干扰素调节因子3基因敲除对伪狂犬病病毒增殖的影响

doi:10.16431/j.cnki.1671-7236.2019.05.001

Abstract

Abstract:

The aim of the study was to investigate the effect of knockout of interferon regulatory factor 3 (IRF3) on the replication of pseudorabies virus (PRV).The IRF3 knockout PK15 cell line was established using lentiviral-mediated CRISPR/Cas9 genome editing technology.The recombinant plasmid pIRF3-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was obtained and infected with PK15 cells.The polyclonal cell line was obtained by puromycin screening.After T7 digestion,PK15-IRF3^-/- monoclonal stable cell line was obtained by limiting dilution method.To verify whether the stable cell line of IRF3 gene was successfully constructed,Real-time PCR and Western blotting were used to detect the expression of PRV-related genes and proteins.Fluorescence microscopy and flow cytometry were used to observe the virus replication.The results showed that the fluorescence intensity of PK15-IRF3^-/- cells was significantly stronger than that of PK15 cells after infection of PRV-GFP in PK15-IRF3^-/- cell line.The PK15-IRF3^-/- virus titer was significantly stronger than that of PK15 cells after infection with PRV-QXX,and the gE protein level of PRV was significantly higher than that of PK15 cells.The same results were obtained by detecting changes of PRV TK gene in the two cells at the mRNA level.Further studies showed that IFN-β mRNA in PK15 cells increased significantly with time after infection with PRV-QXX (P<0.05),but there was no significant change in IFN-β mRNA in PK15-IRF3^-/- cells.The above results indicated that knocking out IRF3 significantly promoted the proliferation of PRV,and IRF3 played an important role in virus replication,providing new methods and strategies for the prevention and control of pseudorabies.

Key words: interferon regulatory factor 3 (IRF3); pseudorabies virus (PRV); CRISPR/Cas9

CLC Number:

ZHANG Shuang, FAN Shuangshuang, CHANG Wenru, DING Guangxu, GUO Ruizhen, DUAN Lifang, DU Yongkun, CHU Beibei, YANG Guoyu, WANG Jiang. Effect of Interferon Regulatory Factor 3 Gene Knockout on Proliferation of Pseudorabies Virus[J]. , 2019, 46(5): 1253-1262.

