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

doi:10.16431/j.cnki.1671-7236.2022.08.039

Abstract

Abstract: 【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 0.02 μmol/L and endoplasmic reticulum stress inhibitor taurodeoxycholic acid (TUDCA) of 0,20,40,80 and 160 μmol/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 TCID₅₀/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 eIF2α phosphorylation 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 p58^IPK was significantly increased at 36 h (P<0.05),and the transcript levels of p58^IPK and 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 0 μmol/L group,cell viability were extremely significantly decreased by 0.01 and 0.02 μmol/L Tg,PRV titers were extremely significantly increased by 0.005 and 0.01 μmol/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.

Key words: Pseudorabies virus; BHK-21 suspension-cultured cells; endoplasmic reticulum stress; unfolded protein response

CLC Number:

S855.3

CHEN Li, NI Minshu, XU Yue, BAO Xi, ZHUANG Tenghan, FENG Lei, GUO Meijin. Effect of PRV Infection on the Endoplasmic Reticulum Stress and Unfolded Protein Response in Suspension-cultured BHK-21 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3235-3244.

References

[1] FERAL K,JAUD M,PHILIPPE C,et al.ER stress and unfolded protein response in leukemia:Friend,foe,or both?[J].Biomolecules,2021,11(2):199.
[2] IVANOVA E A,OREKHOV A N.The role of endoplasmic reticulum stress and unfolded protein response in atherosclerosis[J].International Journal of Molecular Sciences,2016,17(2):193.
[3] 田丽平,冯冠榕,鲁会军,等.冠状病毒感染与内质网应激研究进展[J].病毒学报,2022,38(2):456-459. TIAN L P,FENG G R,LU H J,et al.Research advances in Coronavirus infection and endoplasmic reticulum stress[J].Chinese Journal of Virology,2022,38(2):456-459.(in Chinese)
[4] CHRISTEN V,MEILI N,FENT K.Microcystin-LR induces endoplasmatic reticulum stress and leads to induction of NFkappaB,interferon-alpha,and tumor necrosis factor-alpha[J].Environmental Science and Technology,2013,47(7):3378-3385.
[5] HE B.Viruses,endoplasmic reticulum stress,and interferon responses[J].Cell Death and Differentiation,2006,13(3):393-403.
[6] RON D,WALTER P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nature Reviews Molecular Cell Biology,2007,8(7):519-529.
[7] HETZ C,SAXENA S.ER stress and the unfolded protein response in neurodegeneration[J].Nature Reviews Neurology,2017,13(8):477-491.
[8] HETZ C,ZHANG K,KAUFMAN R J.Mechanisms,regulation and functions of the unfolded protein response[J].Nature Reviews Molecular Cell Biology,2020,21(8):421-438.
[9] 赵冬敏,黄欣梅,章丽娇,等.坦布苏病毒感染诱导雏鸭体内未折叠蛋白反应[J].中国农业科学,2021,54(4):855-863. ZHAO D M,HUANG X M,ZHANG L J,et al.The induction of unfolded protein response in Tembusu virus infected ducklings[J].Scientia Agricultura Sinica,2021,54(4):855-863.(in Chinese)
[10] CHAKRABARTI A,CHEN A W,VARNER J D.A review of the mammalian unfolded protein response[J].Biotechnology and Bioengineering,2011,108(12):2777-2793.
[11] LE THOMAS A,FERRI E,MARSTERS S,et al.Decoding non-canonical mrna decay by the endoplasmic-reticulum stress sensor IRE1α[J].Nature Communications,2021,12(1):7310.
[12] WU S X,YE S S,HONG Y X,et al.Hepatitis B virus small envelope protein promotes hepatocellular carcinoma angiogenesis via endoplasmic reticulum stress signaling to upregulate the expression of vascular endothelial growth factor A[J].Journal of Virology,2022,96(4):e0197521.
[13] MCKAY L G A,THOMAS J,ALBALAWI W,et al.The HCV envelope glycoprotein down-modulates NF-κB signalling and associates with stimulation of the host endoplasmic reticulum stress pathway[J].Frontiers in Immunology,2022,13:831695.
[14] TROUVE P,FEREC C,GENIN E.The interplay between the unfolded protein response,inflammation and infection in cystic fibrosis[J].Cells,2021,10(11):2980.
