鸭抗病毒蛋白(viperin)在转染的BHK-21细胞表达并抑制鸭坦布苏病毒出芽
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摘要
目的探索鸭抗病毒白(viperin)对鸭坦布苏病毒(DTMUV)增殖的影响。方法使用生物学信息学方法分析鸭viperin基因,构建系统发育树。构建原核表达质粒pGEX-6P-viperin和真核表达质粒pEGFP-N1-viperin,分别转化Rosseta感受态细胞和转染BHK-21细胞。用DTMUV分别感染真核表达质粒和空载体质粒的BHK-21细胞,利用实时荧光定量PCR检测细胞沉淀和上清中DTMUV的含量,利用质谱的方法鉴定增强型绿色荧光蛋白(EGFP)单克隆抗体捕获的结合蛋白。结果鸭viperin基因与其他种属viperin基因有显著差异。鸭viperin蛋白能成功在BHK-21细胞表达,鸭viperin抑制DTMUV在BHK-21细胞中的出芽。质谱分析结果显示转染真核表达质粒的细胞中可能存在6种影响抑制DTMUV增殖、出芽的蛋白。结论鸭viperin蛋白可在BHK-21细胞表达,且能抑制鸭坦布苏病毒出芽。
Objective To measure the effect of duck viperin protein on the proliferation of duck Tembusu virus(DTMUV). Methods We analyzed the duck viperin gene using a bioinformatics. Plasmids pGEX-6P-viperin and pEGFP-N1-viperin were constructed and transformed into Rosseta and transfected into BHK-21 cells, respectively. BHK-21 cells transfected with plasmid pEGFP-N1-viperin and empty vector were infected with DTMUV. The content of DTMUV in cell precipitation and supernatant was analyzed by real-time fluorescent quantitative PCR. Finally, the effect of viperin on DTMUV proliferation and the binding proteins captured by EGFP monoclonal antibody were evaluated using mass spectrometry. Results Significant differences were observed between the expression levels of viperin gene in duck compared to other species. Duck viperin was found to inhibit the budding of DTMUV in BHK-21 cells. Further, we identified six proteins that might be involved in the inhibition of the proliferation and budding of DTMUV, possibly indicative of the viperin anti-Tembusu virus pathway in ducks. Conclusion Expression patterns of duck viperin reveal how the budding of duck Tembusu virus is inhibited.
Objective To measure the effect of duck viperin protein on the proliferation of duck Tembusu virus(DTMUV). Methods We analyzed the duck viperin gene using a bioinformatics. Plasmids pGEX-6P-viperin and pEGFP-N1-viperin were constructed and transformed into Rosseta and transfected into BHK-21 cells, respectively. BHK-21 cells transfected with plasmid pEGFP-N1-viperin and empty vector were infected with DTMUV. The content of DTMUV in cell precipitation and supernatant was analyzed by real-time fluorescent quantitative PCR. Finally, the effect of viperin on DTMUV proliferation and the binding proteins captured by EGFP monoclonal antibody were evaluated using mass spectrometry. Results Significant differences were observed between the expression levels of viperin gene in duck compared to other species. Duck viperin was found to inhibit the budding of DTMUV in BHK-21 cells. Further, we identified six proteins that might be involved in the inhibition of the proliferation and budding of DTMUV, possibly indicative of the viperin anti-Tembusu virus pathway in ducks. Conclusion Expression patterns of duck viperin reveal how the budding of duck Tembusu virus is inhibited.
引文
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[15] 唐海波.Viperin抑制狂犬病病毒机理的探索研究[D].广西大学,2015.Tang H.Study on the mechanism of viperin inhibits rabies virus[D].Guangxi University,2015.
[16] 钟自富.鸭viperin蛋白抗新城疫病毒机制的初步研究[D].中国农业科学院,2015.Zhong Z.Preliminary study on antiviral activity of duck viperin protein against newcastle disease virus[D].Chinese Academy of Agricultural Sciences,2015.
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[18] Shah M,Bharadwaj M S K,Gupta A,et al.Chicken viperin inhibits Newcastle disease virus infection in vitro:A possible interaction with the viral matrix protein[J/OL].Cytokine,2019,120:28.40.DOI:10.1016/j.cyto.2019.04.007.
