伪狂犬病毒感染对BHK-21悬浮细胞内质网应激和未折叠蛋白反应的影响

Effect of PRV Infection on the Endoplasmic Reticulum Stress and Unfolded Protein Response in Suspension-cultured BHK-21Cells

【目的】探讨伪狂犬病毒(PRV)感染对BHK-21悬浮细胞内质网应激及未折叠蛋白反应(UPR)的影响。【方法】将悬浮的乳仓鼠肾细胞(BHK-21)以感染复数(MOI)0.01接种PRV,分别在接毒后12、24、36、48和56 h取样,用CCK-8法检测细胞存活率,按Reed-Muench法检测病毒滴度,筛选病毒接种处理的合适时间;以不接毒细胞作对照组,分别在感染后12、24、36和48 h取样,用实时荧光定量PCR法检测内质网应激标志分子葡萄糖调节蛋白78(GRP78)及UPR的3种感受蛋白(PERK、IRE1、ATF6)相关通路中基因的表达变化,Western blotting法检测相关蛋白的表达变化。在接种PRV同时分别加入0、0.001、0.005、0.01和0.02μmol/L内质网应激诱导剂毒胡萝卜素(Tg)和0、20、40、80和160μmol/L内质网应激抑制剂牛磺熊去氧胆酸(TUDCA),48 h收集细胞测定病毒滴度和细胞存活率。【结果】PRV感染56 h细胞存活率低于70%,感染48 h时病毒滴度达到最高(8.1 lg TCID/mL),因此后续试验分别在接毒后12、24、36和48 h取样。与对照组相比,PRV感染36和48 h GRP78转录水平均极显著升高(P<0.01);PERK通路中,转录激活因子4(ATF4)转录水平在PRV感染36、48 h均极显著升高(P<0.01),生长抑制DNA损伤基因34(GADD34)转录水平在PRV感染36 h显著升高(P<0.05)、48 h极显著升高(P<0.01),磷酸化真核翻译起始因子(P-eIF2α)蛋白水平在PRV感染36、48 h均极显著升高(P<0.01);IRE1通路中,PRV感染组细胞36 h后多以剪切形式sXBP1存在,同时下游p58mRNA水平在36 h显著增加(P<0.05),p58和EDEM mRNA水平在48 h均极显著增加(P<0.01);ATF6通路中,PRV感染不同时间ERp57、PDI、Calnexin和Calreticulin的表达均无显著变化(P>0.05)。与0μmol/L组相比,0.01和0.02μmol/L Tg极显著降低细胞存活率(P<0.01),0.005和0.01μmol/L Tg均极显著增加了PRV病毒滴度(P<0.01)。【结论】PRV感染可以诱导BHK-21悬浮细胞内质网应激,激活UPR的PERK和IRE1信号通路,说明PRV利用内质网应激增强其复制。

【Objective】This study was aimed to investigate the effect of Pseudorabies virus(PRV)infection on endoplasmic reticulum stress(ER)and unfolded protein response(UPR)in BHK-21 suspension-cultured cells.【Method】The suspension-cultured baby hamster kidney cells(BHK-21)were infected by PRV at a multiplicity of infection(MOI)of 0.01.Samples were taken at 12,24,36,48 and 56 h after inoculation,respectively.Cell survival rate was detected by CCK-8 method,and virus titer was detected by Reed-Muench method,and the appropriate time of virus inoculation was screened.The uninfected cells were used as control group.Samples were taken at 12,24,36,and 48 h after infection,and the gene expression changes in the pathways related to endoplasmic reticulum stress marker GRP78 and UPR receptor proteins(PERK,IRE1 and ATF6)were detected by Real-time quantitative PCR,the expression of related proteins were detected by Western blotting.Endoplasmic reticulum stress inducer toxocarotene(Tg)of 0,0.001,0.005,0.01 and 0.02μmol/L and endoplasmic reticulum stress inhibitor taurodeoxycholic acid(TUDCA)of 0,20,40,80 and 160μmol/L were added to PRV at the same time.Cells were collected at 48 h to determine virus titer and cell survival rate.【Result】The relative cell viability was less than 70%after 56 h,and the viral titer reached 8.1 lg TCID/mL at 48 h after PRV infection.Therefore,samples within 48 h(12,24,36 and 48 h)post PRV infection were selected to perform the analysis of endoplasmic reticulum stress.Compared with control group,the transcript levels of GRP78 were extremely significantly increased at 36 and 48 h by PRV infection(P<0.01).In the PERK pathway,the transcript level of ATF4 was extremely significantly increased at 36 and 48 h(P<0.01),and the transcript level of GADD34 was significantly increased at 36 h(P<0.05)and extremely significantly increased at 48 h(P<0.01).The level of eIF2αphosphorylation was extremely significantly increased at 36 and 48 h by PRV infection(P<0.01).In the IRE1 pathway,sXBP1(spliced XBP1)was founded from 36 h after PRV infection.The transcript level of p58was significantly increased at 36 h(P<0.05),and the transcript levels of p58and EDEM were extremely significantly increased at 48 h(P<0.01).In the ATF6 pathway,there were no significant changes in the transcript levels of ERp57,PDI,Calnexin,and Calreticulin(P>0.05).Compared with 0μmol/L group,cell viability were extremely significantly decreased by 0.01 and 0.02μmol/L Tg,PRV titers were extremely significantly increased by 0.005 and 0.01μmol/L Tg(P<0.01).【Conclusion】Endoplasmic reticulum stress was induced,and PERK and IRE1 signaling pathways of UPR were activated by PRV infection in suspension-cultured BHK-21 cells.The PRV deployed endoplasmic reticulum stress to enhance its replication.