可调控表达人博卡病毒1型非结构蛋白NS1稳定细胞系的建立及其反式转录激活作用初探
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摘要
人博卡病毒1型(Human bocavirus 1,HBoV1)非结构蛋白NS1是多功能蛋白,对病毒复制有重要作用,同时可诱导宿主细胞凋亡。在研究NS1蛋白功能时,降低NS1蛋白对宿主细胞的毒性作用是急需解决的问题。基于此,文中建立了可调控表达HBoV1非结构蛋白NS1的稳定细胞系。构建NS1重组慢病毒质粒(含可调控启动子),应用转染试剂将NS1重组慢病毒质粒转染至HEK293T细胞。通过嘌呤霉素筛选抗性细胞、多西环素诱导NS1表达,建立可稳定表达NS1-100、NS1-70蛋白的HEK 293T细胞系,利用荧光标记蛋白和Western blotting检测,确定NS1蛋白的表达。并在稳定表达NS1细胞系中转染HBoV1启动子-荧光素酶基因的质粒,分析NS1的反式转录激活活性。结果表明NS1蛋白可在建立的细胞系中稳定表达,且稳定表达NS1蛋白对HBoV1启动子有较强的激活活性,为进一步研究非结构蛋白NS1的功能及人博卡病毒致病机理奠定了良好的基础。
Human bocavirus 1(HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293 T cells using transfection reagent. The HEK 293 T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline.The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.
Human bocavirus 1(HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293 T cells using transfection reagent. The HEK 293 T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline.The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.
引文
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[2]Zhou JY,Peng Y,Peng XY,et al.Human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in Changsha,China.Infl Other Respir Viruses,2018,12(2):279-286.
[3]Silva PE,Figueiredo CA,Luchs A,et al.Human bocavirus in hospitalized children under 5 years with acute respiratory infection,Sao Paulo,Brazil,2010.Arch Virol,2018,163(5):1325-1330.
[4]Salvo M,Lizasoain A,Castells M,et al.Human bocavirus:detection,quantification and molecular characterization in sewage and surface waters in uruguay.Food Environ Virol,2018,10(2):193-200.
[5]Chen AX,Xie GC,Pan D,et al.Three-dimensional culture of human airway epithelium in matrigel for evaluation of human rhinovirus C and bocavirus infections.Biomed Environ Sci,2018,31(2):136-145.
[6]Abdel-Moneim AS,Mahfouz ME,Zytouni DM.Detection of human bocavirus in Saudi healthy blood donors.PLoS ONE,2018,13(2):e0193594.
[7]Cotmore SF,Agbandje-McKenna M,Chiorini JA,et al.The family Parvoviridae.Arch Virol,2014,159(5):1239-1247.
[8]Shen WR,Deng XF,Zou W,et al.Analysis of cis and trans requirements for DNA replication at the right-end hairpin of the human bocavirus 1 genome.JVirol,2016,90(17):7761-7777.
[9]Liu QS,Zhang ZF,Zheng ZH,et al.Human bocavirus NS1 and NS1-70 proteins inhibit TNF-α-mediated activation of NF-κB by targeting p65.Sci Rep,2016,6:28481.
[10]Li JJ,Yang YB,Dong YM,et al.Key elements of the human bocavirus type 1(HBoV1)promoter and its trans-activation by NS1 protein.Virol J,2013,10:315.
[11]Morita E,Nakashima A,Asao H,et al.Human parvovirus B19 nonstructural protein(NS1)induces cell cycle arrest at G1 phase.J Virol,2003,77(5):2915-2921.
[12]Hsu TC,Wu WJ,Chen MC,et al.Human parvovirus B19 non-structural protein(NS1)induces apoptosis through mitochondria cell death pathway in COS-7cells.Scand J Infect Dis,2004,36(8):570-577.
[13]Tzang BS,Chen DY,Tsai CC,et al.Human parvovirus B19 nonstructural protein NS1 enhanced the expression of cleavage of 70 kDa U1-snRNPautoantigen.J Biomed Sci,2010,17:40.
[14]Sanchez JL,Romero Z,Quinones A,et al.Dna binding and cleavage by the human parvovirus B19NS1 nuclease domain.Biochemistry,2016,55(47):6577-6593.
[15]Willwand K,Moroianu A,H?rlein R,et al.Specific interaction of the nonstructural protein NS1 of minute virus of mice(MVM)with[ACCA]2 motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.J Gen Virol,2002,83:1659-1664.
[16]Sheu MJ,Chiu CC,Yang DJ,et al.The root extract of Gentiana macrophylla pall.Alleviates B19-NS1-exacerbated liver injuries in NZB/W F1mice.J Med Food,2017,20(1):56-64.
[17]Wu JQ,Chen X,Ye HY,et al.Nonstructural protein(NS1)of human parvovirus B19 stimulates host innate immunity and blunts the exogenous type Iinterferon signaling in vitro.Virus Res,2016,222:48-52.
[18]Chen DY,Chen YM,Chen HH,et al.Human parvovirus B19 nonstructural protein NS1 activates NLRP3 inflammasome signaling in adultonset Still’s disease.Mol Med Rep,2018,17(2):3364-3371.
[19]Daeffler L,H?rlein R,Rommelaere J,et al.Modulation of minute virus of mice cytotoxic activities through site-directed mutagenesis within the NS coding region.J Virol,2003,77(23):12466-12478.
[20]Xu P,Zhou Z,Xiong M,et al.Parvovirus B19 NS1protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.PLoSPathog,2017,13(3):e1006266.
[21]Yang B,Cai DW,Yu PR,et al.Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9cell cultures.Virus Genes,2009,38(3):478-486.
[22]Yan ZY,Keiser NW,Song Y,et al.A novel chimeric adenoassociated virus 2/human bocavirus 1parvovirus vector efficiently transduces human airway epithelia.Mol Ther,2013,21(12):2181-2194.
[23]Wang ZK,Deng XF,Zou W,et al.Human bocavirus1 is a novel helper for adeno-associated virus replication.J Virol,2017,91(18):e00710-17.