草鱼jam-a分子在胚胎幼鱼期及受GCRV感染PSF细胞的表达分析
|
摘要
草鱼呼肠孤病毒(Grass carp reovirus, GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病,导致高死亡率。草鱼吻端成纤维细胞(Grass carp snout fibroblast cells, PSF)是GCRV的敏感细胞系。JAM-A (Junctional adhesion molecule A)为免疫球蛋白超家族成员,是多种病毒的细胞受体。本研究在前期克隆到草鱼3种jam-a基因,命名为gcjam-a1,gcjam-a2和gcjam-a3。在获取ORF序列的基础上,利用qRT-PCR分析了3种gcjam-a在草鱼胚胎及幼鱼不同发育时期及PSF细胞中受GCRV(GD108株)感染前后的表达模式。结果显示,检测的13个胚胎及幼鱼发育时期中,gcjam-a1在未受精卵中高表达,在受精卵至出膜前的胚胎表达水平均较低;从出膜后1～3 d表达量开始上升;出膜6～15 d均呈高水平表达。gcjam-a2与gcjam-a3在草鱼胚胎及幼鱼发育各阶段表达水平较低。在无病毒感染的PSF细胞中,gcjam-a只有少量表达。受GCRV-GD108感染后,病毒S7基因在PSF细胞中的拷贝数随时间呈显著上调趋势,gcjam-a的表达量也有不同程度的上调,mRNA上调水平为gcjam-a1>gcjam-a2>gcjam-a3。本研究证实了3种gcjam-a基因在PSF细胞中的表达均与GCRV-GD108感染相关,其中,gcjam-a1的表达水平受GCRV-GD108感染影响最大,同时,它在孵化出膜后表达上升,推测它可能与病毒的感染更相关。gcjam-a1可作为下一步GCRV与宿主互作研究中的候选分子。
Grass carps are seriously threatened by GCRV(grass carp reovirus) that can cause high mortality to fingerling and yearling grass carps. Grass carp snout fibroblast cells(PSF) are highly sensitive to GCRV. Junctional adhesion molecule A(JAM-A), an immunoglobulin superfamily member, acts as a viral cell receptor. In our previous study, the cDNA sequences of grass carp jam-a1, jam-a2, and jam-a3(named gcjam-a1, gcjam-a2, and gcjam-a3) were cloned. Based on this, q RT-PCR was used to analyze the expression pattern of gcjam-as at different embryonic and juvenile development stages and in GCRV-GD108-infected PSF cells. The results showed that the expression pattern of gcjam-a1, 2, and 3 differed during the embryonic development stages. m RNA expression of gcjam-a1 could be detected in unfertilized eggs and at a lower level from the fertilized egg stage to 1 dph(day post hatch). However, the m RNA was highly expressed at 1～3 dph and high levels were maintained from 3 dph to the end of the experiment(15 dph). The expression of gcjam-a2 and gcjam-a3 was very low at different embryonic development stages compared to that of gcjam-a1. gcjam-as were only slightly expressed in non-infected PSF cells. After GCRV-GD108 infection, the expression of S7 in PSF cells increased significantly, and the expression of gcjam-as in PSF cells also increased to different levels after GCRV-GD108 infection. Upregulation of the gcjam-as was in the order: gcjam-a1> gcjam-a2>gcjam-a3. The results showed that the expression of jam-as was related to GCRV infection in PSF cells and that the expression of jam-a1 was most influenced by GCRV-GD108 infection. It is also expressed in early embryonic development, suggesting that jam-a1 is the most relevant to GCRV infection. This study will lay the foundation for further research on GCRV receptors.
