内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用

为探究葡萄糖调节蛋白94(GRP94)对伪狂犬病毒(Pseudorabies virus,PRV)增殖的影响，以及GRP94与PRV结构蛋白之间的相互作用。利用慢病毒转染构建GRP94基因过表达的BHK-21细胞，以及CRISPR/Cas9技术构建GRP94基因敲除的BHK-21细胞，检测PRV感染不同细胞的增殖水平，比较内质网应激相关蛋白表达水平，探索GRP94与PRV结构蛋白之间的互作关系。结果表明，GRP94基因过表达细胞株显著有利于PRV增殖，而GRP94基因缺失细胞株对PRV增殖无显著影响。蛋白免疫印迹结果显示，在GRP94基因缺失细胞株中，GRP78表达水平显著上调。免疫共沉淀结果表明，GRP94与gB、gD、gL具有相互作用。GRP94基因过表达有利于PRV增殖，检测未折叠蛋白反应(unfolded protein response, UPR)相关蛋白，研究GRP94在病毒增殖过程中的具体作用，可为PRV与内质网应激提供分子方面的研究基础，于对抗PRV病毒的感染具有重要意义。

Regulation of endoplasmic reticulum molecular chaperone GRP94 on Pseudorabies virus replication

To explore the effect of glucose-regulated protein 94(GRP94) on Pseudorabies virus(PRV) replication and interaction between GRP94 and PRV structural proteins. BHK-21 cells with GRP94 overexpression were constructed by recombinant lentiviruses transfection and BHK-21 cells with GRP94 coding sequences knockout were constructed by CRISPR/Cas9 system. Progeny PRV titers were measured to compare the changes of virus proliferation efficiency in different cell lines. Western blot was performed to detect the expression level of unfolded protein response (UPR) related proteins in different cell lines. The interaction between virus structural proteins and GRP94 was determined by immunoprecipitation analysis. PRV replication was significantly increased in BHK-21-GRP94 cells, while no significant effect on PRV replication was occurred in BHK-21-GRP94-KO cells. The expression level of GRP78 was significantly up-regulated in BHK-21-GRP94-KO cells and interactions between GRP94 and gB, gD, gI were detected by Co-IP. The overexpression of GRP94 was conducive to the proliferation of PRV. By analyzing UPR related proteins, it was of great significance to study the specific role of GRP94 in the process of virus replication, so as to provide a molecular research basis for PRV and ER stress.