To investigate the effects of porcine epidemic diarrhea virus(PEDV)and its non-structural protein 15(NSP15)on cell pyroptosis,this study transfected pGSDMD-p30 plasmid into porcine intestinal epithelial cells(IPEC-J2 cells).The cells were in-fected with PEDV 24 hours after transfection.Cell supernatants and cells were collected at 12 and 24 hours post-infection.The percentage of lactate dehydrogenase(LDH)release was measured to assess pyroptosis,and the transcription level of GSDMD mRNA was analyzed using qPCR.The results showed that LDH release in pGSDMD-p30-transfected IPEC-J2 cells was significantly higher than in the control groups at all time points,indicating pyroptosis occurred.Compared to cells transfected with pGSDMD-p30 plasmid,LDH release and GSDMD mRNA transcription levels were considerably reduced in PEDV-infected cells.The pGSDMD-p30 plasmid was co-transfected with varying doses of PEDV-NSP15 plasmid into HEK293T(pGSDMD-p30+NSP15 group);Plasmids expressing Caspase-1,pGSDMD,and PEDV-NSP15 were co-transfected respectively into HEK293T cells and IPEC-J2 cells(Caspase-1+pGSDMD+NSP15 group).LDH release was measured in the cell supernatants 24 hours after transfection.The results showed that co-transfection with PEDV-NSP15 and pGSDMD-p30 significantly reduced LDH release in HEK293T and IPEC-J2 cells compared to the pGSDMD-p30 group.In addition,co-transfection of Caspase-1+pGSDMD+NSP15 also significantly reduced LDH release compared to the Caspase-1+pGSDMD group.These results indicated that NSP15 can inhibit pyroptosis induced in different manners in different cell types.Furthermore,HEK293T cells were co-transfected with NSP15 and MYC-pGSDMD plasmids and the cells were treated respectively with different doses autophagy inhibitor 3-MA and proteasome inhibitor MG132 for 6 hours.Western blot showed that GSDMD expression levels decreased with increasing doses of NSP15,and 3-MA or MG132 did not reverse this effect.Additionally,qPCR results indicated a significant reduction in GSDMD mRNA transcription levels in cells co-transfected with NSP15.These findings suggest that the degradation of GSDMD by PEDV NSP15 is not achieved through autophagy and proteasome pathways,but by inhibiting the transcription level of GSDMD in cells.Four mutants plasmids of endonuclease activitysites(H226A,H241A,D265A,K282A)in NSP15 were co-transfected with pGSDMD-p30 or pGSDMD plasmids into HEK293T cells,LDH release and western blot was performed after 24 hours.Compared to the p30 group,the results demonstrated that while mutations at H226,H241,and K282 had no significant impact on LDH release or GSDMD expression,the D265 mutation still led to a significant reduction in GSDMD degradation and LDH release.This indicates that H226,H241,and K282 are the key sites for NSP15's endonuclease activity in degrading GSDMD and inhibiting pyroptosis.The above results indicate that PEDV inhibits cell pyroptosis in various cells through its NSP15 protein,and this study first evidence that the protein degrades GSDMD and inhibits cell pyroptosis through its endonuclease active sites H226,H241 and K282,providing a potential target for therapeutic strategies against PED.
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