Grass carps are seriously threatened by GCRV(grass carp reovirus) that can cause high mortality to fingerling and yearling grass carps. Grass carp snout fibroblast cells(PSF) are highly sensitive to GCRV. Junctional adhesion molecule A(JAM-A), an immunoglobulin superfamily member, acts as a viral cell receptor. In our previous study, the cDNA sequences of grass carp jam-a1, jam-a2, and jam-a3(named gcjam-a1, gcjam-a2, and gcjam-a3) were cloned. Based on this, q RT-PCR was used to analyze the expression pattern of gcjam-as at different embryonic and juvenile development stages and in GCRV-GD108-infected PSF cells. The results showed that the expression pattern of gcjam-a1, 2, and 3 differed during the embryonic development stages. m RNA expression of gcjam-a1 could be detected in unfertilized eggs and at a lower level from the fertilized egg stage to 1 dph(day post hatch). However, the m RNA was highly expressed at 1～3 dph and high levels were maintained from 3 dph to the end of the experiment(15 dph). The expression of gcjam-a2 and gcjam-a3 was very low at different embryonic development stages compared to that of gcjam-a1. gcjam-as were only slightly expressed in non-infected PSF cells. After GCRV-GD108 infection, the expression of S7 in PSF cells increased significantly, and the expression of gcjam-as in PSF cells also increased to different levels after GCRV-GD108 infection. Upregulation of the gcjam-as was in the order: gcjam-a1> gcjam-a2>gcjam-a3. The results showed that the expression of jam-as was related to GCRV infection in PSF cells and that the expression of jam-a1 was most influenced by GCRV-GD108 infection. It is also expressed in early embryonic development, suggesting that jam-a1 is the most relevant to GCRV infection. This study will lay the foundation for further research on GCRV receptors.
引文
Antar AAR,Konopka JL,Campbell JA,et al.Junctional adhesion molecule-A is required for hematogenous dissemination of reovirus.Cell Host and Microbe,2009,5(1):59-71
Aurrand-Lions M,Johnson-Leger C,Wong C,et al.Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members.Blood,2001,98(13):3699-3707
Banerjee M,Johnson JE.Activation,exposure and penetration of virally encoded,membrane-active polypeptides during nonenveloped virus entry.Current Protein and Peptide Science,2008,9(1):16-27
Bartesaghi A,Merk A,Borgnia MJ,et al.Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryoelectron microscopy.Nature Structural and Molecular Biology,2013,20(12):1352-1357
Bradfield PF,Nourshargh S,Aurrand-Lions M,et al.JAMfamily and related proteins in leukocyte migration(Vestweber series).Arteriosclerosis,Thrombosis,and Vascular Biology,2005,27(10):2104-2112
Chi YY,Tian YY,Ye X,et al.Molecular properties of grass carp reovirus in southern China and establishment of a duplex PCR detection method.Chinese Journal of Virology,2011,27(4):358-364[迟妍妍,田园园,叶星,等.南方养殖草鱼呼肠孤病毒的分子特性比较及双重PCR检测方法的建立.病毒学报,2011,27(4):358-364]
Danthi P,Guglielmi KM,Kirchner E,et al.From touchdown to transcription:The reovirus cell entry pathway.Cell Entry by Non-Enveloped Viruses,2010,343:91-119
Danthi P,Holm GH,Stehle T,et al.Reovirus receptors,cell entry,and proapoptotic signaling.Advances in Experimental Medicine and Biology,2013,790:42-71
