羊口疮病毒ORF128锚蛋白对HEK293T细胞NF-κB信号通路的调节作用及机制
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摘要
目的研究羊口疮病毒(ORFV)感染HEK293T宿主细胞后,其编码的锚蛋白ORF128蛋白对宿主核因子κB(NF-κB)信号通路的影响及其机制。方法将来自ORFV/QH02/2010株的ORF128 DNA序列分别构建至真核表达载体pCMV-tag2B和pEGFP-N1;在病毒感染细胞期间,利用反转录PCR检测ORF128 mRNA水平、激光共聚焦显微镜检测ORF128蛋白的亚细胞定位;利用双荧光素酶报告基因检测系统分析ORF128对NF-κB信号通路的调节作用, Western blot法检测NF-κBp65的核移位以及NF-κB抑制蛋白α(IκBα)的蛋白磷酸化水平。结果 ORF128蛋白在病毒感染早期表达且定位于宿主细胞核, ORF128蛋白可抑制NF-κB转录因子报告载体荧光素酶活性; ORF128的表达抑制NF-κBp65的核移位和磷酸化的IκBα(p-IκBα)的降解。结论 ORF128蛋白通过抑制p-IκBα蛋白的降解过程,阻止NF-κBp65蛋白核移位,进而抑制宿主NF-κB信号通路的活化。
Objective To elucidate the regulating effect of orf virus(ORFV) encoded ORF128 on NF-κB signaling pathway during the infection of HEK293T cells with ORFV and the underlying mechanism. Methods The ORF128 DNA sequences from ORFV/QH02/2010 strain were constructed into eukaryotic expression vectors pCMV-tag2B and pEGFP-N1. During viral infection of cells, the level of ORF128 mRNA was detected by reverse transcription PCR and the subcellular localization of ORF128 protein by laser confocal microscopy. A dual luciferase reporter assay system was used to analyze the regulating effect of ORF128 on NF-κB signaling pathway, and Western blot analysis to detect the nuclear translocation of NF-κBp65 and the phosphorylation of IκBα protein(p-IκBα). Results ORF128 protein was expressed at the early stage and localized in the cell nuclear during ORFV infection, and it inhibited the expression of NF-κB reporter luciferase activity. The expression of the protein blocked the nuclear translocation of NF-κBp65 and the degradation of p-IκBα. Conclusion ORF128 inhibits the activation of NF-κB signaling pathway by blocking the nuclear translocation of NF-κBp65 and the degradation of p-IκBα.
Objective To elucidate the regulating effect of orf virus(ORFV) encoded ORF128 on NF-κB signaling pathway during the infection of HEK293T cells with ORFV and the underlying mechanism. Methods The ORF128 DNA sequences from ORFV/QH02/2010 strain were constructed into eukaryotic expression vectors pCMV-tag2B and pEGFP-N1. During viral infection of cells, the level of ORF128 mRNA was detected by reverse transcription PCR and the subcellular localization of ORF128 protein by laser confocal microscopy. A dual luciferase reporter assay system was used to analyze the regulating effect of ORF128 on NF-κB signaling pathway, and Western blot analysis to detect the nuclear translocation of NF-κBp65 and the phosphorylation of IκBα protein(p-IκBα). Results ORF128 protein was expressed at the early stage and localized in the cell nuclear during ORFV infection, and it inhibited the expression of NF-κB reporter luciferase activity. The expression of the protein blocked the nuclear translocation of NF-κBp65 and the degradation of p-IκBα. Conclusion ORF128 inhibits the activation of NF-κB signaling pathway by blocking the nuclear translocation of NF-κBp65 and the degradation of p-IκBα.
引文
[1] Haig D M,Mercer A A.Ovine diseases.Orf[J].Vet Res,1998,29(3/4):311-326.
[2] Caravaglio J V,Khachemoune A.Orf virus infection in humans:A review with a focus on advances in diagnosis and treatment[J].J Drugs Dermatol,2017,16(7):684-689.
[3] Morand A,Delaigue S,Morand J J.Review of poxvirus:emergence of monkeypox[J].Med Sante Trop,2017,27(1):29-39.
[4] Mckeever D J,Jenkinson D M,Hutchinson G,et al.Studies of the pathogenesis of orf virus infection in sheep[J].J Comp Pathol,1988,99(3):317-328.
[5] Jenkinson D M,Mcewan P E,Moss V A,et al.Location and spread of Orf virus antigen in infected ovine skin[J].Vet Dermatol,2010,1(4):189-195.
