PK-15细胞敲除TANK结合激酶1基因促进猪伪狂犬病病毒复制的研究
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摘要
TANK结合激酶1(TBK1)是病毒感染时IRF3、IRF7磷酸化及Ⅰ型干扰素表达的关键酶,在抗病毒天然免疫应答和获得性免疫应答中发挥重要作用。为研究TBK1基因敲除对猪伪狂犬病病毒(PRV)复制的影响,首先利用CRISPR/Cas9技术构建猪TBK1基因稳定敲除PK-15细胞系,并利用CCK-8检测敲除TBK1对PK-15细胞活力的影响。然后利用流式细胞术检测PRV-GFP荧光强度,用RT-qPCR检测PRV-gB、PRV-gE、PRV-TK、IL-1β、IFN-β和ISG15的转录水平,用Western blot检测PRV-gB和PRV-gE蛋白表达水平,以及通过滴度测定检测子代病毒的感染力,综合评价TBK1基因稳定敲除PK-15细胞对PRV病毒复制的影响。结果显示:T7E1检测结果显示TBK1基因外显子2区的3个sgRNA靶位点均切出了目的条带,选取编辑效率最高的TBK1-sgRNA1细胞系进行单克隆化培养,获取6株稳定敲除TBK1基因的单克隆细胞,随机选取4号细胞株进行CCK-8检测,结果显示敲除TBK1对PK-15细胞活力无影响。流式检测结果显示PRV-GFP感染阳性细胞占总PK-15细胞的56.89%,PRV-GFP感染阳性细胞占总PK-15-TBK1~(-/-)细胞的77.95%,表明PK-15-TBK1~(-/-)细胞可以促进PRV-GFP复制。RT-qPCR及WB检测显示该细胞系可以促进PRV mRNA转录和蛋白表达。滴度测定结果显示,PRV-QXX在PK-15细胞复制后TCID_(50)为10~(6.8) TCID_(50)·0.1 mL~(-1),而在PK-15-TBK1~(-/-)细胞中复制后TCID_(50)为10~(8.5) TCID_(50)·0.1 mL~(-1)。另外,RT-qPCR结果显示该细胞系可以抑制PRV感染引起的IL-1β、IFN-β和ISG15转录上调。以上结果表明TBK1基因敲除PK-15细胞系促进PRV复制,可能与IL-1β、IFN-β和ISG15转录水平抑制有关。
TANK-binding kinase 1(TBK1) is a key enzyme responsible for IRF3, IRF7 phos-phorylation and type Ⅰ interferons expression during viral infection. Furthermore, TBK1 is an important element in antiviral natural and acquired immune response. To study the effect of TBK1 gene knockout on porcine pseudorabies virus(PRV) replication, in this study, TBK1 knockout PK-15 cell line was constructed by CRISPR/Cas9 technology. Next, the cell viability of PK-15-TBK1~(-/-) cells was monitored by CCK-8 assay. Then, the following indexes were used to comprehensively evaluate the effect of TBK1 gene knockout on PRV replication: fluorescence intensity of PRV-GFP was assayed by flow cytometry; mRNA levels of PRV-gB, PRV-gE, PRV-TK, IL-1β, IFN-β and ISG15 were measured by RT-qPCR; protein expression levels of PRV-gB and PRV-gE were evaluated by Western Blot; infectivity of progeny virus was determined by titer determination. Results were as follows: Firstly, T7 E1 assay results showed that target bands were identified from 3 sgRNA target sites in exon 2 regions of TBK1 gene. TBK1-sgRNA1 cells with highest editing efficiency were selected with limiting dilution method for monoclonal cultivation by inoculating into a 96-pore plate. Then, No.4 cell strain from 6 independent TBK1 stable knockout monoclonal cells was chosen for CCK-8 assay. The results showed that knockout of TBK1 gene had no effect on cell viability. In addition, flow cytometry results showed that positive cells infected with PRV-GFP account for 56.89% of the total PK-15 cells, and those were 77.95% of the total PK-15-TBK1~(-/-) cells, indicating that PK-15-TBK1~(-/-) cells could enhance PRV replication. Moreover, RT-qPCR and WB results showed that PK-15-TBK1~(-/-) cells could up-regulate PRV mRNA transcription and protein translation. Titer determination showed that the TCID_(50) of new progeny virions in PK-15 cells and PK-15-TBK1~(-/-) cells were 10~(6.8) TCID_(50)·0.1 mL~(-1) and 10~(8.5) TCID_(50)·0.1 mL~(-1), respectively. Besides, RT-qPCR results showed that up-regulation transcription of IL-1β, IFN-β and ISG15 induced by PRV infection were resisted in PK-15-TBK1~(-/-) cells. In conclusion, the above results indicate that knockout of TBK1 gene promotes PRV replication in PK-15 cells, which might be linked to the inhibition of transcription of IL-1β, IFN-β and ISG15.