Du F,Su J,Huang R,et al.Cloning and preliminary functional studies of the JAM-A gene in grass carp(Ctenopharyngodon idellus).Fish and Shellfish Immunology,2013,34(6):1476-1484
Guglielmi KM,Kirchner E,Holm GH,et al.Reovirus binding determinants in junctional adhesion molecule-A.Journal of Biological Chemistry,2007,282(24):17930-17940
Harrison SC.Viral membrane fusion.Virology,2015,479-480:498-507
Kim J,Tao Y,Reinisch KM,et al.Orthoreovirus and Aquareovirus core proteins:Conserved enzymatic surfaces,but not protein-protein interfaces.Virus Research,2004,101(1):15-28
Kirchner E,Guglielmi KM,Strauss HM,et al.Structure of reovirus sigma1 in complex with its receptor junctional adhesion molecule-A.PLo S Pathogens,2008,4(12):e1000235
Kobayashi I,Kobayashi-Sun J,Kim AD,et al.Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.Nature,2014,512(7514):319-323
Kurose M,Kakuki T,Takano K,et al.Junctional adhesion molecule-A in head and neck squamous cell Carcinoma.Advances in Oto-Rhino-Laryngology,2016,77:92-97
Li HL,Xu SY,Deng GC,et al.Establishment of cell line PSFfrom the fibroblast cell of grass carp snout tissue and studies of its biological characteristics.Journal of Chinese Academy of Fishery Sciences,1988,1(1):1-8[李焕林,许淑英,邓国成,等.草鱼吻端成纤维细胞系PSF的建立及其生物学特性.中国水产科学研究院学报,1988,1(1):1-8]
Li X,Zeng WW,Wang Q,et al.The study on the proliferation of GCRVⅡin different fish cell lines.Journal of Fisheries of China,2016,40(8):1249-1257[李贤,曾伟伟,王庆,等.草鱼呼肠孤病毒Ⅱ型在不同鱼类细胞中的增殖情况.水产学报,2016,40(8):1249-1257]
Makino A,Shimojima M,Miyazawa T,et al.Junctional adhesion molecule 1 is a functional receptor for feline calicivirus.Journal of Virology,2006,80(9):4482-4490
Nibert ML,Duncan R.Bioinformatics of recent aqua-and orthoreovirus isolates from fish:Evolutionary gain or loss of FAST and fiber proteins and taxonomic implications.PLoSOne,2013,8(7):e68607
Pei C,Ke F,Chen ZY,et al.Complete genome sequence and comparative analysis of grass carp reovirus strain 109(GCRe V-109)with other grass carp reovirus strains reveals no significant correlation with regional distribution.Archives of Virology,2014,159(9):2435-2440
Rangel AA,Rockemann DD,Hetrick FM,et al.Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus.Journal of General Virology,1999,80(Pt 9):2399-2402
Rehder D,Iden S,Nasdala I,et al.Junctional adhesion molecule-A participates in the formation of apico-basal polarity through different domains.Experimental Cell Research,2006,312(17):3389-3403
Schulz WL,Haj AK,Schiff LA.Reovirus uses multiple endocytic pathways for cell entry.Journal of Virology,2012,86(23):12665-12675
Severson EA,Parkos CA.Structural determinants of junctional adhesion molecule A(JAM-A)function and mechanisms of intracellular signaling.Current Opinion in Cell Biology,2009,21(5):701-707
Skehel JJ,Wiley DC.Receptor binding and membrane fusion in virus entry:The influenza hemagglutinin.Annual Review of Biochemistry,2000,69:531-569
Tian YY,Jiao ZZ,Dong JJ,et al.Cloning and expression analysis of jam-a genes in grass carp(Ctenopharyngodon idellus).South China Fisheries Science,2017,13(6):30-40[田园园,焦真真,董浚键,等.草鱼jam-a基因的克隆及表达分析.南方水产科学,2017,13(6):30-40]
