内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用

doi:10.3969/j.issn.1004-1524.2022.11.08

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (11): 2386-2394.DOI: 10.3969/j.issn.1004-1524.2022.11.08

内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用

倪敏舒¹^,²(), 陈丽²^,³^,⁴, 鲍熹²^,³^,⁴, 徐悦²^,³^,⁴, 庄腾寒²^,³^,⁴, 冯磊¹^,²^,³^,⁴^,^*()

1.江苏大学药学院,江苏镇江 212013
2.江苏省农业科学院动物免疫工程研究所,国家兽用生物制品工程技术研究中心,江苏南京 210014
3.江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州 225009
4.江苏省食品质量安全重点实验室省部共建国家重点实验室培育基地,江苏南京 210014

收稿日期:2022-03-07 出版日期:2022-11-25 发布日期:2022-11-29
通讯作者: 冯磊
作者简介:*冯磊,E-mail:fenglei@jaas.ac.cn
倪敏舒(1996—),男,江苏常州人,硕士研究生,主要从事病毒感染研究。E-mail:847497386@qq.com
基金资助:
江苏省重点研发计划(SBE2022780057)

Regulation of endoplasmic reticulum molecular chaperone GRP94 on Pseudorabies virus replication

NI Minshu¹^,²(), CHEN Li²^,³^,⁴, BAO Xi²^,³^,⁴, XU Yue²^,³^,⁴, ZHUANG Tenghan²^,³^,⁴, FENG Lei¹^,²^,³^,⁴^,^*()

1. School of Pharmacy, Jiangsu University, Zhenjiang 212013, Jiangsu, China
2. Institute of Veterinary Immunology & Engineering, National Research Center of Engineering and Technology for Veterinary Biologicals, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
3. Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, China
4. Jiangsu Key Laboratory of Food Quality and Safety, State Key Laboratory Cultivation Base Jointly Built by Provinces and Ministries, Nanjing 210014, China

Received:2022-03-07 Online:2022-11-25 Published:2022-11-29
Contact: FENG Lei

摘要/Abstract

摘要：

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。

关键词: 葡萄糖调节蛋白94, 葡萄糖调节蛋白78, 伪狂犬病毒, 蛋白互作

Abstract:

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.

Key words: glucose-regulated protein 94, glucose-regulated protein 78, Pseudorabies virus, protein interaction

中图分类号:

S859.1

倪敏舒, 陈丽, 鲍熹, 徐悦, 庄腾寒, 冯磊. 内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用[J]. 浙江农业学报, 2022, 34(11): 2386-2394.

NI Minshu, CHEN Li, BAO Xi, XU Yue, ZHUANG Tenghan, FENG Lei. Regulation of endoplasmic reticulum molecular chaperone GRP94 on Pseudorabies virus replication[J]. Acta Agriculturae Zhejiangensis, 2022, 34(11): 2386-2394.

图/表 9

表1 sgRNA序列

Table 1 sgRNA sequence

引物Primer	上游引物Forward primer (5'→3')	下游引物Reverse primer(5'→3')
sgRNA-1	CACCTTTGGTCGCCTCTGTACGA	AAACTCGTACAGAGGCGACCAAA
sgRNA-2	CACCAGGAAGACTTGAGATGTGCA	AAACTGCACATCTCAAGTCTTCCT

图1 GRP94基因过表达细胞形态 A,荧光场;B,自然光场。

Fig.1 Morphology of GRP94 overexpressing cell lines A, Fluorescence; B, Natural light.

图2 细胞单克隆的PCR产物电泳鉴定泳道1~6,阳性细胞单克隆;泳道C,阴性细胞。

Fig.2 PCR products of single cell clones Lane 1-6, PCR products from different single cell clones; Lane C, Negative cells.

图3 单克隆细胞PCR产物与GRP94基因序列的比对结果上方序列为泳道C回收测序结果;下方序列为泳道1回收测序结果。

Fig.3 Comparison of GRP94 gene sequence with PCR product of single cell clones Upper sequence was PCR product sequence of lane C; Lower sequence was PCR product sequence of lane 1.

图4 不同BHK-21细胞中GRP94的表达 *和**分别表示在P<0.1和P<0.05水平差异显著。下同。

Fig.4 Detection of GRP94 expression in different BHK-21 cells * and ** meant significant differences at the levels of P<0.1 and P<0.05, respectively. The same as below.

图5 不同细胞增殖PRV的毒价比较 ***表示在P<0.001水平差异显著。下同。

Fig.5 Viral titers of PRV cultured in different cells *** meant significant difference at the level of P<0.001. The same as below.

图6 不同BHK-21细胞感染PRV后UPR相关蛋白的表达

Fig.6 Expression of UPR related protein in different BHK-21 cells infected with PRV