TANK-binding kinase 1(TBK1) is a key enzyme responsible for IRF3, IRF7 phos-phorylation and type Ⅰ interferons expression during viral infection. Furthermore, TBK1 is an important element in antiviral natural and acquired immune response. To study the effect of TBK1 gene knockout on porcine pseudorabies virus(PRV) replication, in this study, TBK1 knockout PK-15 cell line was constructed by CRISPR/Cas9 technology. Next, the cell viability of PK-15-TBK1~(-/-) cells was monitored by CCK-8 assay. Then, the following indexes were used to comprehensively evaluate the effect of TBK1 gene knockout on PRV replication: fluorescence intensity of PRV-GFP was assayed by flow cytometry; mRNA levels of PRV-gB, PRV-gE, PRV-TK, IL-1β, IFN-β and ISG15 were measured by RT-qPCR; protein expression levels of PRV-gB and PRV-gE were evaluated by Western Blot; infectivity of progeny virus was determined by titer determination. Results were as follows: Firstly, T7 E1 assay results showed that target bands were identified from 3 sgRNA target sites in exon 2 regions of TBK1 gene. TBK1-sgRNA1 cells with highest editing efficiency were selected with limiting dilution method for monoclonal cultivation by inoculating into a 96-pore plate. Then, No.4 cell strain from 6 independent TBK1 stable knockout monoclonal cells was chosen for CCK-8 assay. The results showed that knockout of TBK1 gene had no effect on cell viability. In addition, flow cytometry results showed that positive cells infected with PRV-GFP account for 56.89% of the total PK-15 cells, and those were 77.95% of the total PK-15-TBK1~(-/-) cells, indicating that PK-15-TBK1~(-/-) cells could enhance PRV replication. Moreover, RT-qPCR and WB results showed that PK-15-TBK1~(-/-) cells could up-regulate PRV mRNA transcription and protein translation. Titer determination showed that the TCID_(50) of new progeny virions in PK-15 cells and PK-15-TBK1~(-/-) cells were 10~(6.8) TCID_(50)·0.1 mL~(-1) and 10~(8.5) TCID_(50)·0.1 mL~(-1), respectively. Besides, RT-qPCR results showed that up-regulation transcription of IL-1β, IFN-β and ISG15 induced by PRV infection were resisted in PK-15-TBK1~(-/-) cells. In conclusion, the above results indicate that knockout of TBK1 gene promotes PRV replication in PK-15 cells, which might be linked to the inhibition of transcription of IL-1β, IFN-β and ISG15.
引文
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[6] 刘博涛,宋昌军,刘翊中.伪狂犬病毒致病机理的研究进展[J].西北民族大学学报:自然科学版,2008,29(4):65-68.LIU B T,SONG C J,LIU Y Z.Research progress of pathogenesis of pseudorabies virus[J].Journal of Northwest University for Nationalities:Natural Science,2008,29(4):65-68.(in Chinese)
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[12] LIU S Q,CAI X,WU J X,et al.Phosphorylation of innate immune adaptor proteins MAVS,STING,and TRIF induces IRF3 activation[J].Science,2015,347(6227):aaa2630.