Tian YY,Jiao ZZ,Dong JJ,et al.Grass carp reovirus-GD108fiber protein is involved in cell attachment.Virus Genes,2017,53(4):613-622
Torres-Flores JM,Silva-Ayala D,Espinoza MA,et al.The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.Virology,2015,475:172-178
Vetrano S,Rescigno M,Cera MR,et al.Unique role of junctional adhesion molecule-a in maintaining mucosal homeostasis in inflammatory bowel disease.Gastroenterology,2008,135(1):173-184
Wang JJ,Liu Y,Yu L,et al.Characterization of grass carp(Ctenopharyngodon idellus)ovary cell line and its susceptibility to spring viremia of carp virus.Progress in Fishery Sciences,2016,37(6):56-61[王津津,刘莹,于力,等.草鱼(Ctenopharyngodon idellus)性腺细胞系(GCO)特性和对鲤春病毒的敏感性.渔业科学进展,2016,37(6):56-61]
Wang S.National fisheries economic statistics bulletin of 2016.China Fisheries,2017(7):23-24[王莎.2016年全国渔业经济统计公报.中国水产,2017(7):23-24]
Xu SY,Li HL,Deng GC,et al.The preparation and immune effect of attenuated live vaccine obtained through cell culture for hemorrhage of grass carp.Journal of Fisheries of China,1994,18(2):110-117[许淑英,李焕林,邓国成,等.草鱼出血病细胞培养弱毒疫苗的制备及其免疫效果.水产学报,1994,18(2):110-117]
Ye X,Tian Y,Deng G,et al.Complete genomic sequence of a reovirus isolated from grass carp in China.Virus Research,2012,163(1):275-283
Yeung D,Manias JL,Stewart DJ,et al.Decreased junctional adhesion molecule-A expression during blood-brain barrier breakdown.Acta Neuropathologica,2008,115(6):635-642
Zeng BH,Liao ZY,Xiang X,et al.Effects of bile acids on growth performance,muscle composition and digestive enzyme activities of Ctenopharyngodon idellus.Progress in Fishery Sciences,2017,38(2):99-106[曾本和,廖增艳,向枭,等.胆汁酸对草鱼(Ctenopharyngodon idellus)的生长性能、肌肉营养成分及消化酶活性的影响.渔业科学进展,2017,38(2):99-106]
Zhang A,He L,Wang Y.Prediction of GCRV virus-host protein interactome based on structural motif-domain interactions.BMC Bioinformatics,2017,18(1):145
Zhang J,Zhang M,Sun L.Junctional adhesion molecule A of red drum(Sciaenops ocellatus):A possible immunomodulator and a target for bacterial immune evasion.Veterinary Immunology and Immunopathology,2014,161(1-2):99-107
Zhao R,Lou FR,Ding FH,et al.The nutritional value and processing quality of Ctenopharyngodon idellus,Liza haematocheila,and Centropristis striata.Progress in Fishery Sciences,2016,37(6):62-67[赵睿,娄方瑞,丁福红,等.草鱼(Ctenopharyngodon idellus)、梭鱼(Liza haematocheila)、黑石斑鱼(Centropristis striata)的营养成分及加工品质比较.渔业科学进展,2016,37(6):62-67]
Zhao Z,Vajen T,Karshovska E,et al.Deletion of junctional adhesion molecule A from platelets increases early-stage neointima formation after wire injury in hyperlipidemic mice.Journal of Cellular and Molecular Medicine,2017,21(8):1523-1531
Aurrand-Lions M,Johnson-Leger C,Wong C,et al.Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members.Blood,2001,98(13):3699-3707
Banerjee M,Johnson JE.Activation,exposure and penetration of virally encoded,membrane-active polypeptides during nonenveloped virus entry.Current Protein and Peptide Science,2008,9(1):16-27
Bartesaghi A,Merk A,Borgnia MJ,et al.Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryoelectron microscopy.Nature Structural and Molecular Biology,2013,20(12):1352-1357
Bradfield PF,Nourshargh S,Aurrand-Lions M,et al.JAMfamily and related proteins in leukocyte migration(Vestweber series).Arteriosclerosis,Thrombosis,and Vascular Biology,2005,27(10):2104-2112
