禽呼肠孤病毒σA基因对鸡胚成纤维细胞天然免疫相关基因转录水平的调控影响
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摘要
为了明确禽呼肠孤病毒(Avian reovirus,ARV)σA基因对宿主天然免疫应答的影响,本研究利用实时荧光定量PCR方法检测pCAGEN-σA转染鸡胚成纤维细胞(DF1)后,不同时间点(转染后第3、6、12、24和36小时)DF1细胞中天然免疫相关基因,包括Toll样受体(TLRs)、MDA5、MAVS、My D88、TRIF、IRF-3、IRF7、NF-κB、IFN-α、IFN-β、IL-6、IL-8、IFITM3、Mx1和OASL等基因的转录水平变化。结果,与空载体转染细胞相比,pCAGEN-σA转染细胞中上述这些天然免疫基因的m RNA水平出现不同程度变化(P<0.05或P<0.01)。TLR3应对最快速,其m RNA水平在转染后第3小时和第6小时迅速上升,但9 h后逐渐下降;MDA5和TLR7的m RNA水平,则分别在转染后第6小时和第12小时开始显著升高,并分别在转染后第12小时和第36小时达到峰值(P<0.05或P<0.01);TLR15的m RNA水平整体变化不明显。My D88和MAVS的m RNA水平呈表达上调趋势;TRIF在转染后第6小时表达量迅速达到峰值(2.83倍),随后急速下降。NF-κB呈表达上调趋势;而IRF3/7的表达则被抑制。I型干扰素(IFN-α和IFN-β)呈现相似的表达模式,在转染后第6小时和第24小时表达量增加,其他时间点均为表达量下降; IL-6和IL-8则表达上调,分别在转染后第9小时和第6小时达到峰值(P<0.01)。IFITM3、Mx1、OASL的m RNA水平也显著升高,均在转染后第12小时达到峰值(P<0.01)。上述试验结果表明,在DF-1细胞中过表达σA基因,能显著引起天然免疫相关基因的表达的变化,为进一步阐释ARV的分子致病机制和免疫应答机理奠定了基础。
This study is aimed to assess the influences of avian reovirus(ARV)σA gene on expression of innate immune genes in chicken embryo fibroblasts cells.(Method)Using the quantitative real-time PCR assays,it was performed to determine the transcriptional levels of TLR3,TLR7,TLR15,MDA5,My D88,MAVS,TRIF,IRF3/7,NF-κB,IFN-α,IFN-β,IL-6,IL-8,IFITM3,Mx1 and OASL at 3 h,6 h,12 h,24 h and 36 h post-transfection in p CAGEN-σA-transfected cells.In result,the m RNA transcriptional levels of these innate immune-associated genes were significantly induced(P<0.05 or P<0.01).TLR3 were quickly up-regulated at 3 h and 6 h post-transfection, but gradually reduced at 9 h post-transfection.The m RNA transcriptional levels of MDA5 and TLR7 were obviously promoted at 6 h and 12 h post-transfection,and specially reached the summit maximum values at 12 h and 36 h post-transfection,respectively(P<0.05 or P<0.01);TLR15 did not exhibit obvious expression changes.The m RNA expression levels of My D88 and MAVS was up-regulated within 48 h.Meanwhile,the m RNA expression level of TRIF was peaked at 6 h post-transfection,but subsequently reduced after 12 h post-transfection.The m RNA expression levels of NF-κB were up-regulated during the whole experiment course,meanwhile the IRF3/7 m RNA expression level was inhibited.IFN-α and IFN-β were up-regulated at 3 h and 6 h post-transfection,but were decreased at other time points post-transfection.IL-6 and IL-8 were significantly up-regulated and peaked at 9 h and 24 h post-transfection,respectively(P<0.05 或 P<0.01).The m RNA expression levels of IFITM3,Mx1 and OASL were significantly up-regulated and peaked at 12 h post-transfection(P<0.01).In conclusion,ARV σA gene is able to modulate the increased m RNA expression of the innate immune genes,which would contribute to further understand ARV pathogenesis and host innate immune responses to ARV infection.