图7 免疫共沉淀检测GRP94、GRP78与PRV病毒gB、gD、gN、gI

Fig.7 Immunoprecipitation tests of GRP78, GRP94 and gB, gD, gN, gI

图8 免疫共沉淀检测GRP94、GRP78与PRV病毒gC、gH、gL、gM

Fig.8 Immunoprecipitation tests of GRP78, GRP94 and gC, gH, gL, gM

参考文献 25

[1]	LUGANINI A, DI NARDO G, MUNARON L, et al. Human Cytomegalovirus US21 protein is a viroporin that modulates calcium homeostasis and protects cells against apoptosis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(52): E12370-E12377.
[2]	LI Y, XIA Y C, CHENG X M, et al. Hepatitis B surface antigen activates unfolded protein response in forming ground glass hepatocytes of chronic hepatitis B[J]. Viruses, 2019, 11(4): 386. DOI URL
[3]	WALTER P, RON D. The unfolded protein response: from stress pathway to homeostatic regulation[J]. Science, 2011, 334(6059): 1081-1086. DOI PMID
[4]	BERTOLOTTI A, ZHANG Y H, HENDERSHOT L M, et al. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response[J]. Nature Cell Biology, 2000, 2(6): 326-332. DOI PMID
[5]	SHEN J S, CHEN X, HENDERSHOT L, et al. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals[J]. Developmental Cell, 2002, 3(1): 99-111. DOI PMID
[6]	VAN HUIZEN R, MARTINDALE J L, GOROSPE M, et al. P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2α signaling[J]. Journal of Biological Chemistry, 2003, 278(18): 15558-15564. DOI URL
[7]	MA Y J, HENDERSHOT L M. Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress[J]. Journal of Biological Chemistry, 2003, 278(37): 34864-34873. DOI PMID
[8]	LEE A H, IWAKOSHI N N, GLIMCHER L H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response[J]. Molecular and Cellular Biology, 2003, 23(21): 7448-7459. DOI URL
[9]	HARTL F U, MARTIN J. Molecular chaperones in cellular protein folding[J]. Current Opinion in Structural Biology, 1995, 5(1): 92-102. PMID
[10]	ZHOU Y S, QI B Z, GU Y X, et al. Porcine Circovirus 2 deploys PERK pathway and GRP78 for its enhanced replication in PK-15 cells[J]. Viruses, 2016, 8(2): 56. DOI URL
[11]	HE B. Viruses, endoplasmic reticulum stress, and interferon responses[J]. Cell Death & Differentiation, 2006, 13(3): 393-403.
[12]	LEE A S. Mammalian stress response: induction of the glucose-regulated protein family[J]. Current Opinion in Cell Biology, 1992, 4(2): 267-273. PMID
[13]	ZHAO D M, LIU Q T, HAN K K, et al. Identification of glucose-regulated protein 78 (GRP78) as a receptor in BHK-21 cells for duck tembusu virus infection[J]. Frontiers in Microbiology, 2018, 9: 694. DOI PMID
[14]	CHANG S C, ERWIN A E, LEE A S. Glucose-regulated protein (GRP94 and GRP78) genes share common regulatory domains and are coordinately regulated by common trans-acting factors[J]. Molecular and Cellular Biology, 1989, 9(5): 2153-2162. DOI PMID
[15]	HENDERSHOT L M. Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition[J]. The Journal of Cell Biology, 1990, 111(3): 829-837. DOI URL
[16]	ROSE M D, MISRA L M, VOGEL J P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene[J]. Cell, 1989, 57(7): 1211-1221. DOI URL
[17]	BRUNEAU N, LOMBARDO D. Chaperone function of a Grp 94-related protein for folding and transport of the pancreatic bile salt-dependent lipase[J]. Journal of Biological Chemistry, 1995, 270(22): 13524-13533. DOI URL
[18]	DORNER A J, KRANE M G, KAUFMAN R J. Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells[J]. Molecular and Cellular Biology, 1988, 8(10): 4063-4070. DOI PMID
[19]	DORNER A J, WASLEY L C, KAUFMAN R J. Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells[J]. Journal of Biological Chemistry, 1989, 264(34): 20602-20607. PMID
[20]	SAMBROOK J F. The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum[J]. Cell, 1990, 61(2): 197-199. PMID
[21]	MARZEC M, ELETTO D, ARGON Y. GRP94: an HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum[J]. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2012, 1823(3): 774-787. DOI PMID
[22]	RANDOW F, SEED B. Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability[J]. Nature Cell Biology, 2001, 3(10): 891-896. PMID
[23]	MELNICK J, DUL J L, ARGON Y. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic Reticulum[J]. Nature, 1994, 370(6488): 373-375. DOI URL
[24]	MELNICK J, ARGON Y. Molecular chaperones and the biosynthesis of antigen receptors[J]. Immunology Today, 1995, 16(5): 243-250. PMID
[25]	KAPULKIN V, HIESTER B G, LINK C D. Compensatory regulation among ER chaperones in C. elegans[J]. FEBS Letters, 2005, 579(14): 3063-3068. DOI URL

内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用

Regulation of endoplasmic reticulum molecular chaperone GRP94 on Pseudorabies virus replication

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 25

相关文章 5

编辑推荐

Metrics

本文评价

[1]	李磊, 赵花金, 伍子焘, 李升和, 姜锦鹏, 刘畅. 决明子抗氧化作用机制的网络药理学分析[J]. 浙江农业学报, 2020, 32(10): 1855-1865.
[2]	袁献宇, 杨龙斌, 何赞赞, 毛天骄, 何长生, 占松鹤, 孙裴, 魏建忠, 李郁. 安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征[J]. 浙江农业学报, 2020, 32(1): 43-56.
[3]	易可可, 阴文奇, 周远成, 蒋金蓁, 张白玉, 李中银, 颜其贵. 四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析[J]. 浙江农业学报, 2019, 31(9): 1429-1436.
[4]	刘慧洁, 徐恒, 邱文怡, 李晓芳, 张华, 朱英, 李春寿, 王良超. 转录因子在植物生长发育及非生物逆境响应的作用[J]. 浙江农业学报, 2019, 31(7): 1205-1214.
[5]	耿文学, 唐波, 华涛, 侯继波, 张道华, 许家荣. 免疫增强剂对猪伪狂犬灭活疫苗的免疫增强作用[J]. 浙江农业学报, 2016, 28(11): 1828-1833.