Chi YY,Tian YY,Ye X,et al.Molecular properties of grass carp reovirus in southern China and establishment of a duplex PCR detection method.Chinese Journal of Virology,2011,27(4):358-364[迟妍妍,田园园,叶星,等.南方养殖草鱼呼肠孤病毒的分子特性比较及双重PCR检测方法的建立.病毒学报,2011,27(4):358-364]
Danthi P,Guglielmi KM,Kirchner E,et al.From touchdown to transcription:The reovirus cell entry pathway.Cell Entry by Non-Enveloped Viruses,2010,343:91-119
Danthi P,Holm GH,Stehle T,et al.Reovirus receptors,cell entry,and proapoptotic signaling.Advances in Experimental Medicine and Biology,2013,790:42-71
Du F,Su J,Huang R,et al.Cloning and preliminary functional studies of the JAM-A gene in grass carp(Ctenopharyngodon idellus).Fish and Shellfish Immunology,2013,34(6):1476-1484
Guglielmi KM,Kirchner E,Holm GH,et al.Reovirus binding determinants in junctional adhesion molecule-A.Journal of Biological Chemistry,2007,282(24):17930-17940
Harrison SC.Viral membrane fusion.Virology,2015,479-480:498-507
Kim J,Tao Y,Reinisch KM,et al.Orthoreovirus and Aquareovirus core proteins:Conserved enzymatic surfaces,but not protein-protein interfaces.Virus Research,2004,101(1):15-28
Kirchner E,Guglielmi KM,Strauss HM,et al.Structure of reovirus sigma1 in complex with its receptor junctional adhesion molecule-A.PLo S Pathogens,2008,4(12):e1000235
Kobayashi I,Kobayashi-Sun J,Kim AD,et al.Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.Nature,2014,512(7514):319-323
Kurose M,Kakuki T,Takano K,et al.Junctional adhesion molecule-A in head and neck squamous cell Carcinoma.Advances in Oto-Rhino-Laryngology,2016,77:92-97
Li HL,Xu SY,Deng GC,et al.Establishment of cell line PSFfrom the fibroblast cell of grass carp snout tissue and studies of its biological characteristics.Journal of Chinese Academy of Fishery Sciences,1988,1(1):1-8[李焕林,许淑英,邓国成,等.草鱼吻端成纤维细胞系PSF的建立及其生物学特性.中国水产科学研究院学报,1988,1(1):1-8]
Li X,Zeng WW,Wang Q,et al.The study on the proliferation of GCRVⅡin different fish cell lines.Journal of Fisheries of China,2016,40(8):1249-1257[李贤,曾伟伟,王庆,等.草鱼呼肠孤病毒Ⅱ型在不同鱼类细胞中的增殖情况.水产学报,2016,40(8):1249-1257]
Makino A,Shimojima M,Miyazawa T,et al.Junctional adhesion molecule 1 is a functional receptor for feline calicivirus.Journal of Virology,2006,80(9):4482-4490
Nibert ML,Duncan R.Bioinformatics of recent aqua-and orthoreovirus isolates from fish:Evolutionary gain or loss of FAST and fiber proteins and taxonomic implications.PLoSOne,2013,8(7):e68607
Pei C,Ke F,Chen ZY,et al.Complete genome sequence and comparative analysis of grass carp reovirus strain 109(GCRe V-109)with other grass carp reovirus strains reveals no significant correlation with regional distribution.Archives of Virology,2014,159(9):2435-2440
Rangel AA,Rockemann DD,Hetrick FM,et al.Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus.Journal of General Virology,1999,80(Pt 9):2399-2402
Rehder D,Iden S,Nasdala I,et al.Junctional adhesion molecule-A participates in the formation of apico-basal polarity through different domains.Experimental Cell Research,2006,312(17):3389-3403
Schulz WL,Haj AK,Schiff LA.Reovirus uses multiple endocytic pathways for cell entry.Journal of Virology,2012,86(23):12665-12675