This study is aimed to assess the influences of avian reovirus(ARV)σA gene on expression of innate immune genes in chicken embryo fibroblasts cells.(Method)Using the quantitative real-time PCR assays,it was performed to determine the transcriptional levels of TLR3,TLR7,TLR15,MDA5,My D88,MAVS,TRIF,IRF3/7,NF-κB,IFN-α,IFN-β,IL-6,IL-8,IFITM3,Mx1 and OASL at 3 h,6 h,12 h,24 h and 36 h post-transfection in p CAGEN-σA-transfected cells.In result,the m RNA transcriptional levels of these innate immune-associated genes were significantly induced(P<0.05 or P<0.01).TLR3 were quickly up-regulated at 3 h and 6 h post-transfection, but gradually reduced at 9 h post-transfection.The m RNA transcriptional levels of MDA5 and TLR7 were obviously promoted at 6 h and 12 h post-transfection,and specially reached the summit maximum values at 12 h and 36 h post-transfection,respectively(P<0.05 or P<0.01);TLR15 did not exhibit obvious expression changes.The m RNA expression levels of My D88 and MAVS was up-regulated within 48 h.Meanwhile,the m RNA expression level of TRIF was peaked at 6 h post-transfection,but subsequently reduced after 12 h post-transfection.The m RNA expression levels of NF-κB were up-regulated during the whole experiment course,meanwhile the IRF3/7 m RNA expression level was inhibited.IFN-α and IFN-β were up-regulated at 3 h and 6 h post-transfection,but were decreased at other time points post-transfection.IL-6 and IL-8 were significantly up-regulated and peaked at 9 h and 24 h post-transfection,respectively(P<0.05 或 P<0.01).The m RNA expression levels of IFITM3,Mx1 and OASL were significantly up-regulated and peaked at 12 h post-transfection(P<0.01).In conclusion,ARV σA gene is able to modulate the increased m RNA expression of the innate immune genes,which would contribute to further understand ARV pathogenesis and host innate immune responses to ARV infection.
引文
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[14]王琳.J亚群禽白血病病毒与宿主细胞相互作用机制研究[D].扬州:扬州大学,2018.WANG Lin.Interaction mechanism between subgroup J avian leucosis virus and host cells[D].Yangzhou:Yangzhou University,2018.(in Chinese)
[15]XIE L,XIE Z,HUANG L,et al.Avian reovirusσA andσNS proteins activate the phosphatidylinositol3-kinase-dependent Akt signalling pathway[J].Archieve Virology,2016,161(8):2243-2248.
[16]HE Y,XIE Z,DAI J,et al.Responses of the Toll-like receptor and melanoma differentiation-associated protein 5 signaling pathways to avian infectious bronchitis virus infection in chicks[J].Virology Sinica,2016,31(1):57-68.
[17]CHEN S,LUO G,YANG Z,et al.Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathway[J].Veterinary Research,2016,47(1):74.
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[20]LI C,WEI H,YU L,et al.Nuclear localization of the p17 protein of avian reovirus is correlated with autophagy induction and an increase in viral replication[J].Archive Virology,2015,160(12):3001-3010.
[21]HUANG W,CHIU H,LIAO T,et al.Avian reovirus protein p17 functions as a nucleoporin Tpr suppressor leading to activation of p53,p21 and PTEN and inactivation of PI3K/AKT/mTOR and ERK signaling pathways[J].PLoS One,2015,10(9):e0138627.
[22]CHI P,HUANG W,LAI I,et al.The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10(PTEN)and AMP-activated protein kinase(AMPK),as well as dsRNA-dependent protein kinase(PKR)/eIF2αsignaling pathways[J].Journal of Biology Chemistry,2013,288(5):3571-3584.
[23]LUND J M,ALEXOPOULOU L,Sato A,et al.Recognition of single-stranded RNA viruses by toll-like receptor 7[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101:5598-5603.
[24]MATSUMOTO M,FUNAMI K,OSHIUMI H,et al.Toll-like receptor 3:a link between toll-like receptor,interferon and viruses[J].Microbiology and Immunology,2004,48:147-154.
[25]王国庆.口蹄疫病毒2B蛋白拮抗RIG-I抗病毒作用研究[D].兰州:甘肃农业大学,2015.WANG Guo-qing.The study on FMDV 2B-induced antagonistic effects on RIG-I-mediated antiviral activity[D].Lanzhou:Gansu Agricultural Univeristy,2015.(in Chinese)
[26]YE C,JIA L,SUN Y,et al.Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5[J].Journal of Virology,2014,88(19):11154-11165.
