鲫疱疹病毒ORF31R(CaHV-31R)的特征及其编码蛋白与细胞器共定位
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摘要
鲫疱疹病毒(Carassius auratus herpesvirus, CaHV)是一种引起鲫急性鳃出血症和高死亡率的病毒病原。病毒要借助功能基因与细胞组分(如细胞器)的相互作用才能完成感染与复制。本实验通过生物信息分析、PCR扩增、重组质粒构建及其与细胞器标记质粒共转染鱼细胞(epithelioma papulosum Cyprinid, EPC)的荧光观察,对预测鲫疱疹病毒基因CaHV-31R编码蛋白的特性、亚细胞定位及与细胞器共定位进行研究。结果显示,鲫疱疹病毒编码蛋白CaHV-31R由313个氨基酸组成,含一个跨膜结构域(248～270 aa)和一个RNase E/G家族蛋白的典型结构域(40～182 aa);与5种来源鲤疱疹病毒同源蛋白多重比对发现,与鲤疱疹病毒II型中的日本株ST-J1和中国株SY-C1的同一性较高(分别为100%和80.7%),与鲤疱疹病毒I型代表株CyHV-1的同一性居中(为26.5%),而与鲤疱疹病毒III型中的德国株CyHV-3(或称锦鲤疱疹病毒Koi herpesvirus,KHV)和中国株CyHV-3-GZ11的同一性最低(分别为20.7%和18.2%);经基因扩增和构建真核重组质粒pEGFP-31R,当用其单独转染EPC细胞,绿色荧光信号主要在细胞质中呈弥散分布,少量在细胞质中或在细胞核外围呈点状分布;当用pEGFP-31R与内质网标记质粒pDsRed2-ER或与高尔基体标记质粒pDsRed2-Golgi共转染细胞,绿色荧光信号在胞质中的分布情况与单独转染时不同,分别能与2种有单层膜结构的细胞器—内质网和高尔基体共定位。研究表明,CaHV-31R是鲫疱疹病毒中一个能编码与细胞器共定位蛋白的基因。
Carassius auratus herpesvirus(CaHV) is a pathogen that can cause crucian carp disease with acute gill hemorrhages and high mortality. Interactions of functional genes with cellular components, such as cellular organs,are needed by viruses to complete infection and replication. In the present study, bioinformatic analysis, PCR amplification, gene cloning, constructing recombinant plasmids, and fluorescence observation of the fish cells(epithelioma papulosum Cyprinid, EPC) cotransfected with cellular organs tagged plasmids were used to analyze the characteristics, subcellular localization and colocalization with cellular organs of the protein encoded by the gene CaHV ORF31 R(CaHV-31 R). As the results show, the encoding protein CaHV-31 R was composed of 313 amino acids, which consists of a transmembrane domain(248-270 aa) and a typical domain of RNase E/G protein family(40-182 aa). The multiple alignment with homology proteins from 5 different fish hepesviruses exhibited higher identity(100% and 80.7%) with the Janpanese strain ST-J1 and Chinese strain SY-C1, which were both the members of type II Cyprinid hepesviruses, centered identity(26.5%) with CyHV-1 which was the representative member of type I Cyprinid hepesviruses, and the lowest identity(20.7% and 18.2%) with the Germanic strain CyHV-3(also named Koi herpesvirus, KHV) and Chinese strain(CyHV-3-GZ11), which were both the members of type III Cyprinid hepesviruses. The plasmids pEGFP-31 R was constructed by PCR amplification, gene cloning,and then used to transfect EPC cells. pEGFP-31 R dispersed distribution in cytoplasm and a small part of spotty distribution were found circling around outside the nucleus. CaHV-31 R was used to colocalize with two celluar organs with monolayer membrane in cytoplasm, which was endoplasmic reticulum and Golgi apparatus. The results indicated that CaHV-31 R was a gene of Carassius auratus herpesvirus, and was able to encode a protein that colocalizes with cellular organs.