Severson EA,Parkos CA.Structural determinants of junctional adhesion molecule A(JAM-A)function and mechanisms of intracellular signaling.Current Opinion in Cell Biology,2009,21(5):701-707
Skehel JJ,Wiley DC.Receptor binding and membrane fusion in virus entry:The influenza hemagglutinin.Annual Review of Biochemistry,2000,69:531-569
Tian YY,Jiao ZZ,Dong JJ,et al.Cloning and expression analysis of jam-a genes in grass carp(Ctenopharyngodon idellus).South China Fisheries Science,2017,13(6):30-40[田园园,焦真真,董浚键,等.草鱼jam-a基因的克隆及表达分析.南方水产科学,2017,13(6):30-40]
Tian YY,Jiao ZZ,Dong JJ,et al.Grass carp reovirus-GD108fiber protein is involved in cell attachment.Virus Genes,2017,53(4):613-622
Torres-Flores JM,Silva-Ayala D,Espinoza MA,et al.The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.Virology,2015,475:172-178
Vetrano S,Rescigno M,Cera MR,et al.Unique role of junctional adhesion molecule-a in maintaining mucosal homeostasis in inflammatory bowel disease.Gastroenterology,2008,135(1):173-184
Wang JJ,Liu Y,Yu L,et al.Characterization of grass carp(Ctenopharyngodon idellus)ovary cell line and its susceptibility to spring viremia of carp virus.Progress in Fishery Sciences,2016,37(6):56-61[王津津,刘莹,于力,等.草鱼(Ctenopharyngodon idellus)性腺细胞系(GCO)特性和对鲤春病毒的敏感性.渔业科学进展,2016,37(6):56-61]
Wang S.National fisheries economic statistics bulletin of 2016.China Fisheries,2017(7):23-24[王莎.2016年全国渔业经济统计公报.中国水产,2017(7):23-24]
Xu SY,Li HL,Deng GC,et al.The preparation and immune effect of attenuated live vaccine obtained through cell culture for hemorrhage of grass carp.Journal of Fisheries of China,1994,18(2):110-117[许淑英,李焕林,邓国成,等.草鱼出血病细胞培养弱毒疫苗的制备及其免疫效果.水产学报,1994,18(2):110-117]
Ye X,Tian Y,Deng G,et al.Complete genomic sequence of a reovirus isolated from grass carp in China.Virus Research,2012,163(1):275-283
Yeung D,Manias JL,Stewart DJ,et al.Decreased junctional adhesion molecule-A expression during blood-brain barrier breakdown.Acta Neuropathologica,2008,115(6):635-642
Zeng BH,Liao ZY,Xiang X,et al.Effects of bile acids on growth performance,muscle composition and digestive enzyme activities of Ctenopharyngodon idellus.Progress in Fishery Sciences,2017,38(2):99-106[曾本和,廖增艳,向枭,等.胆汁酸对草鱼(Ctenopharyngodon idellus)的生长性能、肌肉营养成分及消化酶活性的影响.渔业科学进展,2017,38(2):99-106]
Zhang A,He L,Wang Y.Prediction of GCRV virus-host protein interactome based on structural motif-domain interactions.BMC Bioinformatics,2017,18(1):145
Zhang J,Zhang M,Sun L.Junctional adhesion molecule A of red drum(Sciaenops ocellatus):A possible immunomodulator and a target for bacterial immune evasion.Veterinary Immunology and Immunopathology,2014,161(1-2):99-107
Zhao R,Lou FR,Ding FH,et al.The nutritional value and processing quality of Ctenopharyngodon idellus,Liza haematocheila,and Centropristis striata.Progress in Fishery Sciences,2016,37(6):62-67[赵睿,娄方瑞,丁福红,等.草鱼(Ctenopharyngodon idellus)、梭鱼(Liza haematocheila)、黑石斑鱼(Centropristis striata)的营养成分及加工品质比较.渔业科学进展,2016,37(6):62-67]
Zhao Z,Vajen T,Karshovska E,et al.Deletion of junctional adhesion molecule A from platelets increases early-stage neointima formation after wire injury in hyperlipidemic mice.Journal of Cellular and Molecular Medicine,2017,21(8):1523-1531