[13] DALRYMPLE N A,CIMICA V,MACKOW E R.Dengue virus NS proteins inhibit RIG-I/MAVS signaling by blocking TBK1/IRF3 phosphorylation:dengue virus serotype 1 NS4A is a unique interferon-regulating virulence determinant[J].mBio,2015,6(3):e00553-15.
[14] LI M,XIN T,GAO X T,et al.Foot-and-mouth disease virus non-structural protein 2B negatively regulates the RLR-mediated IFN-β induction[J].Biochem Biophys Res Commun,2018,504(1):238-244.
[15] DING Z,FANG L R,JING H Y,et al.Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1[J].J Virol,2014,88(16):8936-8945.
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[17] XUE Q,LIU H S,ZHU Z X,et al.Seneca valley virus 3C protease negatively regulates the type I interferon pathway by acting as a viral deubiquitinase[J].Antiviral Res,2018,160:183-189.
[18] KANG S M,CHOI C,CHOI I,et al.Hepatitis E virus methyltransferase inhibits type I interferon induction by targeting RIG-I[J].J Microbiol Biotechnol,2018,28(9):1554-1562.
[19] VAN OPDENBOSCH N,DE REGGE N,VAN POUCKE M,et al.Effects of interferon on immediate-early mRNA and protein levels in sensory neuronal cells infected with herpes simplex virus type 1 or pseudorabies virus[J].Vet Microbiol,2011,152(3-4):401-406.
[20] SUN Y,LUO Y Z,WANG C H,et al.Control of swine pseudorabies in China:opportunities and limitations[J].Vet Microbiol,2016,183:119-124.
[21] METTENLEITER T C.Immunobiology of pseudorabies (Aujeszky’s disease)[J].Vet Immunol Immunopathol,1996,54(1-4):221-229.
[22] YIN Y,XU Z W,LIU X W,et al.A live gI/gE-deleted pseudorabies virus (PRV) protects weaned piglets against lethal variant PRV challenge[J].Virus Genes,2017,53(4):565-572.
[23] ENQUIST L W,HUSAK P J,BANFIELD B W,et al.Infection and spread of alphaherpesviruses in the nervous system[J].Adv Virus Res,1998,51:237-347.
[24] FERRARI M,GUALANDI G L,CORRADI A,et al.Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus[J].Comparat Immunol Microbiol Infect Dis,1998,21(4):291-303.
[25] WANG J C,SONG Z C,GE A M,et al.Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK&gE genes[J].BMC Vet Res,2018,14(1):287.
[26] SETAS PONTES M,DEVRIENDT B,FAVOREEL H W.Pseudorabies virus triggers glycoprotein gE-mediated ERK1/2 activation and ERK1/2-dependent migratory behavior in T cells[J].J Virol,2015,89(4):2149-2156.
[27] 刘兴楼,马迪,舒赛男,等.人巨细胞病毒感染THP-1源性巨噬细胞对IL-1β表达的影响[J].国际病毒学杂志,2017,24(3):151-155.LIU X L,MA D,SHU S N,et al.Effects of human cytomegalovirus infection in THP-1 derived macrophages on the expression of IL-1β[J].International Journal of Virology,2017,24(3):151-155.(in Chinese)
[28] 甘霖,赵俊,陈敬贤,等.干扰素抗病毒效力影响因素的研究进展[J].微生物与感染,2018,13(1):43-48.GAN L,ZHAO J,CHEN J X,et al.Research progress on the influencing factors of antiviral efficacy of interferon[J].Journal of Microbes and Infections,2018,13(1):43-48.(in Chinese)
[29] 温正旺,陈洁,金龙腾,等.TLR3-TBK1-IRF-3信号通路在EV71手足口病脑损伤中的机制研究[J].浙江临床医学,2016,18(11):1995-1997.WEN Z W,CHEN J,JIN L T,et al.Study on the mechanism of TLR3-TBK1-IRF-3 signaling pathway in brain injury caused by EV71 hand-foot-and-mouth disease[J].Zhejiang Clinical Medical Journal,2016,18(11):1995-1997.(in Chinese)
[30] LIN H W,CHANG T J,YANG D J,et al.Regulation of virus-induced inflammatory response by β-carotene in RAW264.7 cells[J].Food Chem,2012,134(4):2169-2175.