[6] Pasparakis M.Regulation of tissue homeostasis by NF-kappaB signalling:implications for inflammatory diseases[J].Nat Rev Immunol,2009,9(11):778-788.
[7] Nestle F O,Di Meglio P,Qin J Z,et al.Skin immune sentinels in health and disease[J].Nat Rev Immunol,2009,9(10):679-691.
[8] Zhang Q,Lenardo M J,Baltimore D.30 years of NF-κB:A blossoming of relevance to human pathobiology[J].Cell,2017,168(1/2):37-57.
[9] Hiscott J,Nguyen T L,Arguello M,et al.Manipulation of the nuclear factor-kappa B pathway and the innate immune response by viruses[J].Oncogene,2006,25(51):6844-6867.
[10] Le Negrate G.Viral interference with innate immunity by preventing NF-κB activity[J].Cell Microbiol,2012,14(2):168-181.
[11] Christian F,Smith E L,Carmody R J.The regulation of NF-κB subunits by phosphorylation[J/OL].Cells,2016,5(1):E12.DOI:10.3390/cells5010012.
[12] 王平忠,于海涛,白雪帆,等.NF-κB信号通路在病毒感染中的作用[J].细胞与分子免疫学杂志,2011,27(8):933-934.Wang P,Yu H,Bai X,et al.The role of NF-κB signaling pathway in viral infection[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2011,27(8):933-934.
[13] Nagendraprabhu P,Khatiwada S,Chaulagain S,et al.A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling[J/OL].Plos Pathog,2017,13(12):e1006779.DOI:10.1371/journal.ppat.1006779.eCollection 2017 Dec.
[14] Ning Z,Zheng Z,Hao W,et al.The N terminus of Orf virus-encoded protein 002 inhibits acetylation of NF-κB p65 by preventing Ser(276) phosphorylation[J/OL].PLoS One,2013,8(3):e58854.DOI:10.1371/journal.pone.0058854.Epub 2013 Mar 11.
[15] Diel D G,Luo S,Delhon G,et al.Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence[J].J Virol,2011,85(5):2037-2049.
[16] Brady G,Haas D A,Farrell P J,et al.Molluscum Contagiosum virus protein MC005 inhibits NFκB activation by targeting NEMO-regulated IKK activation[J/OL].J Virol,2017,91(15):e00545-17.DOI:10.1128/JVI.00545-17.Print 2017 Aug 1.
[17] Khatiwada S,Delhon G,Nagendraprabhu P,et al.A parapoxviral virion protein inhibits NF-κB signaling early in infection[J/OL].PLoS Pathog,2017,13(8):e1006561.DOI:10.1371/journal.ppat.1006561.eCollection 2017 Aug.
[18] Brady G,Haas D A,Farrell P J,et al.Poxvirus protein MC132 from Molluscum Contagiosum virus inhibits NF-κB activation by targeting p65 for degradation[J].J Virol,2015,89(16):8406-8415.
[19] Di Pilato M,Mejías-Pérez E,Sorzano C O S,et al.Distinct roles of vaccinia virus NF-κB inhibitor proteins A52,B15 and K7 in the immune response[J/OL].J Virol,2017,91(13):e00575-17.DOI:10.1128/JVI.00575-17.Print 2017 Jul 1.
[20] Odon V,Georgana I,Holley J,et al.Novel class of viral ankyrin proteins targeting the host E3 ubiquitin ligase Cullin-2[J/OL].J Virol,2018,92(23).e01374-18.DOI:10.1128/JVI.01374-18.Print 2018 Dec 1.
[21] Liu R,Moss B.Vaccinia virus C9 ankyrin-repeat/F-box protein is a newly identified antagonist of the type I interferon-induced antiviral state[J/OL].J Virol,2018,92(9):e00053-18.DOI:10.1128/JVI.00053-18.Print 2018 May 1.
[22] 陈轶霞,龙玲,王明明.痘病毒ANK/F-box蛋白的生物学特性及泛素化相关功能[J].病毒学报,2017,33(4):155-161.Chen Y,Long L,Wang M.Biological characteristics of poxvirus ANK/ f-box protein and its functions related to ubiquitination[J].Bing Du Xue Bao,2017,33(4):155-161.
[23] Herbert M H,Squire C J,Mercer A A.Poxviral ankyrin proteins[J].Viruses,2015,7(2):709-738.
[24] Burles K,van Buuren N,Barry M.Ectromelia virus encodes a family of ankyrin/F-box proteins that regulate NF-κB[J].Virology,2014,468/469/470:351-362.
