热休克蛋白HSP90B1影响牛病毒性腹泻病毒复制的研究

doi:10.11843/j.issn.0366-6964.2023.02.025

摘要/Abstract

摘要： 旨在探究热休克蛋白90 β家族成员1（heat shock protein 90 beta family member 1，HSP90B1）对牛病毒性腹泻病毒（bovine viral diarrhea virus，BVDV）复制的影响。本研究通过实时荧光定量聚合酶链式反应（quantitative real time polymerase chain reaction，qRT-PCR）和Western blot检测BVDV感染MDBK细胞后HSP90B1 mRNA和蛋白的表达情况，利用CRISPR/Cas9技术构建HSP90B1 KO细胞，计数检测敲除HSP90B1对细胞生长的影响，BVDV TC株感染HSP90B1 KO和对照组Scramble细胞后，使用qRT-PCR、免疫荧光、病毒滴度以及细胞病变效应（cytopathic effects，CPE）检测BVDV的复制情况。结果表明，qRT-PCR检测显示BVDV感染24 h时与空白组相比HSP90B1 mRNA转录水平显著升高（P<0.05），36 h后极显著升高（P<0.01），同时Western blot显示BVDV感染组HSP90B1蛋白的表达水平较未感染组明显增加；Western blot检测显示，与MDBK细胞相比HSP90B1 KO细胞中的HSP90B1蛋白表达量明显降低；细胞计数显示，相同生长时间的Scramble、HSP90B1 KO细胞与MDBK细胞的数量未见差异；qRT-PCR结果显示，与对照组相比，HSP90B1 KO细胞中BVDV 5'UTR RNA水平在BVDV感染12 h后显著降低（P<0.05），36 h后极显著降低（P<0.01）；免疫荧光检测结果显示，与对照组相比，HSP90B1 KO细胞中的绿色荧光明显减少；BVDV感染后病毒滴度与对照组相比，12 h后HSP90B1 KO的病毒滴度显著降低（P<0.05），36 h后极显著降低（P<0.01），CPE显示，在感染12 h后对照组细胞出现明显CPE，而HSP90B1 KO细胞仅显示出少量CPE，感染36 h后HSP90B1 KO细胞出现明显CPE，此时对照组细胞出现大量CPE并有细胞脱落。以上结果表明BVDV诱导MDBK细胞中HSP90B1的表达；利用CRISPR/Cas9技术成功敲除MDBK细胞的HSP90B1基因，构建HSP90B1 KO细胞，试验表明敲除HSP90B1能够抑制BVDV的复制。

关键词: 热休克蛋白, 牛病毒性腹泻病毒, CRISPR/Cas9, 复制

Abstract: The present study was aimed to investigate the effect of heat shock protein 90 beta family member 1 (HSP90B1) on the replication of bovine viral diarrhea virus (BVDV). The expression of HSP90B1 mRNA and protein in MDBK cells after BVDV infection were detected by using quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. HSP90B1 KO cells were constructed using CRISPR/Cas9 technology. The effect of knockdown of HSP90B1 on cell growth was detected by counting. After infecting with BVDV TC strain, BVDV replication in HSP90B1 KO and control Scramble cells were detected using qRT-PCR, immunofluorescence, viral titer and cytopathic effects (CPE). The qRT-PCR results showed that the transcription level of HSP90B1 mRNA was significantly increased at 24 h after BVDV infection compared with the blank group (P<0.05), and was significantly increased after 36 h (P<0.01). Meanwhile, Western blot results showed that the expression level of HSP90B1 protein of BVDV infection group was significantly increased compared with the uninfected group; Western blot results showed that the expression of HSP90B1 protein in HSP90B1 KO cells was significantly lower than that in MDBK cells; There was no difference in the number of cells; qRT-PCR results showed that BVDV 5'UTR RNA levels in HSP90B1 KO cells were significantly reduced after 12 h of BVDV infection (P<0.05) and highly significantly reduced after 36 h compared with the control group (P< 0.01); the results of immunofluorescence detection showed that the green fluorescence in HSP90B1 KO cells was significantly reduced compared with the control group; viral titer after BVDV infection was significantly lower in HSP90B1 KO compared to control (P<0.05) after 12 h and highly significantly lower after 36 h (P<0.01), CPE showed that control cells showed The CPE showed that control cells showed significant CPE after 12 h of infection, while HSP90B1 KO cells showed only a small amount of CPE, and HSP90B1 KO cells showed significant CPE after 36 h of infection, at which time control cells showed a large amount of CPE with cell shedding. The above results show that BVDV induces the expression of HSP90B1 in MDBK cells; the HSP90B1 gene in MDBK cells was successfully knocked out by CRISPR/Cas9 technology, and HSP90B1 KO cells were constructed, experiments show that knockout of HSP90B1 can inhibit BVDV replication.

