猪急性腹泻综合征冠状病毒诱导细胞自噬并增强病毒复制的研究

Swine acute diarrhea syndrome coronavirus induced autophagy and enhanced virus replication

为研究猪急性腹泻综合征冠状病毒(SADS-CoV)感染细胞是否诱导自噬及自噬对病毒复制的影响,本研究将SADS-CoV(MOI 0.1)接种Vero E6细胞培养36 h后,经透射电子显微镜观察可见,SADS-CoV感染的细胞中出现了包裹胞浆的自噬体样双层膜结构,证实SADS-CoV可以诱导Vero E6细胞发生自噬。构建pEGFP-LC3的真核表达质粒并经双酶切及测序鉴定正确后转染Vero E6细胞,24 h后以SADS-CoV感染,24 h后经激光共聚焦显微镜观察可见,与阴性对照组细胞相比,感染SADS-CoV的细胞出现绿色荧光的点状聚集。进一步将SADS-CoV(MOI 0.1)感染Vero E6细胞不同时间后通过western blot检测自噬相关蛋白LC3-Ⅱ和p62表达水平的变化,结果显示,与阴性对照组相比,SADS-CoV感染组LC3-Ⅱ的表达水平升高,LC3-Ⅱ/GAPDH比值呈上升趋势,而p62蛋白表达水平则显著降低(P<0.05),表明SADS-CoV感染能够诱导Vero E6细胞产生完整的自噬应答。为了探究自噬对病毒复制的影响,本研究分别采用自噬抑制剂3-MA和自噬诱导剂Rapamycin处理Vero E6细胞后以SADS-CoV感染,于感染后不同时间收获细胞,通过western blot和病毒滴度(TCID_(50))测定检测SADS-CoV N蛋白表达水平的变化。结果显示,与仅感染病毒的对照组相比,采用3-MA处理Vero E6细胞后SADS-CoV N蛋白的表达水平及TCID_(50)均显著降低(P<0.05),而采用Rapamycin处理后SADS-CoV N蛋白的表达水平及TCID_(50)则均显著升高(P<0.05)。针对ATG5基因设计3条特异性的ATG5 si RNA(siATG5-1/2/3),将干扰效率较好的siATG5-1转染Vero E6细胞后再以SADS-CoV感染,24 h后检测病毒滴度并利用western blot检测SADS-CoV N蛋白表达水平的变化。结果显示,与未转染siATG5的3组阴性细胞相比,转染细胞中SADS-CoV N蛋白的表达水平及病毒滴度均显著降低(P<0.05)。本研究首次表明SADS-CoV感染可以诱导宿主细胞自噬并促进病毒的复制,该结果为深入研究SADS-CoV感染与其致病机理以及防控该病毒的感染奠定了基础。

The objective of the study was to explore whether autophagy was induced by swine acute diarrhea syndrome coronavirus(SADS-CoV)infection and its impact on virus replication during autophagy.Vero E6 cells were infected with SADS-CoV at a MOI of 0.1 and then observed by transmission electron microscope.The results showed that the SADS-CoV infected cells produced an autophagosome like double-layer membrane containing cytoplasm,proving that SADS-CoV could induce autophagy in Vero E6 cells.A eukaryotic expression plasmid of pEGFP-LC3 was constructed and identified by enzyme digestion analysis and sequencing,the resulting plasmid was transfected into Vero E6 cells after infection with SADS-CoV,and then observed by confocal laser microscope.Compared with the negative control group,the SADS-CoV infected cells showed a dot aggregation of green fluorescence.Western blot analysis of the autophagy related proteins LC3-II and p62 in SADS-CoV-infected Vero E6 cells showed an increase in LC3-II band intensity,an elevated LC3-II/GAPDH ratio,and a significant decrease in p62 protein expression(P<0.05),indicating a robust autophagic response to SADS-CoV infection in Vero E6 cells.To explore the effect of autophagy on viral replication,we used autophagy inhibitor 3-MA and autophagy inducer Rapamycin to detect the expression level of SADS-CoV N protein by western blot and the viral titers(TCID_(50)).The results showed that compared with the control group infected only with SADS-CoV,the expression level of SADS-CoV N protein and the viral titers were significantly decreased after Vero E6 cells were treated with 3-MA significantly decreased(P<0.05).After treatment with Rapamycin,the expression level of SADS-CoV N protein and the viral titers were significantly increased(P<0.05).Additionally,three specific ATG5 siRNAs(siATG5 siRNA-1/2/3)were designed for ATG5 gene.The siATG5-1,with higher knockdown efficiency,was transfected into Vero E6 cells and then infected with SADS-CoV to detect the change of the expression level of SADS-CoV N protein.The findings revealed a significant decrease in the expression level of SADS-CoV N protein and the viral titers(P<0.05)compared to 3 control group cells,indicating the knockdown of ATG5 reduced viral replication.This study shows for the first time that SADS-CoV infection can induce autophagy and promote virus replication,which has positive significance for further study of SADS-CoV infection and its pathogenic mechanism as well as prevention and control of disease.