[31] LIU H M,LI S S,YANG X,et al.Porcine ISG15 modulates the antiviral response during pseudorabies virus replication[J].Gene,2018,679:212-218.
[2] 王雪,张毓川,陈玮.TANK结合激酶1在抗病毒免疫应答中的作用研究进展[J].浙江大学学报:医学版,2016,45(5):550-557.WANG X,ZHANG Y C,CHEN W.Research progress on the role of TANK-binding kinase 1 in anti-virus innate immune response[J].Journal of Zhejiang University:Medical Sciences,2016,45(5):550-557.(in Chinese)
[3] TENOEVER B R,SHARMA S,ZOU W,et al.Activation of TBK1 and IKKε kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity[J].J Virol,2004,78(19):10636-10649.
[4] TAKAOKA A,TANIGUCHI T.Cytosolic DNA recognition for triggering innate immune responses[J].Adv Drug Deliv Rev,2008,60(7):847-857.
[5] 韩斌.TBK-1增强HBcAg核酸疫苗特异性免疫应答的实验研究[D].杭州:浙江大学,2011.HAN B.Experimental study on TBK-1 enhancing specific immune responses to HBcAg nucleic acid vaccine[D].Hangzhou:Zhejiang University,2011.(in Chinese)
[6] 刘博涛,宋昌军,刘翊中.伪狂犬病毒致病机理的研究进展[J].西北民族大学学报:自然科学版,2008,29(4):65-68.LIU B T,SONG C J,LIU Y Z.Research progress of pathogenesis of pseudorabies virus[J].Journal of Northwest University for Nationalities:Natural Science,2008,29(4):65-68.(in Chinese)
[7] BRUKMAN A,ENQUIST L W.Suppression of the interferon-mediated innate immune response by pseudorabies virus[J].J Virol,2006,80(13):6345-6356.
[8] WANG J,ZENG L,ZHANG L,et al.Cholesterol 25-hydroxylase acts as a host restriction factor on pseudorabies virus replication[J].J Gen Virol,2017,98(6):1467-1476.
[9] AIN Q U,CHUNG J Y,KIM Y H.Current and future delivery systems for engineered nucleases:ZFN,TALEN and RGEN[J].J Control Release,2015,205:120-127.
[10] 舒磊磊,甲芝莲,吴勇浒,等.CRISPR/Cas9技术的应用性研究[J].畜牧兽医学报,2016,47(7):1316-1323.SHU L L,JIA Z L,WU Y H,et al.The application research of CRISPR/Cas9[J].Acta Veterinaria et Zootechnica Sinica,2016,47(7):1316-1323.(in Chinese)
[11] FITZGERALD K A,MCWHIRTER S M,FAIA K L,et al.IKKε and TBK1 are essential components of the IRF3 signaling pathway[J].Nat Immunol,2003,4(5):491-496.
[12] LIU S Q,CAI X,WU J X,et al.Phosphorylation of innate immune adaptor proteins MAVS,STING,and TRIF induces IRF3 activation[J].Science,2015,347(6227):aaa2630.
[13] DALRYMPLE N A,CIMICA V,MACKOW E R.Dengue virus NS proteins inhibit RIG-I/MAVS signaling by blocking TBK1/IRF3 phosphorylation:dengue virus serotype 1 NS4A is a unique interferon-regulating virulence determinant[J].mBio,2015,6(3):e00553-15.
[14] LI M,XIN T,GAO X T,et al.Foot-and-mouth disease virus non-structural protein 2B negatively regulates the RLR-mediated IFN-β induction[J].Biochem Biophys Res Commun,2018,504(1):238-244.
