclear translocation by indirect immunofluorescence experiments.The results showed that the nuclear translocation of p-STAT1 and p-STAT2 were partly inhibited in the presence of F334 L.Therefore,it is concluded that F334 L can specifically interact with IRF9 and the nuclear translocation of ISGF3 was blocked to some extent.Subsequently,to explore the detailed molecular mechanism of IRF9 degradation,we co-transfected F334 L and IRF9 eukaryotic expression plasmid into HEK-293 T cells.Then,the cells were treated with ubiquitin-proteasome inhibitor(MG132),autophagy inhibitor(Chloroquine),and pan apoptosis inhibitor(Z-VAD-FMK)respectively.We found that the decrease of IRF9 protein induced by ASFV F334 L was through the ubiquitin-proteasome pathway by Western blot.Next,in order to illustrate the modified form of polyubiquitin chain involved in the degradation of IRF9 protein,F334 L and IRF9 were co-transfected with His-K48-Ub or His-K63-Ub plasmids into HEK-293 T cells.Our data showed that the degradation of IRF9 mediated by ASFV F334 L depends on K48 polyubiquitin chain modification.In conclusion,we found that ASFV F334 L can antagonize the type ? interferon signal transduction pathway.ASFV F334 L mediated IRF9 to undergo K48 polyubiquitin chain modification and degraded IRF9 through the ubiquitin-proteasome pathway,which inhibited the nuclear translocation of ISGF3 and ultimately inhibited ISRE promoter activity and the expression of ISGs.This study describes a new mechanism for ASFV to escape the host innate immune response,and provides a new theoretical basis for understanding the relationship between ASFV pathogenesis and the host innate immune system.