猪流行性腹泻病毒非结构蛋白NSP15抑制细胞焦亡的分子机制研究

Molecular mechanism of PEDV non structural protein 15 inhibiting pyroptosis

为探究猪流行性腹泻病毒(PEDV)及其NSP15蛋白对细胞焦亡的影响及初步的分子作用机制,本研究将表达pGSDMD-p30的质粒转染猪小肠上皮细胞(IPEC-J2细胞),24 h后将PEDV感染该细胞,于感染后12 h和24 h分别收集细胞上清液和细胞,采用LDH试剂盒测定各时间点细胞上清中乳酸脱氢酶(LDH)的释放比例;采用qPCR检测细胞中GSDMD mRNA的转录水平。结果显示,各时间点,转染表达pGSDMD-p30质粒的IPEC-J2细胞中LDH的释放比例均极显著升高,表明细胞发生焦亡,且与转染p GSDMD-p30质粒的细胞相比,PEDV感染的细胞中LDH的释放比例及GSDMD mRNA的转录水平均极显著降低。将表达pGSDMD-p30的质粒分别与不同剂量的PEDV-NSP15质粒共转染HEK293T细胞(pGSDMD-p30+NSP15组);将表达Caspase-1、pGSDMD和表达NSP15的质粒分别共转染HEK293T和IPEC-J2细胞(Caspase-1+pGSDMD+NSP15组)。上述细胞于24 h后均测定各组细胞上清液中LDH的释放比例。结果显示,与pGSDMD-p30组(共转染表达pGSDMD-p30与空载体的细胞)相比,pGSDMD-p30+NSP15组HEK293T细胞及IPEC-J2细胞中LDH的释放比例均显著降低;与Caspase-1+pGSDMD对照组细胞相比,Caspase-1+pGSDMD+NSP15组细胞中LDH的释放比例极显著降低。上述结果表明,NSP15在不同细胞中均可以抑制GSDMD-p30及GSDMD+Caspase-1两种方式引起的细胞焦亡,提示PEDV可能通过NSP15抑制细胞的焦亡。将自噬抑制剂3-MA和蛋白酶体途径抑制剂MG132分别加入不同剂量的NSP15质粒与MYC-pGSDMD质粒共转染的HEK293T细胞中,6 h后采用western blot检测细胞中GSDMD的表达水平;将上述质粒共转染HEK293T细胞,24 h后通过qPCR检测细胞中GSDMD mRNA的转录水平。结果显示,随着NSP15转染剂量的增加,细胞中GSDMD的表达水平逐渐减少,加入3-MA和MG132并不影响GSDMD的表达水平;相比于共转染空载体和NSP15质粒的对照组,MYC-pGSDMD+NSP15组细胞中GSDMD mRNA的转录水平极显著降低。上述结果表明,PEDV NSP15降解GSDMD的过程不是通过自噬和蛋白酶体途径实现的。将4个核酸内切酶活性位点突变的NSP15质粒分别与pGSDMD-p30或pGSDMD质粒共转染HEK293T细胞,24 h后采用LDH试剂盒测定各组细胞上清中LDH的释放比例;采用western blot检测各组细胞中GSDMD的表达水平。结果显示,与p30组相比,H^(226)、H^(241)和K^(282)突变组细胞上清中LDH的释放比例均无显著变化,而D^(265)突变组细胞上清中LDH的释放比例极显著降低;H^(226)、H^(241)和K^(282)突变组细胞中GSDMD的表达水平与阴性对照组细胞中GSDMD的表达水平相当,而D^(265)突变组细胞中GSDMD的表达水平明显减少。上述结果表明,PEDV通过其NSP15蛋白抑制各种细胞的细胞焦亡,且本研究首次证明该蛋白通过其核酸内切酶活性位点H^(226)、H^(241)和K^(282)降解GSDMD并抑制细胞焦亡,为PED的治疗和开发抗PED的药物提供参考。

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 infected 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 p GSDMD-p30plasmid,LDH release and GSDMD m RNA transcription levels were considerably reduced in PEDV-infected cells.The p GSDMD-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(H^(226)A,H^(241)A,D^(265)A,K^(282)A)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 H^(226),H^(241),and K^(282)had no significant impact on LDH release or GSDMD expression,the D^(265)mutation still led to a significant reduction in GSDMD degradation and LDH release.This indicates that H^(226),H^(241),and K^(282)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 NSP15protein,and this study first evidence that the protein degrades GSDMD and inhibits cell pyroptosis through its endonuclease active sites H^(226),H^(241)and K^(282),providing a potential target for therapeutic strategies against PED.