DDX1影响猪流行性腹泻病毒复制的机制研究

doi:10.16431/j.cnki.1671-7236.2023.04.027

Abstract

Abstract: 【Objective】 The purpose of this study was to explore the function of RNA helicase DEAD-box family protein DDX1 in porcine ileal epithelial cells (IPI-2I) infected by Porcine epidemic diarrhea virus (PEDV),and lay a foundation for revealing the biological function of DDX1.【Method】 According to the sequence of porcine DDX1 gene in GenBank (accession No.XM_021087822.1),specific primers were designed.The full length of DDX1 gene was amplified by PCR and ligated into pMD19-T cloning vector.After successful identification by double enzyme digestion,the DDX1 gene was ligated into pcDNA3.1 expression vector.After the transformation of the product,the positive colonies were screened.IPI-2I cells were cultured,and the cells that had been passaged twice were spread in a six-hole plate for follow-up experiment.The recombinant plasmids of the positive colonies were transfected into the six-well plate for 24 h.The protein was collected,and the transfection efficiency of DDX1 was detected by Western blotting.IPI-2I cells transfected with pcDNA3.1-DDX1 or PBS (control) were infected with PEDV with a multiple of infection (MOI) of 1.0 to establish a cell model of PEDV infection.Total protein and RNA were collected 12 h after PEDV infection.The expression of PEDV N protein at cell level was detected by indirect immunofluorescence assay,the expression of PEDV N gene mRNA was detected by Real-time quantitative PCR,and the expression of PEDV N protein was detected by Western blotting.Bioinformatics software was used to predict the phosphorylation site of DDX1 peotein,and immunoprecipitation test was used to detect the phosphorylation level of DDX1 after PEDV infection in IPI-2I cells for 12 h.【Result】 The target DDX1 gene band of 2 223 bp was successfully obtained by PCR amplification.The results of double digestion identification showed that pMD19-T-DDX1 cloning vector and pcDNA3.1-DDX1 eukaryotic expression vector were successfully constructed.The expression of DDX1 protein in the cells transfected with pcDNA3.1-DDX1 was extremely significantly higher than that in PBS group (P＜0.01),indicating that DDX1 transfection was successful.The results of indirect immunofluorescence assay showed that compared with PBS group,there were a large number of green fluorescence signals in PEDV infection group,indicating that the IPI-2I cell model infected by PEDV was successfully constructed.In IPI-2I cells transfected with pcDNA3.1-DDX1 recombinant plasmid,compared with PBS group,the fluorescence intensity of PEDV N protein significantly decreased,the mRNA and protein expression of PEDV N gene were extremely significantly decreased (P＜0.01).Bioinformatics analysis showed that DDX1 protein had multiple phosphorylation sites,including 17 serine,12 threonine and 6 tyrosine.The results of immunoprecipitation test showed that the level of DDX1 phosphorylation in PEDV infected group was extremely significantly higher than that in uninfected group (P＜0.01).【Conclusion】 In this study,the eukaryotic expression vector of pcDNA3.1-DDX1 was successfully constructed.It was found that 12 h after PEDV infection,DDX1 inhibited the replication of PEDV and increased the phosphorylation level,indicating that DDX1 protein might inhibit the replication of PEDV through phosphorylation during PEDV infection.

Key words: DDX1; Porcine epidemic diarrhea virus (PEDV); phosphorylation

CLC Number:

S852.65^＋1

HUO Mingkai, GUAN Feihu, QIU Runhui, WEI Chunyan, YU Haitao, ZHU Jiale, ZHANG Hui. Study on the Mechanism of DDX1 Inhibiting Porcine Epidemic Diarrhea Virus Replication[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1556-1566.