[19] 王楠楠,刘芳,许漩,等.坦布苏病毒荧光定量RT-PCR检测方法的建立及初步应用[J].中国兽医科学,2015,45(1):15-19.Wang N,Liu F,Xu X,et al.Development and application of SYBR GreenⅠfluorescence quantitative RT-PCR for detection of Tembusu virus[J].Zhongguo Shou Yi Ke Xue,2015,45(1):15-19.
[20] Helbig K J,Beard M R.The role of viperin in the innate antiviral response[J].J Mol Biol,2014,426(6):1210-1219.
[21] Lindqvist R,?verby A K.The Role of viperin in antiflavivirus responses[J].DNA Cell Biol,2018,37(9):725-730.
[22] Wang H J,Li X F,Liu L,et al.The emerging duck flavivirus is not pathogenic for primates and is highly sensitive to mammalian interferon antiviral signaling[J].J Virol,2016,90(14):6538-6548.
[23] Panayiotou C,Lindqvist R,Kurhade C,et al.Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation[J/OL].J Virol,2018,92(7):e02054-17.DOI:10.1128/JVI.02054-17.Print 2018 Apr 1.
[24] Nasr N,Maddocks S,Turville S G,et al.HIV-1 infection of human macrophages directly induces viperin which inhibits viralproduction[J].Blood,2012,778-788.
[25] Wang Z,Yu X,Niu X,et al.Screening of the proteins interacting with NS1 of TMUV by yeast two-hybrid system and the identification of the function of the interacted protein[J].Infect Genet Evol,2018,63:277-284.
[26] Helbig K J,Eyre N S,Yip E,et al.The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A[J].Hepatology,2011,54(5):1506-1517.
[27] Sun X,Wang S,Lin X,et al.Proteome analysis of duck Tembusu virus (DTMUV)-infected BHK-21 cells[J/OL].Proteomics,2017,17(12):1700033.DOI:10.1002/pmic.201700033.
[28] Li G,Feng T,Pan W,et al.DEAD-box RNA helicase DDX3X inhibits DENV replication via regulating type one interferon pathway[J].Biochem Biophys Res Commun,2015,456(1):327-332.
[2] Lindqvist R,?verby A K.The Role of viperin in antiflavivirus responses[J].DNA Cell Biol,2018,37(9):725-730.
[3] Van der Hoek K H,Eyre N S,Shue B,et al.viperin is an important host restriction factor in control of Zika virus infection[J/OL].Sci Rep,2017,7(1):4475.DOI:10.1038/s41598-017- 04138-1.
[4] 陈洁滢,孙亚楠,邵文涵,等.番鸭viperin基因的克隆表达及其抗MDRV的作用研究[J].中国兽医杂志,2018,54(4):14-17.Chen J,Sun Y,Shao W,et al.Cloning and expression of viperin gene in Muscovy duck and its antiviral effect against MDRV[J].Zhongguo Shou Yi Za Zhi,2018,54(4):14-17.
[5] Khanlari Z,Sabahi F,Hosseini S Y,et al.HCV NS3 blocking effect on IFN induced ISGs like viperin and IL28 with and without NS4A[J/OL].Hepat Mon,2014,14(6):e17822.DOI:10.5812/ hepatmon.17822.eCollection 2014 Jun.
[6] Saitoh T,Satoh T,Yamamoto N,et al.Antiviral protein viperin promotes Toll-like receptor 7- and Toll-like receptor 9-mediated typeⅠinterferon production in plasmacytoid dendritic cells[J].Immunity,2011,34(3):352-363.
[7] Seo J Y,Yaneva R,Cresswell P.Viperin:a multifunctional,interferon-inducible protein that regulates virus replication[J].Cell Host Microbe,2011,10(6):534-539.
[8] Ng L F P,Hiscox J A.Viperin poisons viral replication.Cell host microbe[J],2018,24(2):181-183.
[9] Dumbrepatil A B,Ghosh S,Zegalia K A,et al.Viperin interacts with the kinase IRAK1 and the E3 ubiquitin ligase TRAF6,coupling innate immune signaling to antiviral ribonucletide synthesis[J].J Biol Chem,2019,294(17):6888-6898.