[15] MULVEY M,ARIAS C,MOHR I.Maintenance of endoplasmic reticulum (ER) homeostasis in herpes simplex virus type 1-infected cells through the association of a viral glycoprotein with PERK,a cellular ER stress sensor[J].Journal of Virology,2007,81(7):3377-3390.
[16] 刘宇婷,李国新,王斌.疱疹病毒与内质网应激[J].生物工程学报,2021,37(1):67-77. LIU Y T,LI G X,WANG B.Herpesvirus and endoplasmic reticulum stress[J].Chinese Journal of Biotechnology,2021,37(1):67-77.(in Chinese)
[17] YIN H,ZHAO L,JIANG X,et al.DEV induce autophagy via the endoplasmic reticulum stress related unfolded protein response[J].PLoS One,2017,12(12):e0189704.
[18] MULLER T,HAHN E C,TOTTEWITZ F,et al. Pseudorabies virus in wild swine:A global perspective[J].Archives of Virology,2011,156(10):1691-1705.
[19] TAN L,YAO J,YANG Y,et al.Current status and challenge of Pseudorabies virus infection in China[J].Virologica Sinica,2021,36(4):588-607.
[20] SUN Y,LUO Y,WANG C H,et al.Control of swine pseudorabies in China:Opportunities and limitations[J].Veterinary Microbiology,2016,183:119-124.
[21] ZHOU M,WU X,JIANG D,et al.Characterization of a moderately pathogenic Pseudorabies virus variant isolated in China,2014[J].Infection Genetics Evolution,2019,68:161-171.
[22] KLUPP B G.Pseudorabies virus infections[J].Pathogens,2021,10(6):719.
[23] 任春芝,路彩霞,韦英益,等.辣蓼黄酮乙酸乙酯部分对PRV体外诱导RAW264.7细胞分泌炎性因子的影响[J].中国畜牧兽医,2021,48(3):1132-1140. REN C Z,LU C X,WEI Y Y,et al.Effect of ethyl acetate fraction of flavonoids from Polygonum hydropiper L.on cytokines secretion in RAW264.7 cells induced by Pseudorabies virus[J].China Animal Husbandry&Veterinary Medicine,2021,48(3):1132-1140.(in Chinese)
[24] DELVA J L,NAUWYNCK H J,METTENLEITER T C,et al.The attenuated Pseudorabies virus vaccine strain bartha k61:A brief review on the knowledge gathered during 60 years of research[J].Pathogens,2020,9(11):897.
[25] YANG S,ZHU J,ZHOU X,et al.Induction of the unfolded protein response (UPR) during Pseudorabies virus infection[J].Veterinary Microbiology,2019,239:108485.
[26] REED L J,MUENCH H.A simple method of estimating fifty percent endpoints[J].American Journal of Epidemiology,1938,27:493-497.
[27] KOHLI E,CAUSSE S,BAVEREL V,et al.Endoplasmic reticulum chaperones in viral infection:Therapeutic perspectives[J].Microbiology and Molecular Biology Reviews,2021,85(4):e0003521.
[28] BAGCHI P.Endoplasmic reticulum in viral infection[J].International Review of Cell and Molecular Biology,2020,350:265-284.
[29] WANG J,SONG Z,GE A,et al.Safety and immunogenicity of an attenuated chinese Pseudorabies variant by dual deletion of TK&gE genes[J].BMC Veterinary Research,2018,14(1):287.
[30] YANG S,PEI Y,ZHAO A.Itraq-based proteomic analysis of porcine kidney epithelial PK-15 cells infected with Pseudorabies virus[J].Scientific Reports,2017,7:45922.
[31] XUE M,FU F,MA Y,et al.The PERK arm of the unfolded protein response negatively regulates Transmissible gastroenteritis virus replication by suppressing protein translation and promoting type Ⅰ interferon production[J].Journal of Virology,2018,92:e00431-18.
[32] XU C,WANG M,SONG Z,et al.Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro[J].Virus Research,2018,248:44-52.
[33] CATANZARO N,MENG X J.Induction of the unfolded protein response (UPR) suppresses Porcine reproductive and respiratory syndrome virus (PRRSV) replication[J].Virus Research,2020,276:197820.
[34] HE W,XU H,GOU H,et al.CSFV infection up-regulates the unfolded protein response to promote its replication[J].Frontiers in Microbiology,2017,8:2129.
[35] GLADUE D P,LARGO E,HOLINKA L G,et al. Classical swine fever virus p7 protein interacts with host protein CAMLG and regulates calcium permeability at the endoplasmic reticulum[J].Viruses,2018,10(9):460.

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

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