Carassius auratus herpesvirus(CaHV) is a pathogen that can cause crucian carp disease with acute gill hemorrhages and high mortality. Interactions of functional genes with cellular components, such as cellular organs,are needed by viruses to complete infection and replication. In the present study, bioinformatic analysis, PCR amplification, gene cloning, constructing recombinant plasmids, and fluorescence observation of the fish cells(epithelioma papulosum Cyprinid, EPC) cotransfected with cellular organs tagged plasmids were used to analyze the characteristics, subcellular localization and colocalization with cellular organs of the protein encoded by the gene CaHV ORF31 R(CaHV-31 R). As the results show, the encoding protein CaHV-31 R was composed of 313 amino acids, which consists of a transmembrane domain(248-270 aa) and a typical domain of RNase E/G protein family(40-182 aa). The multiple alignment with homology proteins from 5 different fish hepesviruses exhibited higher identity(100% and 80.7%) with the Janpanese strain ST-J1 and Chinese strain SY-C1, which were both the members of type II Cyprinid hepesviruses, centered identity(26.5%) with CyHV-1 which was the representative member of type I Cyprinid hepesviruses, and the lowest identity(20.7% and 18.2%) with the Germanic strain CyHV-3(also named Koi herpesvirus, KHV) and Chinese strain(CyHV-3-GZ11), which were both the members of type III Cyprinid hepesviruses. The plasmids pEGFP-31 R was constructed by PCR amplification, gene cloning,and then used to transfect EPC cells. pEGFP-31 R dispersed distribution in cytoplasm and a small part of spotty distribution were found circling around outside the nucleus. CaHV-31 R was used to colocalize with two celluar organs with monolayer membrane in cytoplasm, which was endoplasmic reticulum and Golgi apparatus. The results indicated that CaHV-31 R was a gene of Carassius auratus herpesvirus, and was able to encode a protein that colocalizes with cellular organs.
引文
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[22] Davison A J, Kurobe T, Gatherer D, et al. Comparative genomics of carp herpesviruses[J]. Journal of Virology,2013, 87(5):2908-2922.
[23]方进,邓院生,王俊,等.急性病毒性鲫鳃出血病的病理变化[J].中国水产科学,2016, 23(2):336-343.Fang J, Deng Y S, Wang J, et al. Pathological changes of acute viral hemorrhages in the gills of crucian carp[J].Journal of Fishery Sciences of China, 2016, 23(2):336-343(in Chinese).
[24]桂朗,李正秋,张奇亚.牙鲆一株弹状病毒病原的分离与鉴定[J].水生生物学报,2007, 31(3):345-353.Gui L, Li Z Q, Zhang Q Y. Isolation and characterization of a rhabdovirus from diseased flounder Paralichthys olivaceus[J]. Acta Hydrobiologica Sinica, 2007, 31(3):345-353(in Chinese).
[25] Zhang Q Y, Li Z Q, Gui J F. Studies on morphogenesis and cellular interactions of Rana grylio virus in an infected fish cell line[J]. Aquaculture, 1999, 175(3-4):185-197.
[26] Belasco J G. Ribonuclease E:Chopping knife and sculpting tool[J]. Molecular Cell, 2017, 65(1):3-4.
[27] Alt-Bara S, Carpousis A J. RNA degradosomes in bacteria and chloroplasts:classification, distribution and evolution of RNase E homologs[J]. Molecular Microbiology, 2015,97(6):1021-1135.
[28] Johnson D C, Baines J D. Herpesviruses remodel host membranes for virus egress[J]. Nature Reviews Microbiology, 2011,9(5):382-394.
[2]张奇亚,桂建芳.水生病毒学[M].北京:高等教育出版社,2008:122-128.Zhang Q Y, Gui J F. Aquatic Virology[M]. Beijing:Higher Education Press, 2008:122-128(in Chinese).
[3]Walker P J, Winton J R. Emerging viral diseases of fish and shrimp[J]. Veterinary Research, 2010, 41(6):51.
[4]桂建芳,朱作言.水产动物重要经济性状的分子基础及其遗传改良[J].科学通报,2012, 57(15):1751-1760.Gui J F, Zhu Z Y. Molecular basis and genetic improvement of economically important traits in aquaculture animals[J]. Chinese Science Bulletin,2012, 57(15):1751-1760(in Chinese).
[5]Murray A G. Epidemiology of the spread of viral diseases under aquaculture[J]. Current Opinion in Virology,2013, 3(1):74-78.
[6]OIE. Manual of diagnostic tests for aquatic animals[EB/OL]. 2016. http://www.oie.int/international-standardsetting/aquatic-manual/access-online/.
[7]张奇亚,桂建芳.水产动物的病毒基因组及其病毒与宿主的相互作用[J].中国科学:生命科学,2015, 58(2):156-169.Zhang Q Y, Gui J F. Virus genomes and virus-host interactions in aquaculture animals[J]. Science China Life Sciences, 2015, 58(2):156-169(in Chinese).
[8]Gui L, Chinchar V G, Zhang Q Y. Molecular basis of pathogenesis of emerging viruses infecting aquatic animals[J]. Aquaculture and Fisheries, 2018, 3(1):1-5.
[9]Wang J, Gui L, Chen Z Y, et al. Mutations in the Cterminal region affect subcellular localization of crucian carp herpesvirus(CaHV)GPCR[J]. Virus Genes, 2016,52(4):484-494.