References

[1] JUNG K,SAIF L J.Porcine epidemic diarrhea virus infection:Etiology,epidemiology,pathogenesis and immunoprophylaxis[J].Veterinary Journal,2015,204(2):134-143.
[2] 刘政龙,王金良,沈志强.猪流行性腹泻病毒研究进展[J].中国畜牧兽医,2015,42(9):2506-2511. LIU Z L,WANG J L,SHEN Z Q.Research progress on Porcine epidemic diarrhea virus[J].China Animal Husbandry & Veterinary Medicine,2015,42(9):2506-2511.(in Chinese)
[3] CAO L,GE X,GAO Y,et al.Porcine epidemic diarrhea virus inhibits dsRNA-induced interferon-β production in porcine intestinal epithelial cells by blockade of the RIG-Ⅰ-mediated pathway[J].Virology Journal,2015,12:127.
[4] LOO Y M,GALE M JR.Immune signaling by RIG-Ⅰ-like receptors[J].Immunity,2011,34(5):680-692.
[5] 郭存财,刘炎,黄耀伟.猪流行性腹泻病毒与宿主抗病毒天然免疫抑制[J].中国生物化学与分子生物学报,2016,32(9):967-975. GUO C C,LIU Y,HUANG Y W.Inhibitory mechanism of host antiviral innate immunity by Porcine epidemic diarrhea virus[J].Chinese Journal of Biochemistry and Molecular,2016,32(9):967-975.(in Chinese)
[6] 王新.DDX3调控NF-κB信号通路的机制研究[D].南京:南京农业大学,2017. WANG X.The mechanism studies of DDX3 in regulating NF-κB signaling pathway[D].Nanjing:Nanjing Agricultural University,2017.(in Chinese)
[7] ROACK S,LINDER P.DEAD-box proteins:The driving forces behind RNA metabolism[J].Nature Reviews, Molecular Cell Biology,2004,5(3):232-241.
[8] GODBOUT R,PACKER M,BIE W.Overexpression of a DEAD box protein (DDX1) in neuroblastoma and retinoblastoma cell lines[J].The Journal of Biological Chemistry,1998,273(33):21161-21168.
[9] HAN C,LIU Y,WAN G,et al.The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression[J].Cell Reports,2014,8(5):1447-1460.
[10] HE L,CHEN Y,WU Y,et al.Nucleic acid sensing pattern recognition receptors in the development of colorectal cancer and colitis[J].Cellular and Molecular Life Sciences,2017,74(13):2395-2411.
[11] 张华,韩超峰,曹雪涛.天然免疫受体RIG-Ⅰ的翻译后修饰及其功能调控[J].中国免疫学杂志,2015,31(7):1002-1007. ZHANG H,HAN C F,CAO X T.Post-translational modification and functional regulation of innate immune receptor RIG-Ⅰ[J].Chinese Journal of Immunology,2015,31(7):1002-1007.(in Chinese)
[12] KUMAR R,MEHTA D,MISHRA N,et al.Role of host-mediated post-translational modifications (PTMs) in RNA virus pathogenesis[J].International Journal of Molecular Sciences,2021,22(1):323.
[13] 梁前进,王鹏程,白燕荣.蛋白质磷酸化修饰研究进展[J].科技导报,2012,30(31):73-79. LIANG Q J,WANG P C,BAI Y R.Summarization on the progress in protein phosphorylation[J].Science & Technology Review,2012,30(31):73-79.(in Chinese)
[14] HUNTER T.Signaling——2000 and beyond[J].Cell,2000,100(1):113-127.
[15] 陆颖,速雪丽,杜琛,等.猪流行性腹泻病毒反向遗传学及其研究进展[J].中国畜牧兽医,2020,47(6):1694-1701. LU Y,SU X L,DU C,et al.Reverse genetics and its research progress of Porcine epidemic diarrhea virus[J].China Animal Husbandry & Veterinary Medicine,2020,47(6):1694-1701.(in Chinese)
[16] 许淑娟,谢晶莹,冯若飞.DEAD-box RNA解旋酶家族在天然免疫应答信号通路中的作用[J].微生物学报,2021,61(3):587-595. XU S J,XIE J Y,FENG R F.DEAD-box RNA helicase family in innate immune response signaling pathway[J].Acta Microbiologica Sinica,2021,61(3):587-595.(in Chinese)
[17] HAMMOND J A,LAMICHHANEA R,MILLAR D P,et al.A DEAD-box helicase mediates an RNA structural transition in the HIV-1 Rev response element[J].Journal of Molecular Biology.2017,429(5):697-714.
[18] XUE Q,LIU H,ZENG Q,et,al.The DEAD-Box RNA helicase DDX1 interacts with the viral protein 3D and inhibits Foot-and-mouth disease virus replication[J].Virologica Sinica,2019,34(6):610-617.
[19] ZHOU Y,WU W,XIE L,et al.Cellular RNA helicase DDX1 is involved in Transmissible gastroenteritis virus nsp14-induced interferon-beta production[J].Frontiers in Immunology,2017,8(9):940-952.
[20] LIN Z,WANG J,ZHU W,et al.Chicken DDX1 acts as an RNA sensor to mediate IFN-β signaling pathway activation in antiviral innate immunity[J].Frontiers in Immunology,2021,12(6):742-754.
[21] ZHU L,GAO T,HUANG Y,et al.Ebola virus VP35 hijacks the PKA-CREB1 pathway for replication and pathogenesis by AKIP1 association[J].Nature Communications,2022,13(1):2256.
[22] LANG L,SHAY C,ZHAO X,et al.Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics[J].Journal of Experimental & Clinical Cancer Research,2017,36(1):112-121.
[23] BETANCOR G,JIMENEZ-GUARDENO J M,LYNHAM S,et,al.MX2-mediated innate immunity against HIV-1 is regulated by serine phosphorylation[J].Nature Microbiology,2021,6(8):1031-1042.

