小反刍兽疫病毒诱导的内质网应激对山羊肾细胞PERK信号通路和细胞凋亡的影响

Effect of endoplasmic reticulum stress induced by peste des petits ruminants virus on PERK signaling pathway and apoptosis in goat kidney cells

病毒感染可引起宿主细胞产生内质网应激(endoplasmic reticulum stress,ERS)和未折叠蛋白反应(unfolded protein response,UPR),导致内质网稳态失衡。为了深入探讨小反刍兽疫病毒(peste des petits ruminants virus,PPRV)感染诱导的ERS对宿主细胞UPR信号通路、病毒复制和细胞凋亡的影响,采用MTT试验测定、间接免疫荧光试验(IFA)和Western blot观察PPRV在山羊肾细胞(LDG-2)的增殖,采用Western blot和实时荧光定量PCR(qRT-PCR)观察PPRV感染对GRP78、PERK及其下游信号分子、细胞凋亡相关蛋白Bcl-2和Bax表达水平的影响。结果显示,PPRV感染36 h后,细胞存活率显著降低并产生明显细胞病变,PPRV-N蛋白表达水平呈上升趋势,且在病毒感染30 h后显著高于细胞对照;与此同时,GRP78、p-eIF2α和GADD153表达水平、p-PERK/PERK和p-eIF2α/eIF2α比值均显著升高。用4-PBA阻断PPRV诱导的ERS,PPRV感染细胞中PPRV-N蛋白、GRP78、p-eIF2α和GADD153表达水平及p-eIF2α/eIF2α、p-PERK/PERK比值均显著降低。在PPRV感染30和36 h,与细胞凋亡相关的Bcl-2表达下调,Bax表达上调,Bcl-2/Bax比值则显著降低。由此可见,PPRV感染可激活PERK/eIF2α/GADD153信号通路,缓解病毒诱导的ERS,促进病毒复制;阻断PPRV诱导的ERS,可抑制PERK信号通路和病毒复制;PPRV感染和持续的ERS可诱导宿主细胞凋亡。

Viral infection can induce endoplasmic reticulum stress(ERS)and unfolded protein reaction(UPR)in host cells.This study aims to further explore the effects of ERS induced by pest des petits ruminants virus(PPRV)infection on UPR signaling pathway,virus replication and apoptosis of host cells.MTT assay,indirect immunofluorescence assay(IFA)and Western blot were used to observe the proliferation of PPRV in goat kidney cells(LDG-2).Western blot and real-time fluorescence quantitative PCR(qRT-PCR)were used to observe the effects of PPRV infection on the expression levels of GRP78,PERK and its downstream signal molecules,apoptosis-related proteins Bcl-2 and Bax.The result indicated that the cell survival rate was significantly declined with evident cytopathic effect at 36 h post-infection,and the expression level of PPRV-N protein tended to be elevated,and was significantly higher than that of cell control at 30 h post-infection.Meanwhile,the expression levels of GRP78,p-eIF2αand GADD153,the ratio of p-PERK/PERK and p-eIF2α/eIF2αwere significantly increased.Moreover,the expression levels of PPRV-N protein,GRP78,peIF2αand GADD153,the ratio of p-eIF2α/eIF2αand p-PERK/PERK were significantly decreased in PPRV-infected cells due to 4-PBA treatment.The expression level of apoptosis-related Bcl-2 was down-regulated,Bax was up-regulated,and the ratio of Bcl-2/Bax was significantly decreased.Therefore,the activation of PERK/eIF2α/GADD153 signaling pathway could be induced by PPRV infection resulting in alleviating of virus-induced ERS,which is beneficial to viral replication.Blocking PPRV-induced ERS could inhibit the activation of PERK signaling pathway and virus replication.PPRV infection and prolonged ERS can induce apoptosis of LDG-2 cells.