[10] Shen G,Wang K,Wang S,et al.Herpes simplex virus 1 counteracts viperin via its virion host shutoff protein UL41[J].J Virol,2014,88(20):12163-12166.
[11] Jumat M R,Huong T N,Ravi L I,et al.viperin protein expression inhibits the late stage of respiratory syncytial virus morphogenesis[J].Antiviral Res,2015,114:11-20.
[12] McGillivary G,Jordan Z B,Peeples M E,et al.Replication of respiratory syncytial virus is inhibited by the host defense molecule viperin[J].J Innate Immun,2013,5(1):60-71.
[13] Vanwalscappel B,Tada T,Landau N R.Toll-like receptor agonist R848 blocks Zika virus replication by inducing the antiviral protein viperin[J].Virology,2018,522:199-208.
[14] Lindqvist R,Upadhyay A,?verby A K.Tick-borne flaviviruses and the type Ⅰ interferon response[J/OL].Viruses,2018,10(7):E340.DOI:10.3390/v10070340.
[15] 唐海波.Viperin抑制狂犬病病毒机理的探索研究[D].广西大学,2015.Tang H.Study on the mechanism of viperin inhibits rabies virus[D].Guangxi University,2015.
[16] 钟自富.鸭viperin蛋白抗新城疫病毒机制的初步研究[D].中国农业科学院,2015.Zhong Z.Preliminary study on antiviral activity of duck viperin protein against newcastle disease virus[D].Chinese Academy of Agricultural Sciences,2015.
[17] Helbig K J,Carr J M,Calvert J K,et al.Viperin is induced following Dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin[J/OL].PLoS Negl Trop Dis,2013,7(4):e2178.DOI:10.1371/journal.pntd.0002178.Print 2013.
[18] Shah M,Bharadwaj M S K,Gupta A,et al.Chicken viperin inhibits Newcastle disease virus infection in vitro:A possible interaction with the viral matrix protein[J/OL].Cytokine,2019,120:28.40.DOI:10.1016/j.cyto.2019.04.007.
[19] 王楠楠,刘芳,许漩,等.坦布苏病毒荧光定量RT-PCR检测方法的建立及初步应用[J].中国兽医科学,2015,45(1):15-19.Wang N,Liu F,Xu X,et al.Development and application of SYBR GreenⅠfluorescence quantitative RT-PCR for detection of Tembusu virus[J].Zhongguo Shou Yi Ke Xue,2015,45(1):15-19.
[20] Helbig K J,Beard M R.The role of viperin in the innate antiviral response[J].J Mol Biol,2014,426(6):1210-1219.
[21] Lindqvist R,?verby A K.The Role of viperin in antiflavivirus responses[J].DNA Cell Biol,2018,37(9):725-730.
[22] Wang H J,Li X F,Liu L,et al.The emerging duck flavivirus is not pathogenic for primates and is highly sensitive to mammalian interferon antiviral signaling[J].J Virol,2016,90(14):6538-6548.
[23] Panayiotou C,Lindqvist R,Kurhade C,et al.Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation[J/OL].J Virol,2018,92(7):e02054-17.DOI:10.1128/JVI.02054-17.Print 2018 Apr 1.
[24] Nasr N,Maddocks S,Turville S G,et al.HIV-1 infection of human macrophages directly induces viperin which inhibits viralproduction[J].Blood,2012,778-788.
[25] Wang Z,Yu X,Niu X,et al.Screening of the proteins interacting with NS1 of TMUV by yeast two-hybrid system and the identification of the function of the interacted protein[J].Infect Genet Evol,2018,63:277-284.
[26] Helbig K J,Eyre N S,Yip E,et al.The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A[J].Hepatology,2011,54(5):1506-1517.
[27] Sun X,Wang S,Lin X,et al.Proteome analysis of duck Tembusu virus (DTMUV)-infected BHK-21 cells[J/OL].Proteomics,2017,17(12):1700033.DOI:10.1002/pmic.201700033.
[28] Li G,Feng T,Pan W,et al.DEAD-box RNA helicase DDX3X inhibits DENV replication via regulating type one interferon pathway[J].Biochem Biophys Res Commun,2015,456(1):327-332.