GRP78在伪狂犬病病毒感染过程中的作用研究

The role of GRP78 in the infection of pseudorabies virus

为探究热休克蛋白70家族成员葡萄糖调节蛋白78(GRP78)在伪狂犬病病毒(PRV)感染宿主过程中的作用,本研究利用PRV Min-A株感染PK-15细胞后,采用western blot和间接免疫荧光试验(IFA)分别检测GRP78蛋白的表达和亚细胞定位情况,western blot结果显示,与对照组相比,PRV感染组的GRP78蛋白水平未见明显变化;IFA结果显示,PRV感染组细胞膜上GRP78的荧光强度明显增加。进一步通过生物素标记细胞膜蛋白,然后利用磁珠分离细胞膜蛋白,经western blot检测结果显示,PRV感染组细胞膜中GRP78的蛋白水平显著高于未感染PRV的对照组(P<0.05)。可见PRV感染不影响细胞内GRP78蛋白的表达,但能够增加GRP78在细胞膜中的含量。利用GRP78多克隆抗体(pAb)与PK-15细胞共孵育1 h后感染PRV,经空斑试验检测细胞上清中的病毒滴度,结果显示,与对照组相比,GRP78 p Ab孵育组的病毒滴度未见明显变化,表明GRP78并不是PRV侵入宿主细胞的关键因子。通过IFA检测GRP78与PRV在细胞中的共定位情况,结果显示,GRP78与PRV在细胞膜上无共定位,但二者在细胞质中有共定位。分别在PK-15细胞中下调或者过表达GRP78后感染PRV,采用western blot和空斑试验分别检测细胞内PRV的蛋白合成和细胞上清中的病毒滴度,结果显示,GRP78下调后PRV蛋白和病毒滴度显著低于对照组(P<0.05),而过表达GRP78组的病毒滴度明显高于对照组,表明GRP78参与调节PRV的释放。本研究首次表明GRP78作为一种重要的宿主因子参与调节PRV从宿主细胞内的释放,该结果为进一步研究PRV与宿主细胞的相互作用提供了参考依据。

To explore the role of glucose-regulated protein,78ku(GRP78)on the infection of pseudorabies virus(PRV),in this study,PK-15 cells were infected with PRV-MinA strain,and then the protein expression level and sub-cellular localization of GRP78 was detected by western blot and indirect immunofluorescence assay(IFA),respectively.The results showed that,compared with the control group,the protein level of GRP78 in PRV infection group shows no difference,while the IFA results showed that the fluorescence intensity of GRP78 in the PRV-infected cell membrane is increased.The cell membrane protein was further separated by magnetic beads after labeling with biotin,and the amount of GRP78 protein was detected by western blot.It was found that PRV infection significantly increases the protein level of GRP78 in the cell membrane.These results show that PRV infection do not influence the expression of GRP78 in cytoplasm,but that increases the amount of GRP78 expression in cell membrane.PK-15 cells were pre-incubated with rabbit polyclonal antibody(pAb)against GRP78 for 1 hour,and then the pretreated cells were infected by PRV.Cell supernatants were collected and the virus titration were performed via plaque formation assay.The results showed that there is no significant difference in virus titer in GRP78 pAb pre-treated group in comparison of control group,which indicating that GRP78 is not the key factor for PRV entering host cells.Furthermore,the co-localization of GRP78 and PRV in cells was detected by IFA,and it was found that the co-localization of GRP78 and PRV is observed in the cytoplasm but not in the cell membrane.The GRP78 protein was silenced or overexpressed in PK-15 cells,respectively,and the effect of GRP78 on PRV replication was detected.The results showed that GRP78 knockdown significantly decreases the intercellular expression of PRV protein and virus titer in supernatant in comparison of control group,while the overexpression of GRP78 significantly increases the virus titer.These results suggest that GRP78 is involved in the regulation of PRV release.In conclusion,this study suggests that GRP78 is an important host factor involved in regulating the release of PRV from host cell for the first time,which provides a reference for further studies on the interaction between PRV and host cells.