[15] DING Z,FANG L R,JING H Y,et al.Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1[J].J Virol,2014,88(16):8936-8945.
[16] OTSUKA M,KATO N,MORIYAMA M,et al.Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses[J].Hepatology,2005,41(5):1004-1012.
[17] XUE Q,LIU H S,ZHU Z X,et al.Seneca valley virus 3C protease negatively regulates the type I interferon pathway by acting as a viral deubiquitinase[J].Antiviral Res,2018,160:183-189.
[18] KANG S M,CHOI C,CHOI I,et al.Hepatitis E virus methyltransferase inhibits type I interferon induction by targeting RIG-I[J].J Microbiol Biotechnol,2018,28(9):1554-1562.
[19] VAN OPDENBOSCH N,DE REGGE N,VAN POUCKE M,et al.Effects of interferon on immediate-early mRNA and protein levels in sensory neuronal cells infected with herpes simplex virus type 1 or pseudorabies virus[J].Vet Microbiol,2011,152(3-4):401-406.
[20] SUN Y,LUO Y Z,WANG C H,et al.Control of swine pseudorabies in China:opportunities and limitations[J].Vet Microbiol,2016,183:119-124.
[21] METTENLEITER T C.Immunobiology of pseudorabies (Aujeszky’s disease)[J].Vet Immunol Immunopathol,1996,54(1-4):221-229.
[22] YIN Y,XU Z W,LIU X W,et al.A live gI/gE-deleted pseudorabies virus (PRV) protects weaned piglets against lethal variant PRV challenge[J].Virus Genes,2017,53(4):565-572.
[23] ENQUIST L W,HUSAK P J,BANFIELD B W,et al.Infection and spread of alphaherpesviruses in the nervous system[J].Adv Virus Res,1998,51:237-347.
[24] FERRARI M,GUALANDI G L,CORRADI A,et al.Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus[J].Comparat Immunol Microbiol Infect Dis,1998,21(4):291-303.
[25] WANG J C,SONG Z C,GE A M,et al.Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK&gE genes[J].BMC Vet Res,2018,14(1):287.
[26] SETAS PONTES M,DEVRIENDT B,FAVOREEL H W.Pseudorabies virus triggers glycoprotein gE-mediated ERK1/2 activation and ERK1/2-dependent migratory behavior in T cells[J].J Virol,2015,89(4):2149-2156.
[27] 刘兴楼,马迪,舒赛男,等.人巨细胞病毒感染THP-1源性巨噬细胞对IL-1β表达的影响[J].国际病毒学杂志,2017,24(3):151-155.LIU X L,MA D,SHU S N,et al.Effects of human cytomegalovirus infection in THP-1 derived macrophages on the expression of IL-1β[J].International Journal of Virology,2017,24(3):151-155.(in Chinese)
[28] 甘霖,赵俊,陈敬贤,等.干扰素抗病毒效力影响因素的研究进展[J].微生物与感染,2018,13(1):43-48.GAN L,ZHAO J,CHEN J X,et al.Research progress on the influencing factors of antiviral efficacy of interferon[J].Journal of Microbes and Infections,2018,13(1):43-48.(in Chinese)
[29] 温正旺,陈洁,金龙腾,等.TLR3-TBK1-IRF-3信号通路在EV71手足口病脑损伤中的机制研究[J].浙江临床医学,2016,18(11):1995-1997.WEN Z W,CHEN J,JIN L T,et al.Study on the mechanism of TLR3-TBK1-IRF-3 signaling pathway in brain injury caused by EV71 hand-foot-and-mouth disease[J].Zhejiang Clinical Medical Journal,2016,18(11):1995-1997.(in Chinese)
[30] LIN H W,CHANG T J,YANG D J,et al.Regulation of virus-induced inflammatory response by β-carotene in RAW264.7 cells[J].Food Chem,2012,134(4):2169-2175.
[31] LIU H M,LI S S,YANG X,et al.Porcine ISG15 modulates the antiviral response during pseudorabies virus replication[J].Gene,2018,679:212-218.