[10] ICTV. The ICTV online(10th)report[EB/OL]. 2017.http://www.ictvonline.org/virusTaxonomy.asp.
[11] Michel B, Fournier G, Lieffrig F, et al. Cyprinid herpesvirus 3[J]. Emerging Infectious Diseases, 2010, 16(12):1835-1843.
[12] Gotesman M, Kattlun J, Bergmann S M, et al. CyHV-3:The third cyprinid herpesvirus[J]. Diseases of Aquatic Organisms,2013, 105(2):163-174.
[13] Goodwin A. Herpesviruses in fish[R]. Southern Regional Aquaculture Center, US, 2012.
[14] Waltzek T B, Kelley G O, Stone D M, et al. Koi herpesvirus represents a third cyprinid herpesvirus(CyHV-3)in the family Herpesviridae[J]. Journal of General Virology, 2005, 86(6):1659-1667.
[15] Reed A N, Izume S, Dolan B P, et al. Identification of B cells as a major site for cyprinid herpesvirus 3 latency[J].Journal of Virology,2014, 88(16):9297-9309.
[16] Gao F X, Wang Y, Zhang Q Y, et al. Distinct herpesvirus resistances and immune responses of three gynogenetic clones of gibel carp revealed by comprehensive transcriptomes[J]. BMC Genomics,2017, 18:561.
[17] Li W, Lee X Z, Weng S P, et al. Whole-genome sequence of a novel Chinese cyprinid herpesvirus 3 isolate reveals the existence of a distinct European genotype in East Asia[J]. Veterinary microbiology, 2015, 175(2-4):185-194.
[18] Li L J, Luo Y Z, Gao Z X, et al. Molecular characterisation and prevalence of a new genotype of Cyprinid herpesvirus 2 in mainland China[J]. Canadian Journal of Microbiology,2015, 61(6):381-387.
[19]夏思瑶,王浩,Podok P,等.鲤疱瘆病毒Ⅱ型对异育银鲫背鳍细胞的显微形态与免疫基因表达水平的影响[J].水产学报,2016,40(12):1915-1922.Xia S Y, Wang H, Podok P, et al. The expression analysis of immune genes and microscopic morphology of CCF cells in response to Cyprinid herpesvirus 2 infection[J]. Journal of Fisheries of China, 2016, 40(12):1915-1922(in Chinese).
[20]桂朗,张奇亚.中国水产动物病毒学研究概述[J].水产学报,2019,43(1):168-187.Gui L, Zhang QY. A brief review on aquatic animal virology researches in China[J]. Journal of Fisheries of China, 2019,43(1):168-187(in Chinese).
[21] Zeng X T, Chen Z Y, Deng Y S, et al. Complete genome sequence and architecture of crucian carp Carassius auratus herpesvirus(CaHV)[J]. Archives of Virology, 2016,161(12):3577-3581.
[22] Davison A J, Kurobe T, Gatherer D, et al. Comparative genomics of carp herpesviruses[J]. Journal of Virology,2013, 87(5):2908-2922.
[23]方进,邓院生,王俊,等.急性病毒性鲫鳃出血病的病理变化[J].中国水产科学,2016, 23(2):336-343.Fang J, Deng Y S, Wang J, et al. Pathological changes of acute viral hemorrhages in the gills of crucian carp[J].Journal of Fishery Sciences of China, 2016, 23(2):336-343(in Chinese).
[24]桂朗,李正秋,张奇亚.牙鲆一株弹状病毒病原的分离与鉴定[J].水生生物学报,2007, 31(3):345-353.Gui L, Li Z Q, Zhang Q Y. Isolation and characterization of a rhabdovirus from diseased flounder Paralichthys olivaceus[J]. Acta Hydrobiologica Sinica, 2007, 31(3):345-353(in Chinese).
[25] Zhang Q Y, Li Z Q, Gui J F. Studies on morphogenesis and cellular interactions of Rana grylio virus in an infected fish cell line[J]. Aquaculture, 1999, 175(3-4):185-197.
[26] Belasco J G. Ribonuclease E:Chopping knife and sculpting tool[J]. Molecular Cell, 2017, 65(1):3-4.
[27] Alt-Bara S, Carpousis A J. RNA degradosomes in bacteria and chloroplasts:classification, distribution and evolution of RNase E homologs[J]. Molecular Microbiology, 2015,97(6):1021-1135.
[28] Johnson D C, Baines J D. Herpesviruses remodel host membranes for virus egress[J]. Nature Reviews Microbiology, 2011,9(5):382-394.