[25] Lamb S A,Rahman M M,Mcfadden G.Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence[J].Virology,2014,464/465:134-145.
[26] Mohamed M R,Rahman M M,Lanchbury J S,et al.Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses[J].Proc Natl Acad Sci U S A,2009,106(22):9045-9050.
[27] Mohamed M R,Rahman M M,Rice A,et al.Cowpox virus expresses a novel ankyrin repeat NF-kappaB inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis[J].J Virol,2009,83(18):9223-9236.
[28] Tulman E R,Afonso C L,Lu Z,et al.The genome of canarypox virus[J].J Virol,2004,78(1):353-366.
[29] Sonnberg S,Seet B T,Pawson T,et al.Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes[J].Proc Natl Acad Sci U S A,2008,105(31):10955-10960.
[30] 冯园,贾怀杰,何小兵,等.口疮病毒B4R锚蛋白抑制细胞NF-κB信号通路的研究[J].中国兽医科学,2016,46(11):1412-1417.Feng Y,Jia H,He X,et al.Poxvirus ankyrin protein B4R from orf virus inhibits cell NF-κB signaling pathway[J].Zhong Guo Shou Yi Ke Xue,2016,46(11):1412-1417.
[31] Diel D G,Luo S,Delhon G,et al.A nuclear inhibitor of NF-κB encoded by a poxvirus[J].J Virol,2011,85(1):264-275.
[32] Guo C J,Chen W J,Yuan L Q,et al.The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase β[J].J Gen Virol,2011,92(Pt 7):1561-1570.
[33] Chen D,Zheng Z,Xiao B,et al.Orf virus 002 protein targets ovine protein S100A4 and inhibits NF-κB signaling[J/OL].Front Microbiol,2016,7:E59.DOI:10.3389/fmicb.2016.01389.eCollection 2016.
[2] Caravaglio J V,Khachemoune A.Orf virus infection in humans:A review with a focus on advances in diagnosis and treatment[J].J Drugs Dermatol,2017,16(7):684-689.
[3] Morand A,Delaigue S,Morand J J.Review of poxvirus:emergence of monkeypox[J].Med Sante Trop,2017,27(1):29-39.
[4] Mckeever D J,Jenkinson D M,Hutchinson G,et al.Studies of the pathogenesis of orf virus infection in sheep[J].J Comp Pathol,1988,99(3):317-328.
[5] Jenkinson D M,Mcewan P E,Moss V A,et al.Location and spread of Orf virus antigen in infected ovine skin[J].Vet Dermatol,2010,1(4):189-195.
[6] Pasparakis M.Regulation of tissue homeostasis by NF-kappaB signalling:implications for inflammatory diseases[J].Nat Rev Immunol,2009,9(11):778-788.
[7] Nestle F O,Di Meglio P,Qin J Z,et al.Skin immune sentinels in health and disease[J].Nat Rev Immunol,2009,9(10):679-691.
[8] Zhang Q,Lenardo M J,Baltimore D.30 years of NF-κB:A blossoming of relevance to human pathobiology[J].Cell,2017,168(1/2):37-57.
[9] Hiscott J,Nguyen T L,Arguello M,et al.Manipulation of the nuclear factor-kappa B pathway and the innate immune response by viruses[J].Oncogene,2006,25(51):6844-6867.
[10] Le Negrate G.Viral interference with innate immunity by preventing NF-κB activity[J].Cell Microbiol,2012,14(2):168-181.
[11] Christian F,Smith E L,Carmody R J.The regulation of NF-κB subunits by phosphorylation[J/OL].Cells,2016,5(1):E12.DOI:10.3390/cells5010012.
[12] 王平忠,于海涛,白雪帆,等.NF-κB信号通路在病毒感染中的作用[J].细胞与分子免疫学杂志,2011,27(8):933-934.Wang P,Yu H,Bai X,et al.The role of NF-κB signaling pathway in viral infection[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2011,27(8):933-934.
[13] Nagendraprabhu P,Khatiwada S,Chaulagain S,et al.A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling[J/OL].Plos Pathog,2017,13(12):e1006779.DOI:10.1371/journal.ppat.1006779.eCollection 2017 Dec.
[14] Ning Z,Zheng Z,Hao W,et al.The N terminus of Orf virus-encoded protein 002 inhibits acetylation of NF-κB p65 by preventing Ser(276) phosphorylation[J/OL].PLoS One,2013,8(3):e58854.DOI:10.1371/journal.pone.0058854.Epub 2013 Mar 11.