[27]竺李莉.SFTS布尼亚病毒非结构蛋白NSs抑制MAVS介导天然免疫机制的研究[D].南京:南京大学,2015.ZHU Li-li.Study on SFTS virus nonstructural protein NSs inhibits MAVS-mediated innate immune[D].Nanjing:Nanjing University,2015.(in Chinese)
[28]DONG J,XU S,WANG J,et al.Porcine reproductive and respiratory syndrome virus 3C protease cleaves the mitochondrial antiviral signalling complex to antagonize IFN-beta expression[J].Journal of General Virology,2015,96(10):3049-3058.
[29]HUANG C,ZHANG Q,GUO X,et al.Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-kappa B essential modulator[J].Journal of Virology,2014,88(18):10934-10945.
[2]HUANG L,XIE Z,XIE L,et al.A duplex real-time PCRassay for the detection and quantification of avian reovirus and Mycoplasma synoviae[J].Virology Journal,2015,12(1):22-27.
[3]黄莉,谢芝勋,谢丽基,等.禽呼肠孤病毒强毒株和弱毒疫苗株感染Vero细胞的蛋白质组学研究[J].畜牧兽医学报,2016,47(02):331-339.HUANG Li,XIE Zhi-xun,XIE Li-ji,et al.Proteomics analysis of vero cells infected by avian reovirus virulent strain and attenuated vaccine strain[J].Acta Veterinaria et Zootechnica Sinica,2016,47(02):331-339.(in Chinese)
[4]BENAVENTE J,MARTINEZ-COSTAS J.Avian reovirus:structure and biology[J].Virus Reserch,2007,123(2):105-119.
[5]MARTINEZ-COSTAS J,GONZALEZ-LOPEZ C,VAKHARIA VN,et al.Possible involvement of the double-stranded RNA-binding core protein sigmaA in the resistance of avian reovirus to interferon[J].Journal of Virology,2000,74(3):1124-1131.
[6]VAZQUEZ-IGLESIAS L,LOSTALE-SEIJO I,MARTINEZ-COSTASJ,et al.Avian reovirus sigmaA localizes to the nucleolus and enters the nucleus by a nonclassical energy-and carrier-independent pathway[J].Journal of Virology,2009,83(19):10163-10175.
[7]GONZALEZ-LOPEZ C,MARTINEZ-COSTAS J,ESTEBAN M,et al.Evidence that avian reovirus sigmaA protein is an inhibitor of the double-stranded RNA-dependent protein kinase[J].Journal of General Virology,2003,84(Pt 6):1629-1639.
[8]JACOBS S R,DAMANIA B.NLRs,inflammasomes,and viral infection[J].Journal of Leukocyte Biology,2012,92(3):469-477.
[9]IMANI F,JACOBS B L.Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein[J].Proceedings of the National Academy of Sciences of the United States of America,1988,85(21):7887-7891.
[10]胡勇.流感病毒NS1蛋白以及SARS冠状病毒N蛋白参与的免疫逃逸机制研究[D].北京:中国人民解放军军事医学科学院,2017.HU Yong.The immune escape mechanism of influenza virus NS1 protein and SARS coronavirus N protein[D].Beijing:The PLA Academy of Military Medical Sciences,2017.(in Chinese)
[11]张昆丽.禽呼肠孤病毒感染对体外细胞和SPF鸡细胞因子mRNA转录影响的初步研究[D].南宁:广西大学,2014.ZHANG Kun-li.A preliminary research on the influence of avian reovirus infection on cytokines mRNA transcription in vitro and in SPF chickens[D].Nanning:Guangxi University,2014.(in Chinese)
[12]黄莉,谢芝勋,蓝元喜,等.禽呼肠孤病毒感染对SPF鸡外周血淋巴细胞中天然免疫相关基因转录的影响[J].畜牧兽医学报,2017,48(01):116-123.HUANG Li,XIE Zhi-xun,LAN Yuan-xi,et al.Effects of avian reovirus infection on expression of innate immune genes in peripheral blood lymphocytes of SPF chickens[J].Acta Veterinaria et Zootechnica Sinica,2017,48(01):116-123.(in Chinese)
[13]黄莉,谢芝勋,蓝元喜,等.禽呼肠孤病毒感染后Toll样受体在SPF鸡不同器官中的表达变化[J].中国兽医科学,2017,47(02):202-206.HUANG Li,XIE Zhi-xun,LAN Yuan-xi,et al.Expression profiles of toll-like receptors in different organs of SPF chickens infected with avian reovirus[J].Chinese Veterinary Science,2017,47(02):202-206.(in Chinese)
[14]王琳.J亚群禽白血病病毒与宿主细胞相互作用机制研究[D].扬州:扬州大学,2018.WANG Lin.Interaction mechanism between subgroup J avian leucosis virus and host cells[D].Yangzhou:Yangzhou University,2018.(in Chinese)
[15]XIE L,XIE Z,HUANG L,et al.Avian reovirusσA andσNS proteins activate the phosphatidylinositol3-kinase-dependent Akt signalling pathway[J].Archieve Virology,2016,161(8):2243-2248.