References

[1] 龙晓婷,刘俊磊,郭洪,等.伪狂犬病毒在潜伏感染猪体内的组织分布[J].畜牧兽医学报,2008,39(5):645-651. LONG X T,LIU J L,GUO H,et al.Distribution of pseudorabies virus in latently infected pigs[J].Acta Veterinaria et Zootechnica Sinica,2008,39(5):645-651.(in Chinese)
[2] 青宁生.动物病毒学家——殷震[J].微生物学报,2018,58(5):955-956. QING N S.An animal virologist——Yin Z[J].Acta Microbiologica Sinica,2018,58(5):955-956.(in Chinese)
[3] GUZ Q,HOUC C,SUN H F,et al.Emergence of highly virulent pseudorabies virus in Southern China[J].Canadian Journal of Veterinary Research,2015,79(3):221-228.
[4] WU R,BAI C Y,SUN J Z,et al.Emergence of virulent pseudorabies virus infection in Northern China[J].Journal of Veterinary Science,2013,14(3):363-365.
[5] ZHAO G N,JIANG D S,LI H.Interferon regulatory factors:At the crossroads of immunity,metabolism,and disease[J].Biochimica et Biophysica Acta,2015,1852(2):365-378.
[6] JUANG Y T,LOWTHER W,KELLUM M,et al.Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3[J].Proceedings of the National Academy of Sciences,1998,95(17):9837-9842.
[7] BELLINGHAM J,GREGORY-EVANS K,GREGORY-EVANS C Y,et al.Mapping of human interferon regulatory factor 3(IRF3) to chromosome 19q13.3-13.4 by an intragenic polymorphic marker[J].Annals of Human Genetics,1998,62(Pt 3):231-234.
[8] TANIGUCHI T,OGASAWARA K,TAKAOKA A,et al.IRF family of transcription factors as regulators of host defense[J].Annual Review of Immunology,2001,19(1):623-655.
[9] OZATO K,TAILOR P,KUBOTA T.The interferon regulatory factor family in host defense:Mechanism of action[J].Journal of Biological Chemistry,2007,282(28):20065-20069.
[10] LIN R,HEYLBROECK C,PITHA P M,et al.Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation,transactivation potential,and proteasome-mediated degradation[J].Molecular and Cellular Biology,1998,18(5):2986-2996.
[11] 李帅,赵一霏,沈国顺.一例猪伪狂犬病的诊断与防控建议[J].黑龙江畜牧兽医,2018,22:122-124. LI S,ZHAO Y Z,SHEN G S.A diagnosis and prevention and control of a case of pseudorabies in pigs[J]. Heilongjiang Animal Science and Veterinary Medicine,2018,22:122-124.(in Chinese)
[12] SAITO T,GALE M JR.Principles of intracellular viral recognition[J].Current Opinion Immunology,2007,19(1):17-23.
[13] WANG H,YANG H,SHIVALILA C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[14] KORKMAZ G,LOPES R,UGALDE A P,et al.Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9[J].Nature Biotechnology,2016,34(2):192-198.
[15] MAZ A,PERETZ M,AHARONY A,et al.Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells[J]. Nature Cell Biology,2018,20(5):610-619.
[16] KLANN T S,BLACK J B,CHELLAPPAN M,et al.CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome[J].Nature Biotechnology,2017,35(6):561-568.
[17] NELSON C A,FREMONT D H,VIRGIN H W,et al.Discovery of a proteinaceous cellular receptor for a norovirus[J].Science,2016,353(6302):933-936.
[18] HAGA K,FUJIMOTO A,TAKAI-TODAKA R,et al.Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(41):E6248-E6255.
[19] VIRREIRA WINTER S,ZYCHLINSKY A,BARDOEL B W.Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity[J].Scientific Reports,2016,6:242-244.
[20] ZHANG R,MINER J J,GORMAN M J,et al.A CRISPR screen defines a signal peptide processing pathway required by flaviviruses[J].Nature,2016,535(7610):164-168.
[21] MA H,DANG Y,WU Y,et al.A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell death[J].Cell Reports,2015,12(4):673-683.
[22] MARCEAU C D,PUSCHNIK A S,MAJZOUB K,et al.Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens[J].Nature,2016,535(7610):159-163.
[23] SIDIK S M,HUET D,GANESAN S M A,et al.A genome-wide CRISPR screen in toxoplasma identifies essential apicomplexan genes[J].Cell,2016,166(6):1423-1435.
[24] MODELL J W,JIANG W,MARRAFFINI L A.CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity[J].Nature,2017,544(7648):101-104.
[25] MOCH C,FROMANT M,BLANQUET S,et al.DNA binding specificities of Escherichia coli Cas1-Cas2 integrase drive its recruitment at the CRISPR locus[J].Nucleic Acids Research,2017,45(5):2714-2723.
[26] WRIGHT A V,LIU J J,KNOTT G J,et al.Structures of the CRISPR genome integration complex[J]. Science,2017,357(6356):1113-1118.
[27] SHALEM O,SANJANA N E,HARTENIAN E,et al.Genome-scale CRISPR-Cas9 knockout screening in human cells[J].Science,2014,343(6166):84-87.
[28] SHALEM O,SANJANA N E,ZHANG F.High-throughput functional genomics using CRISPR-Cas9[J].Nature Reviews Genetics,2015,16(5):299-311.
[29] MILLER L C,ZANELLA E L,WATERS W R,et al.Cytokine protein expression levels in tracheobronchial lymph node homogenates of pigs infected with pseudorabies virus[J].Clinical Vaccine Zmmanol,2010,17(5):728-734.
[30] VAN DER SANDEN S M,WU W,DYBDAHL-SISSOKO N,et al.Engineering enhanced vaccine cell lines to eradicate vaccine-preventable diseases:The polio end game[J].Journal of Virology,2015,90(4):1694-1704.
[31] BIKARD D,EULER C W,JIANG W,et al.Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials[J].Nature Biotechnology,2014,32(11):1146-1150.