Key words: heat shock protein, bovine viral diarrhea virus, CRISPR/Cas9, replication

中图分类号:

S852.659.6

陈俊贞, 权冉, 付强, 葛丽娟, 袁圆圆, 张成远, 李建林, 史慧君. 热休克蛋白HSP90B1影响牛病毒性腹泻病毒复制的研究[J]. 畜牧兽医学报, 2023, 54(2): 683-693.

CHEN Junzhen, QUAN Ran, FU Qiang, GE Lijuan, YUAN Yuanyuan, ZHANG Chengyuan, LI Jianlin, SHI Huijun. Study on the Effect of Heat Shock Protein HSP90B1 on the Replication of Bovine Viral Diarrhea Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 683-693.

参考文献 27

[1]	PETERHANS E, BACHOFEN C, STALDER H, et al. Cytopathic bovine viral diarrhea viruses (BVDV):emerging pestiviruses doomed to extinction[J]. Vet Res, 2010, 41(6):44.
[2]	DIAO N C, GONG Q L, LI J M, et al. Prevalence of bovine viral diarrhea virus (BVDV)in yaks between 1987 and 2019 in mainland China:a systematic review and meta-analysis[J]. Microb Pathog, 2020, 144:104185.
[3]	YEŞILBAǦ K, FÖRSTER C, OZYIǦIT M O, et al. Characterisation of bovine viral diarrhea virus (BVDV) isolates from an outbreak with haemorrhagic enteritis and severe pneumonia[J]. Vet Microbiol, 2014, 169(1-2):42-49.
[4]	张国华, 吴丹丹, 周玉龙, 等. 牛病毒性腹泻病毒黑龙江分离株的分离鉴定[J]. 中国预防兽医学报, 2015, 37(10):805-807.ZHANG G H, WU D D, ZHOU Y L, et al. Isolation and identification of bovine viral diarrhea virus from Heilongjiang Province[J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(10):805-807. (in Chinese)
[5]	张国奇, 李尤简, 肖红冉, 等. 牛病毒性腹泻病毒纳米抗体文库的构建与鉴定[J]. 西北农业学报, 2015, 24(3):20-25.ZHANG G Q, LI Y J, XIAO H R, et al. Construction and characterization of a nanobody library against BVDV[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2015, 24(3):20-25. (in Chinese)
[6]	IYER K, CHAND K, MITRA A, et al. Diversity in heat shock protein families:functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle[J]. Cell Stress Chaperones, 2021, 26(5):743-768.
[7]	ELETTO D, DERSH D, ARGON Y. GRP94 in ER quality control and stress responses[J]. Semin Cell Dev Biol, 2010, 21(5):479-485.
[8]	RANDOW F, SEED B. Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability[J]. Nat Cell Biol, 2001, 3(10):891-896.
[9]	BLOYET L M, WELSCH J, ENCHERY F, et al. HSP90 chaperoning in addition to phosphoprotein required for folding but not for supporting enzymatic activities of measles and Nipah virus L polymerases[J]. J Virol, 2016, 90(15):6642-6656.
[10]	GELLER R, VIGNUZZI M, ANDINO R, et al. Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance[J]. Genes Dev, 2007, 21(2):243-251.
[11]	ROTHAN H A, ZHONG Y W, SANBORN M A, et al. Small molecule grp94 inhibitors block dengue and Zika virus replication[J]. Antiviral Res, 2019, 171:104590.
[12]	LANDI A, VERMEIRE J, IANNUCCI V, et al. Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-inducedCD4 down-regulation[J]. Retrovirology, 2014, 11:118.
[13]	LI Z H, CHEN F Z, YE S Y, et al. Comparative proteome analysis of porcine jejunum tissues in response to a virulent strain of porcine epidemic diarrhea virus and its attenuated strain[J]. Viruses, 2016, 8(12):323.
