Preliminary Study On The Mechanism Of Porcine ENPP1 Involvement In Pseudorabies Virus Infection

Porcine pseudorabies(PR)is a viral infection caused by infection with pseudorabies virus(PRV).After 2011,PRV was widespread in China.Meanwhile the traditional attenuated vaccine,Bartha-K61,had failed to achieve full protection for PR.This article aims to explore the mechanism of action of Ectonucleotide pyrophosphotase phosphodiesterase 1(ENPP1)in the natural immune response triggered by PRV infection in order to lay a theoretical foundation for prevention and control of PRV.Porcine cyclic GMP-AMP(cGAMP)can be catalyzed by the porcine cyclic GMP-AMP synthase(cGAS).It acts as a second messenger in the natural immune response triggered by PRV,and activates the interferon(IFN)signaling pathway eventually.ENPP1 is a type II transmembrane protein and is able to hydrolyze c GAMP.It is speculated that there is a certain link between ENPP1 and PRV-infected natural immunity.In this study,quantitative real-time PCR(qRT-PCR)was used to detect the expression of porcine ENPP1 mRNA in different tissues and cells.It was found that porcine ENPP1 mRNA expression was higher in muscle and PK15 cells.Firstly,To determine whether ENPP1 affects the natural immune response of PRV-infected host cells,three variants of ENPP1 over-expression vectors for the ENPP1 carboxy-terminal fusion Flag-tag were constructed to investigate the effect of ENPP1 domain on PRV infection.Three variants of ENPP1 was wild-type(ENPP1 WT),the truncated type deletions of the nucleotidase-like domain(ENPP1 aa.1-472),and the truncated type that simultaneously lacked the catalytic domain and nucleoidase-like domain(ENPP1 aa.1-123).The ENPP1 overexpression vectors were used to identify the effect of the ENPP1 domain on PRV infection.Secondly,RNA interference(RNAi)was used to detect the change of natural immune response triggered by PRV-infected host cells after ENPP1 knockdown.To further elucidate the specific molecular mechanism of ENPP1 involved in PRV infection,wild-type ENPP1 and its two truncated mutants were overexpressed in HEK293 cells,and then infected with PRV or transfected with cGAMP.utilizing immunoblot analysis and enzyme-linked immuno sorbent assay(ELISA)examined the activation of interferon regulatory factor 3(IRF3)and the secretion of interferon ?(IFN-?)separately.The results showed that the recombinant PRV strain(PRV-GFP)infected 3D4/21 cells with overexpression of ENPP1 WT and ENPP1 aa.1-472 after 36 h,the fluorescence intensity enhanced significantly compared to the control cells.However,there was no significant change in the cells that overexpressed ENPP1 aa.1-123.To confirm the enhancement of PRV infection by ENPP1 overexpression,a WT PRV strain(PRV QXX)was used to infect the cells for 24 h.Then detected the viral titer and IFN mRNA expression amount.3D4/21 cells overexpressed either ENPP1 WT or ENPP1 aa.1-472 displayed an increased viral titer,and the expression of interferon ?(IFN-?)mRNA was significantly increased in virus-infected cells.Whereas the overexpression of the empty vector or ENPP1 aa.1-123 had no effect on PRV QXX infection and IFN mRNA qutity.These results demonstrated that ENPP1 overexpression promotes PRV infection.Compared with the control,PRV QXX infected 3D4/21 cells expressed ENPP1 WT and ENPP1 aa.1-472,the IFN-? and nuclear factor ?B(NF-?B)promoter activity was significantly attenuated.While overexpression of the ENPP1 aa.1-123 variant had no effect on IFN-? and NF-?B promoter activity.The above results indicated that ENPP1 overexpression promotes PRV infection and inhibits the transcription of IFN-? and NF-?B,which dependented on the catalytic domain of ENPP1.Next,PK15 cells were transiently transfected with a control siRNA(siControl)and three different siRNAs specific for ENPP1 mRNA(si ENPP1-1,siENPP1-2,and siENPP1-3)for 24,48,or 72 h.Then detected ENPP1 mRNA expression level by q RT-PCR.The results showed that the knockdown efficiency of ENPP1 mRNA was higher in si ENPP1-2 and si ENPP1-3 transfected cells than in cells transfected with siControl or siENPP1-1.Compared to the control cells,the fluorescence intensity of ENPP1-knockdown cells infected PRV-GFP for 36 h was significantly decreased both in siENPP1-1 and siENPP1-2.The downregulation of ENPP1 also attenuated PRV QXX infection,determined with virus titration.In the mock-PRV-QXX-infected PK15 cells,ENPP1 knockdown by si ENPP1-2 or si ENPP1-3 transfection did not affect IFN-? mRNA levels.However,PRV QXX infection induced significantly higher levels of IFN-? mRNA in the cells transfected with siENPP1-2 or siENPP1-3 than in cells transfected with siControl.Although PRV QXX infection increased the promoter activities of IFN-? and NF-?B in the siControl-transfected cells,significant increased in the IFN-? and NF-?B promoter activities were detected in the ENPP1-knockdown cells.All these results suggest that ENPP1 knockdown inhibits PRV replication and enhances the transcription of IFN-? and NF-?B.Finally,ENPP1 WT,ENPP1 aa.1-472,ENPP1 aa.1-123 were overexpressed in HEK293 cells.The molecular mechanism study of ENPP1 during PRV infection showed that the tendency of IFN-? secretion and IRF3 activation in cells uninfected with PRV QXX or untransfected cGAMP was consistent with the empty vector.However,after PRV QXX infection for 24 h or cGAMP transfection for 6 h,compared with empty vector or ENPP1 aa.1-123 overexpressed cells,in the ENPP1 WT and ENPP1 aa.1-472 overexpressed cells infected with PRV QXX or transfected cGAMP,IRF3 phosphorylation was inhibited significantly,and IFN-? secretion was reduced.All the aforementioned results above indicate that ENPP1 inhibits IRF3 phosphorylation and decreases IFN-? secretion in cells by PRV-infected or cGAMP-transfected.The catalytic domain of ENPP1 palys a key role in the process.In summary,porcine ENPP1 is involved in the natural immune response triggered by PRV infection.ENPP1 inhibits IRF3 phosphorylation and decreases IFN-? secretion which is induced by PRV or cGAMP.The catalytic domain palys a key role in the process of ENPP1 involved in natural immune response caused by PRV infection.The results of this study will provide basic theoretical basis for the prevention and control of PRV.