[16]HE Y,XIE Z,DAI J,et al.Responses of the Toll-like receptor and melanoma differentiation-associated protein 5 signaling pathways to avian infectious bronchitis virus infection in chicks[J].Virology Sinica,2016,31(1):57-68.
[17]CHEN S,LUO G,YANG Z,et al.Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathway[J].Veterinary Research,2016,47(1):74.
[18]LIN K,HSU A,SHIEN J,et al.Avian reovirus sigma Cenhances the mucosal and systemic immune responses elicited by antigen-conjugated lactic acid bacteria[J].Vaccine,2012,30(33):5019-5029.
[19]ZHANG Z,LIN W,LI X,et al.Critical role of eukaryotice longation factor 1 alpha 1(EEF1A1)in avian reovirus sigma-C-induced apoptosis and inhibition of viral growth[J].Archive Virology,2015,160(6):1449-1461.
[20]LI C,WEI H,YU L,et al.Nuclear localization of the p17 protein of avian reovirus is correlated with autophagy induction and an increase in viral replication[J].Archive Virology,2015,160(12):3001-3010.
[21]HUANG W,CHIU H,LIAO T,et al.Avian reovirus protein p17 functions as a nucleoporin Tpr suppressor leading to activation of p53,p21 and PTEN and inactivation of PI3K/AKT/mTOR and ERK signaling pathways[J].PLoS One,2015,10(9):e0138627.
[22]CHI P,HUANG W,LAI I,et al.The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10(PTEN)and AMP-activated protein kinase(AMPK),as well as dsRNA-dependent protein kinase(PKR)/eIF2αsignaling pathways[J].Journal of Biology Chemistry,2013,288(5):3571-3584.
[23]LUND J M,ALEXOPOULOU L,Sato A,et al.Recognition of single-stranded RNA viruses by toll-like receptor 7[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101:5598-5603.
[24]MATSUMOTO M,FUNAMI K,OSHIUMI H,et al.Toll-like receptor 3:a link between toll-like receptor,interferon and viruses[J].Microbiology and Immunology,2004,48:147-154.
[25]王国庆.口蹄疫病毒2B蛋白拮抗RIG-I抗病毒作用研究[D].兰州:甘肃农业大学,2015.WANG Guo-qing.The study on FMDV 2B-induced antagonistic effects on RIG-I-mediated antiviral activity[D].Lanzhou:Gansu Agricultural Univeristy,2015.(in Chinese)
[26]YE C,JIA L,SUN Y,et al.Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5[J].Journal of Virology,2014,88(19):11154-11165.
[27]竺李莉.SFTS布尼亚病毒非结构蛋白NSs抑制MAVS介导天然免疫机制的研究[D].南京:南京大学,2015.ZHU Li-li.Study on SFTS virus nonstructural protein NSs inhibits MAVS-mediated innate immune[D].Nanjing:Nanjing University,2015.(in Chinese)
[28]DONG J,XU S,WANG J,et al.Porcine reproductive and respiratory syndrome virus 3C protease cleaves the mitochondrial antiviral signalling complex to antagonize IFN-beta expression[J].Journal of General Virology,2015,96(10):3049-3058.
[29]HUANG C,ZHANG Q,GUO X,et al.Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-kappa B essential modulator[J].Journal of Virology,2014,88(18):10934-10945.