Inhibition Of RIG-Ⅰ Receptor-mediated IFN-β Production By Porcine Sapelovirus Production

Porcine sapelovirus(PSV)is a single-stranded positive RNA virus of the genus Sapelovirus in the family Picornaviridae,which can cause multi-system syndromes such as porcine poliomyelitis,reproductive disorders,diarrhea and pneumonia,as well as as asymptomatic infections.Since the outbreak of porcine diarrhea disease in UK pig farms in the 1960 s,the virus has spread rapidly throughout the UK and to countries around the world such as China,Korea,Brazil and Spain,causing some economic losses to the pig industry.The virus invades the organism and eventually induces interferon production by activating the corresponding cascade reactions,allowing the organism to establish an antiviral state,but the virus has evolved over time and can also adopt strategies to resist the antiviral response to interferon.Studies have shown that numerous small RNA viruses can achieve immune escape through corresponding strategies,so it is unclear whether PSV has also evolved corresponding strategies to resist the host’s innate immunity.To elucidate the relationship between PSV infection and innate immunity and its mechanisms,this paper focuses on the role of PSV in RLRs receptor-mediated IFN-βproduction and its mechanisms.The main components of this study are as follows:1.Study of the growth characteristics of PSV on different cellsTo investigate the growth characteristics of PSV on ST and LLC-PK1 cells,the cells were first inoculated with 0.1 MOI of PSV,and the results showed that PSV infection of both types of cells could produce significant cytopathic effects,with PSV infection of ST and LLC-PK1 cells showing significant cytopathic effects after 12 h.PSV lesions were more pronounced on ST cells.The viral titers of PSV on ST and LLC-PK1 cells were also determined by applying TCID50,and a one-step growth curve was plotted.The results showed that the viral titer increased with time and reached a peak at 24 h of infection,followed by a decrease in viral titer with time.To understand whether PSV can infect HEK-293 T cells,cells were inoculated with 0.1 MOI of PSV and significant cytopathic lesions were found 18 h after viral infection.PSV was uploaded on HEK-293 T cells for 3 generations,and specific target bands were detected using PCR and Western blot assays,indicating that PSV can stably pass on HEK-293 T cells.Meanwhile,q RT-PCR was applied to determine the m RNA expression level of PSV on HEK-293 T cells,and a one-step growth curve was plotted.The results showed that the copy number of the virus peaked after 18 h of PSV infection on HEK-293 T cells,and then the copy number of the virus decreased with time.Finally,we analyzed the viral proteinexpression level using Western blot,and the results showed that the PSV VP1 protein expression increased with time.The above experimental results indicate that PSV can infect ST,LLC-PK1 and HEK-293 T cells and proliferate stably on them.2.Study on the mechanism of IFN-β production inhibition by PSVTo investigate IFN-β production and the signaling pathway of its action after PSV infection of cells,it was first found by a dual luciferase reporter gene assay that PSV infection of LLC-PK1 and HEK-293 T cells failed to directly activate IFN-β promoter activity and also antagonized Poly(I:C)-induced IFN-βpromoter activity in a dose-dependent manner,indicating that PSV infection antagonizes IFN-βproduction.Whereas Poly(I:C),a key inducer of the RIG-Ⅰ-like receptor pathway,activates the RIG-Ⅰ-like receptor pathway to make it produce IFN-β,we therefore hypothesize that PSV antagonizes Poly(I:C)-induced IFN-β production by blocking certain signaling molecules in the RIG-Ⅰ-like receptor pathway.To elucidate the mechanism by which PSV antagonizes IFN-β production,HEK-293 T cells were inoculated and not inoculated with 0.5 MOI of PSV,and then the expression plasmids of a series of signaling molecules in the RIG-Ⅰ signaling pathway(including: RIG-ⅠN,MDA5,MAVS,TBK1,IKKε,IRF3/5D)were cotransferred into HEK-293 T cells.The dual luciferase reporter gene assay showed that PSV inhibited IFN-β production induced by IRF3/5D and its upstream signaling molecules.As an important transcription factor in the RIG-Ⅰ signaling pathway,an important indicator of its activation is the phosphorylation of IRF3.We next investigated the effect of PSV infection on IRF3 phosphorylation.Western bolt The results showed that Poly(I:C)stimulation significantly enhanced IRF3 phosphorylation,but PSV infection significantly inhibited Poly(I:C)-induced IRF3 phosphorylation.Meanwhile,PSV infection did not alter the expression of total IRF3 protein.Then we further investigated the effect of PSV on the phosphorylation of IRF3 induced by signaling molecules in the RIG-Ⅰ signaling pathway by transfecting plasmids of each signaling molecule RIG-ⅠN,MDA5,MAVS,TBK1,and IKKεinto HEK-293 T cells,and each transfection group infected cells with PSV at 0.5 MOI.western bolt results showed that PSV was able to significantly inhibit RIG-Ⅰ and MDA5-induced IRF3 phosphorylation without affecting MAVS,TBK1 and IKKε-induced IRF3 phosphorylation,indicating that PSV inhibited IFN-β production by suppressing RIG-ⅠN,MDA5-induced IRF3 phosphorylation.3.Study on the mechanism of PSV 3C protein inhibition of RIG-Ⅰ receptor-mediated IFN-β productionTo further investigate the key proteins of PSV that inhibit IFN-β production on HEK-293 T cells,we constructed eukaryotic expression plasmids of each PSV protein and stained the eukaryotic expression vector of each PSV protein into HEK-293 T cells by calcium phosphate method,and identified the successful expression of each PSV protein pellet by Western blot.We then screened the key proteins of PSV that inhibit IFN-β production by dual luciferase reporter gene assay,and the results showed that the 3C,3D,2A and 3A proteins of PSV could significantly inhibit the activity of IFN-β promoter,indicating that they play an important role in the inhibition of IFN-β production by PSV.The dual-luciferase reporter gene assay showed that the 3C protein was able to significantly inhibit IFN-β activity induced by Poly(I:C)in a dose-dependent manner,indicating that PSV antagonism of IFN-β production is mainly acted by the 3C protein.Finally,we selected the 3C protein that inhibited the most significant activity of the IFN-β promoter for the follow-up study.To investigate the mechanism of PSV 3C protein antagonizing IFN-β production,we found that PSV3 C protein inhibited the production of IFN-β induced by TBK1 and its upstream signaling molecules in the RIG-Ⅰ signaling pathway by a dual luciferase reporter gene assay;Western blot assay results showed that PSV 3C protein overexpression inhibited MDA5-induced IRF3 phosphorylation levels without affecting RIG-Ⅰ,MAVS,TBK1 and IKKε-induced IRF3 phosphorylation,indicating that PSV 3C protein inhibits IFN-β production by suppressing MDA5-induced IRF3 phosphorylation;overexpression of 3C protein in HEK-293 T cells was found to inhibit exogenous MDA5 protein levels in a dose-dependent manner In addition,the results of immunoprecipitation assay showed that PSV 3C protein could interact with MDA5.These data suggest that PSV 3C protein inhibits the phosphorylation of IRF3 by interacting with MDA5,which in turn inhibits IFN-β production.In summary,this study confirmed that PSV can infect and stably proliferate on ST,LLC-PK1 and HEK-293 T cells.PSV infection can antagonize IFN-β production by the following mechanisms: PSV inhibits IFN-β production induced by IRF3/5D and its upstream signaling molecules,and PSV can also inhibit RIG-ⅠN,MDA5-induced This suggests that PSV can target RIG-ⅠN and MDA5 to block the RIG-ⅠN signaling pathway and thus inhibit IFN-β production;further studies revealed that PSV 3C protein is a key factor in inhibiting IFN-β production,and inhibits IFN-β production by inhibiting MDA5-induced phosphorylation of IRF3,interacting directly with MDA5 and degrading it.-βproduction.