Effect of Interferon Regulatory Factor 3 Gene Knockout on Proliferation of Pseudorabies Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZHANG Jingxu, XU Yu, LIANG Haiying, ZENG Zhiyong, TANG Deyuan, WANG Bin, XU Songping, WAN Juan, ZHU Yang. Phylogenetic Evolution and Epitope Analysis of Pseudorabies Virus Guizhou Isolates and Vaccine Strains [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1093-1106.
[2]	MENG Jiejie, SONG Yue, FAN Wenjie, YANG Le, XING Jiayou, WANG Jiang, CHU Beibei, YANG Guoyu, WANG Mengdi. Effect of Toll-like Receptor 7 Gene Knockout on Proliferation of Vesicular Stomatitis Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2011-2021.
[3]	XIE Sihao, GOU Hongchao, BIAN Zhibiao, LI Bin, CAI Rujian, ZANG Yingan, LI Chunling. Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1934-1941.
[4]	XU Jing, YANG Guang, JIANG Meiqi, DING Xiangbin, GUO Yiwen, HU Debao, LI Xin, GUO Hong, ZHANG Linlin. Research Advances on CRISPR/Cas9 Technology in Livestock and Poultry Breeding [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1374-1383.
[5]	LI Xiaojiao, HE Yanhua, ZHU Xinyu, ZOU Xian, LUO Chenglong. Research Progress on Application of CRISPR/Cas9 Technology in Pigs and Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4665-4673.
[6]	XU Xin, LIU Mingjun. Research Progress on Application of CRISPR/Cas9 Genome Editing Systems in Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4129-4138.
[7]	XING Jiayou, FAN Wenjie, WANG Yumin, LU Xiuxiang, SONG Yue, CHU Beibei, WANG Jiang, WANG Mengdi. Effect of Tamoxifen on the Proliferation of Porcine Pseudorabies Virus in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4503-4512.
[8]	MA Jing, ZHANG Yanyan, JIA Xinyue, MA Xun, WANG Zhengrong, BO Xinwen. Research Progress of CRISPR/Cas9 System in Parasite Genome Editing [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3713-3725.
[9]	YANG Sha, HAO Haisheng, DU Weihua, PANG Yunwei, ZHAO Shanjiang, ZOU Huiying, ZHU Huabin, YANG Yuze, ZHAO Xueming. Research Progress of CRISPR/dCas9 Technology in Gene Expression Regulation [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 53-59.
[10]	YANG Mingzhu, CHEN Xiaochun, SONG Jiacheng, HOU Yaxin, LI Junping. Research Progress on Construction and Application of Recombinant Animal Herpesvirus Live Vector Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 338-351.
[11]	WANG Huan, ZHU Huabin, LI Junliang, ZOU Huiying, ZHAO Shanjiang. Targeted Editing of Hornless Pc Site in Holstein Bulls Using Tild-CRISPR/Cas9 [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3343-3353.
[12]	QU Zhehui, ZHANG Xiwen, ZHAO Yu, ZHENG Quanfang, LIAN Huixiang, ZHAO Yunhuan, GUO Xiaoqiu. Genetic Variation Analysis of Major Virulence Genes of Pseudorabies Virus Epidemic Strains in Southern Henan Province [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 3019-3029.
[13]	XU Changjiang, WANG Xiaopeng, XU Kui, ZHANG Xiuling, XIANG Guangming, ZHAO Haiquan, MU Yulian, LIN Xiao, LI Kui. Establishment of pAPN Gene Knockout IPI-2I Cell Lines Mediated by CRISPR/Cas9 System [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2282-2290.
[14]	CHEN Fuguang, LU Tongyan, LI Shaowu. Bioinformatic Analysis of CRISPR/Cas System in Flavobacterium psychrophilum [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1927-1938.
[15]	MA Fangxue, AN Yanan, WANG Chao, LI Yuan, LUAN Wenjing, WANG Xuefei, NI Lihui, ZHOU Hong, LIANG Junchao, YU Lu. Construction of FXR Deletion Strain Based on CRISPR/Cas9 Technology and Its Effect on HeLa Cell Proliferation [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 450-456.