Regulation Of African Swine Fever Virus PS273R Protease Mechanistic Studies Of The IFN-I Signaling Pathway

African swine fever(ASF)is a highly contagious viral disease caused by the African swine fever virus(ASFV).Since the first case of ASF emerged in China in2018,it has spread rapidly across the mainland provinces.Studies have shown that ASFV infection has a wide range of clinical signs and is highly susceptible to fulminant hemorrhagic fever,allowing affected domestic and wild pigs to progress from subclinical disease to death within a short period.To date,no effective treatment or vaccine has been developed to prevent the disease and the epidemiological situation is becoming worse as the virus continues to spread and evolve,seriously affecting the development of the pig farming industry in China.Clinically affected pigs show varying degrees of disease,which is inextricably linked to the pathogenicity of ASFV and the immune system of susceptible animals,and there is still a lack of knowledge about the immune escape mechanisms involved.In the early stages of viral infection Type I interferon(IFN-I)is one of the cytokines produced by the body that can rapidly and effectively fight the virus.ASFV is a giant double-stranded DNA virus with a highly complex structure and a large genome,which has predisposed it to evolve more escape routes to antagonize IFN-I production and eliminate its effects.The pS273R protein,a cysteine protease encoded by the S273 R gene,is an important component of the ASFV inner core capsid and plays an important role in viral proliferation by cleaving the internal core capsid protein precursor to produce a mature inner core capsid protein,which is involved in the assembly of viral particles.Its regulatory role in the host immune response has not been fully resolved to date.Therefore,the role of ASFV pS273R in the regulation of the IFN-I signaling pathway and its mechanism of action has been investigated in depth in this thesis.1.Mechanism of antagonism of IFN-I production by ASFV pS273RFirstly,the pS273R protease was found to inhibit the fluorescence activity of the double-stranded DNA mimic poly(d A:d T)induced IFN-β in porcine-derived PK-15 cells in a dose-dependent manner using a dual luciferase reporter system.It was hypothesized that pS273R might target a key effector molecule that cleaves the IFN-I pathway.To screen for possible targets,ASFV pS273R was cotransfected with eight porcine-derived signaling molecule expression plasmids constructed in our laboratory into HEK-293 T cells.Western blot results showed that pS273R protease cleaved the DNA pattern recognition receptor DHX9(DEx H-box helicase 9)protein in a dosedependent manner The DHX9 protein plays a variety of functions in the host innate immune system and is essentially a DEx H-box RNA decapping enzyme,with the ability to recognize double-stranded DNA reported to include a core decapping domain(helicase)at the N-terminal end,a C-terminal repeat arginine and a glycine-glycine domain(RGG-Domain).In this study,a series of DHX9 eukaryotic expression plasmids with multiple mutations and truncated mutations were constructed to further demonstrate that the pS273R protease can cleave the DHX9 protein at multiple points and that the region of action is the RGG-Domain located at the C-terminus of the DHX9 protein.Finally,a dual-luciferase assay showed that the cleaved product was able to inhibit IFN-β production compared to the full-length DHX9 protein.The above findings reveal that the ASFV pS273R protease can act on DHX9 and block the signaling pathway that mediates the production of IFN-I.2.Mechanisms by which ASFV pS273R antagonizes IFN-I signalingBased on the confirmation that ASFV pS273R inhibits IFN-I production,we further investigated whether it could antagonize the production of IFN-I-activated ISGs and thus interfere with the establishment of the host’s antiviral state.Further validation by quantitative real-time PCR(RT-q PCR)showed that the pS273R protease dosedependently reduced the m RNA transcript levels of IFN-α-activated Interferonstimulated genes(ISGs).Compared with wild-type pS273R,the loss of protease activity of pS273R-C232 A protein had a diminished inhibitory effect on IFN-αactivation,indicating that the protease activity of pS273R is involved in its antagonism of IFN-I signaling.In an in-depth investigation of its mechanism of action,target proteins were screened by Western blot,and it was found that pS273R specifically cleaved TYK2 protein and was dependent on its protease activity.In addition,pS273R also degraded STAT2 protein expression,and WB assay results by grey-scale analysis indicated that the protease activity of pS273R was also involved in regulating the degradation.In an assay to identify TYK2 cleavage sites,pS273R was shown to recognize two consecutive P1 sites,Glycine(G)residues at positions 610 and 611 respectively,both of which are located in the pseudokinase structural domain of TYK2.Compared to the full-length TYK2,the N-terminal product TYK2-(1-610aa)was significantly less able to induce ISRE promoter activity after TYK2 cleavage,but the C-terminal product TYK2-(612-1185aa)was not significantly changed,suggesting that TYK2 was not an effector target.Taken together,the above experimental results suggest that the ASFV pS273R protease can negatively regulate the IFN-I signaling pathway by downregulating STAT2 expression.In summary,our results show that the ASFV pS273R protease inhibits type I IFN production by targeting the RGG structural domain of DHX9 for multipoint cleavage;at the same time,pS273R also inhibits IFN-I signaling by degrading STAT2 protein;furthermore,the protease activity of pS273R is essential for inhibiting IFN-I production and signaling,revealing a novel strategy for ASFV immunosuppression.