Study on the Mechanism of DDX1 Inhibiting Porcine Epidemic Diarrhea Virus Replication

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[2]	SONG Shiying, GUO Weilu, XIA Xuefeng, ZHANG Xue, BI Zhenwei, ZHANG Xuehan, FAN Baochao, DONG Hailong, LI Bin. Expression and Purification of Porcine IFN-δ5 and Its Effect Analysis Against PEDV Infection [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3171-3179.
[3]	LIU Yutong, YAO Yanting, LI Ruiling, HAN Wenqi, SHI Ting, WANG Yidan, REN Yupeng. Analysis of Genetic Variation of Porcine Epidemic Diarrhea Virus in Parts of Southwest China from 2020 to 2021 [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2279-2290.
[4]	ZHANG Yanwei, CAI Chunbo, SUN Di, AN Jiaqi, HUANG Xiaoyu, NIU Jin, CUI Zixu, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Study on Expression Characteristics of Myofiber Types and Energy Metabolism Related Genes in Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1223-1232.
[5]	ZHANG Mingliang, GENG Yachun, LIAN Kaiqi, MA Lei, CHANG Ying, WANG Shuangshan, ZHANG Fuliang. Cloning,Bioinformatics Analysis and Truncated Fragment Expression for Truncated M Gene of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1479-1487.
[6]	ZHANG Tianai, LI Tingting, TAO Xiaoli, LI Tengfei, LIN Jiafeng, HU Siman, LIU Wenxiu, TONG Wei, LI Yonggang. Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation and Identification of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4383-4391.
[7]	BAI Weiqin, KA Chula, WU Zhiyong, MIAO Miao, TA Na, GE Riletu. Expression and Identification of B-cell Epitope Embedded S-layer Protein of Porcine Epidemic Diarrhea Virus S2 Gene in Lactobacillus paracasei [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4004-4011.
[8]	LI Jiesen, SUN Ronghang, KUANG Yanqi, CHEN Luman, LIU Qing, GUO Xiaofeng. Optimal Expression of Porcine Epidemic Diarrhea Virus S1D Protein and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4641-4651.
[9]	WANG Zhiping, ZHANG Yunjing, SUN Yujie, XU Xin, WANG Zhiyan, HUANG Baicheng, TIAN Kegong. Development and Evaluation of an Indirect ELISA for Detection of Porcine Epidemic Diarrhea Virus IgA in Pig Serum and Milk Captured by Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2248-2255.
[10]	LU Ying, SU Xueli, DU Chen, WANG Ruomu, ZHONG Lian, CHEN Ying, WEI Zuzhang, HUANG Weijian, OUYANG Kang. Reverse Genetics and Its Research Progress of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1694-1701.
[11]	SHEN Xuehuai, ZHAI Yinjian, YIN Lei, PAN Xiaocheng, ZHANG Danjun, ZHAO Ruihong, DAI Yin, HU Xiaomiao, ZHOU Xueli, HOU Hongyan. Culture of Piglet Intestinal Epithelial Cells in vitro and the Infectivity of Porcine Epidemic Diarrhea Virus on the Cells [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 4051-4058.
[12]	XIANG Meng, ZHANG Huanrong, REN Yupeng, ZHANG Keke. Sequencing and Genetic Variation Analysis of the Whole Gene of Epidemic Strain of PEDV in Pengzhou,Sichuan Province [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 209-220.
[13]	XU Bingbing, ZHANG Jiaxin, WANG Ruijun, WANG Zhiying, LI Xuewu, SONG Guoxin, ZHANG Meina, GUO Fuqiang, ZHANG Junjie, ZHANG Yanjun, SU Rui, LIU Zhihong, LI Jinquan. Research Progress on Mammalian Sperm Mitochondria [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2651-2657.
[14]	JIANG Xinhua, ZHOU Hetian, LUO Feng, YANG Danfeng, ZHANG Zhiqing, CHEN Songchang, DENG Shunzhou. Molecular Epidemiological Investigation of Porcine Epidemic Diarrhea Virus in Jiangxi Province in 2017 [J]. , 2019, 46(5): 1474-1482.
[15]	HE Haijian, WU Yuan, WANG Luyan, LI Qunjin, BA Shaobo, SHI Lin, SHAO Chunyan, SUN Jing, JIANG Sheng, WANG Xiaodu. Eukaryotic Expression of Recombinant Baculovirus of PEDV Isolated Strain S1 Gene [J]. , 2019, 46(3): 832-839.