[14]	KHACHATOORIAN R, FRENCH S W. Chaperones in hepatitis C virus infection[J]. World J Hepatol, 2016, 8(1):9-35.
[15]	梁霄勇, 季新成, 冉多良. 牛病毒性腹泻病毒TC株的分离鉴定及E0基因的序列分析[J]. 畜牧与兽医, 2016, 48(7):36-40.LIANG X Y, JI X C, RAN D L. Isolation and identification of bovine viral diarrhea virus TC strain and sequence analysis of E0 gene[J]. Animal Husbandry & Veterinary Medicine, 2016, 48(7):36-40. (in Chinese)
[16]	田瑞鑫. 牛miR-29b过表达影响牛病毒性腹泻病毒感染BALB/c小鼠的作用研究[D]. 乌鲁木齐:新疆农业大学, 2019.TIAN R X. Analysis of the effects of bovine miR-29b overexpression on BALB/c mice infected with bovine viral diarrhea virus[D]. Urumqi:Xinjiang Agricultural University, 2019. (in Chinese)
[17]	付强, 陈俊贞, 郭妍婷, 等. 应用CRISPR/Cas9技术敲除SERPINF2基因对牛病毒性腹泻病毒复制的影响[J]. 生物技术通报, 2021, 37(5):267-272.FU Q, CHEN J Z, GUO Y T, et al. Effects of knockout of gene SERPINF2 via CRISPR/Cas9 on the replication of bovine viral diarrhea virus[J]. Biotechnology Bulletin, 2021, 37(5):267-272. (in Chinese)
[18]	FU Q, SHI H J, CHEN C F. Roles of bta-miR-29b promoter regions DNA methylation in regulating miR-29b expression and bovine viral diarrhea virus NADL replication in MDBK cells[J]. Arch Virol, 2017, 162(2):401-408.
[19]	VAN LEUR S W, HEUNIS T, MUNNUR D, et al. Pathogenesis and virulence of Flavivirus infections[J]. Virulence, 2021, 12(1):2814-2838.
[20]	SENGUPTA N, GHOSH S, VASAIKAR S V, et al. Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection[J]. PLoS One, 2014, 9(3):e90211.
[21]	ZHONG C Y, LI J Z, MAO L, et al. Proteomics analysis reveals heat shock proteins involved in caprine parainfluenza virus type 3 infection[J]. BMC Vet Res, 2019, 15(1):151.
[22]	LIANG Y S, ZHANG G G, LI Q H, et al. TRIM26 is a critical host factor for HCV replication and contributes to host tropism[J]. Sci Adv, 2021, 7(2):eabd9732.
[23]	MLADINICH M C, CONDE J N, SCHUTT W R, et al. Blockade of autocrine CCL5 responses inhibits Zika virus persistence and spread in human brain microvascular endothelial cells[J]. MBio, 2021, 12(4):e01962-21.
[24]	ISKEN O, POSTEL A, BRUHN B, et al. CRISPR/Cas9-mediated knockout of DNAJC14 verifies this chaperone as a pivotal host factor for RNA replication of pestiviruses[J]. J Virol, 2019, 93(5):e01714-18.
[25]	宋爽, 赵柏林, 曲萍, 等. 牛病毒性腹泻病毒、牛传染性支气管炎病毒和口蹄疫病毒多重TaqMan荧光定量PCR检测方法的建立[J]. 中国预防兽医学报, 2017, 39(2):133-136.SONG S, ZHAO B L, QU P, et al. Establishment of multiplex TaqMan real-time PCR for the detection of bovine viral diarrhea virus, infectious bovine rhinotracheitis virus and foot-mouth disease virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(2):133-136. (in Chinese)
[26]	JORDAN R, WANG L J, GRACZYK T M, et al. Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells[J]. J Virol, 2002, 76(19):9588-9599.
[27]	JORDAN R, NIKOLAEVA O V, WANG L J, et al. Inhibition of host ER glucosidase activity prevents Golgi processing of virion-associated bovine viral diarrhea virus E2 glycoproteins and reduces infectivity of secreted virions[J]. Virology, 2002, 295(1):10-19.