[15] Diel D G,Luo S,Delhon G,et al.Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence[J].J Virol,2011,85(5):2037-2049.
[16] Brady G,Haas D A,Farrell P J,et al.Molluscum Contagiosum virus protein MC005 inhibits NFκB activation by targeting NEMO-regulated IKK activation[J/OL].J Virol,2017,91(15):e00545-17.DOI:10.1128/JVI.00545-17.Print 2017 Aug 1.
[17] Khatiwada S,Delhon G,Nagendraprabhu P,et al.A parapoxviral virion protein inhibits NF-κB signaling early in infection[J/OL].PLoS Pathog,2017,13(8):e1006561.DOI:10.1371/journal.ppat.1006561.eCollection 2017 Aug.
[18] Brady G,Haas D A,Farrell P J,et al.Poxvirus protein MC132 from Molluscum Contagiosum virus inhibits NF-κB activation by targeting p65 for degradation[J].J Virol,2015,89(16):8406-8415.
[19] Di Pilato M,Mejías-Pérez E,Sorzano C O S,et al.Distinct roles of vaccinia virus NF-κB inhibitor proteins A52,B15 and K7 in the immune response[J/OL].J Virol,2017,91(13):e00575-17.DOI:10.1128/JVI.00575-17.Print 2017 Jul 1.
[20] Odon V,Georgana I,Holley J,et al.Novel class of viral ankyrin proteins targeting the host E3 ubiquitin ligase Cullin-2[J/OL].J Virol,2018,92(23).e01374-18.DOI:10.1128/JVI.01374-18.Print 2018 Dec 1.
[21] Liu R,Moss B.Vaccinia virus C9 ankyrin-repeat/F-box protein is a newly identified antagonist of the type I interferon-induced antiviral state[J/OL].J Virol,2018,92(9):e00053-18.DOI:10.1128/JVI.00053-18.Print 2018 May 1.
[22] 陈轶霞,龙玲,王明明.痘病毒ANK/F-box蛋白的生物学特性及泛素化相关功能[J].病毒学报,2017,33(4):155-161.Chen Y,Long L,Wang M.Biological characteristics of poxvirus ANK/ f-box protein and its functions related to ubiquitination[J].Bing Du Xue Bao,2017,33(4):155-161.
[23] Herbert M H,Squire C J,Mercer A A.Poxviral ankyrin proteins[J].Viruses,2015,7(2):709-738.
[24] Burles K,van Buuren N,Barry M.Ectromelia virus encodes a family of ankyrin/F-box proteins that regulate NF-κB[J].Virology,2014,468/469/470:351-362.
[25] Lamb S A,Rahman M M,Mcfadden G.Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence[J].Virology,2014,464/465:134-145.
[26] Mohamed M R,Rahman M M,Lanchbury J S,et al.Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses[J].Proc Natl Acad Sci U S A,2009,106(22):9045-9050.
[27] Mohamed M R,Rahman M M,Rice A,et al.Cowpox virus expresses a novel ankyrin repeat NF-kappaB inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis[J].J Virol,2009,83(18):9223-9236.
[28] Tulman E R,Afonso C L,Lu Z,et al.The genome of canarypox virus[J].J Virol,2004,78(1):353-366.
[29] Sonnberg S,Seet B T,Pawson T,et al.Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes[J].Proc Natl Acad Sci U S A,2008,105(31):10955-10960.
[30] 冯园,贾怀杰,何小兵,等.口疮病毒B4R锚蛋白抑制细胞NF-κB信号通路的研究[J].中国兽医科学,2016,46(11):1412-1417.Feng Y,Jia H,He X,et al.Poxvirus ankyrin protein B4R from orf virus inhibits cell NF-κB signaling pathway[J].Zhong Guo Shou Yi Ke Xue,2016,46(11):1412-1417.
[31] Diel D G,Luo S,Delhon G,et al.A nuclear inhibitor of NF-κB encoded by a poxvirus[J].J Virol,2011,85(1):264-275.
[32] Guo C J,Chen W J,Yuan L Q,et al.The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase β[J].J Gen Virol,2011,92(Pt 7):1561-1570.
[33] Chen D,Zheng Z,Xiao B,et al.Orf virus 002 protein targets ovine protein S100A4 and inhibits NF-κB signaling[J/OL].Front Microbiol,2016,7:E59.DOI:10.3389/fmicb.2016.01389.eCollection 2016.