WSSV与IHHNV竞争对虾鳃细胞膜受体的研究
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摘要
本研究对白斑综合征病毒(White Spot Syndrome Virus,WSSV)与传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Haematopoietic Necrosis Virus,IHHNV)能否竞争虾鳃细胞膜上的NM23蛋白受体进行探索。先用蔗糖梯度离心法提纯WSSV的全蛋白,利用病毒覆盖蛋白印迹技术(VOPBA)与对虾鳃细胞膜NM23蛋白作用,将疑似蛋白条带进行LC-MS/MS分析,初步筛选出3种WSSV蛋白,分别为WSSV013、Wsv497和Wsv035,再构建这3种蛋白的原核表达载体,通过VOPBA和免疫共沉淀(co-immunoprecipitation)验证了Wsv497、Wsv035能与NM23蛋白相互作用,WSSV013不能与NM23蛋白相互作用。初步推测WSSV与IHHNV可能竞争对虾鳃细胞膜上的NM23蛋白受体,该结果为今后研究两种病毒竞争细胞膜受体和虾病毒蛋白作用机制提供理论基础。
White spot syndrome and infectious hypodermal and haematopoietic necrosis are primary viral diseases of prawns. Outbreaks of white spot syndrome virus(WSSV) and infectious hypodermal and haematopoietic necrosis virus(IHHNV) arise worldwide and have caused serious economic losses in recent years, including in China. Some researchers found that, given the same conditions, WSSV-infected prawn display lower levels of mortality than IHHNV-infected prawn. Our laboratory previously demonstrated that IHHNV capsid protein(CP) has the ability to combine with NM23 protein in prawn gill membrane. NM23, a kind of nucleoside diphosphate kinase(NDPK), has various biological functions and is located mainly in the cytoplasm and nucleus, but is also evident in the cell membrane. We conducted an experiment to investigate whether WSSV and IHHNV compete with NM23 protein receptor sites on the cell membrane of shrimp gill tissue. First, we purified the total WSSV proteins using sucrose gradient centrifugation, and then we used a virus overlay protein binding assay(VOPBA) to detect protein-protein interaction between the total proteins and NM23. Next, three suspected positive proteins were selected by the combined LC-MS/MS technique: WSSV013, Wsv497 and Wsv035. Wsv035, also known as VP110, is one of the capsule membrane proteins of WSSV and is encoded by ORF035. It contains a membrane structure domain and a RGD locus(namely, the Arg-Gly-Asp motif). Some scholars, using fluorescence assay and competitive inhibition tests, found Wsv035 could stick to the host cell: Wsv035 RGD can form RGDT peptides at the cell surface and it plays a role in WSSV infection. There have been few studies of Wsv497 and WSSV013 proteins, however, and thus their distribution, structure and functions are not yet clear. Therefore, we constructed prokaryotic expression vectors of these three proteins, for purifying WSSV013, Wsv497 and Wsv035 in order to interact with NM23 protein, using VOPBA and co-immunoprecipitation, respectively. The results showed that Wsv497 and Wsv035 have the ability to interact with NM23, while WSSV013 showed an invalid effect on NM23. Accordingly, we surmise that WSSV and IHHNV have the ability to compete with NM23 protein receptors on the cell membrane of prawn gill tissue. This study adds to theories about the mechanism of competition between WSSV and IHHNV for receptor sites on the cell membrane of prawn gill tissue, and lays a foundation for further studies of protein interactions in the context of WSSV and prawn culture.
White spot syndrome and infectious hypodermal and haematopoietic necrosis are primary viral diseases of prawns. Outbreaks of white spot syndrome virus(WSSV) and infectious hypodermal and haematopoietic necrosis virus(IHHNV) arise worldwide and have caused serious economic losses in recent years, including in China. Some researchers found that, given the same conditions, WSSV-infected prawn display lower levels of mortality than IHHNV-infected prawn. Our laboratory previously demonstrated that IHHNV capsid protein(CP) has the ability to combine with NM23 protein in prawn gill membrane. NM23, a kind of nucleoside diphosphate kinase(NDPK), has various biological functions and is located mainly in the cytoplasm and nucleus, but is also evident in the cell membrane. We conducted an experiment to investigate whether WSSV and IHHNV compete with NM23 protein receptor sites on the cell membrane of shrimp gill tissue. First, we purified the total WSSV proteins using sucrose gradient centrifugation, and then we used a virus overlay protein binding assay(VOPBA) to detect protein-protein interaction between the total proteins and NM23. Next, three suspected positive proteins were selected by the combined LC-MS/MS technique: WSSV013, Wsv497 and Wsv035. Wsv035, also known as VP110, is one of the capsule membrane proteins of WSSV and is encoded by ORF035. It contains a membrane structure domain and a RGD locus(namely, the Arg-Gly-Asp motif). Some scholars, using fluorescence assay and competitive inhibition tests, found Wsv035 could stick to the host cell: Wsv035 RGD can form RGDT peptides at the cell surface and it plays a role in WSSV infection. There have been few studies of Wsv497 and WSSV013 proteins, however, and thus their distribution, structure and functions are not yet clear. Therefore, we constructed prokaryotic expression vectors of these three proteins, for purifying WSSV013, Wsv497 and Wsv035 in order to interact with NM23 protein, using VOPBA and co-immunoprecipitation, respectively. The results showed that Wsv497 and Wsv035 have the ability to interact with NM23, while WSSV013 showed an invalid effect on NM23. Accordingly, we surmise that WSSV and IHHNV have the ability to compete with NM23 protein receptors on the cell membrane of prawn gill tissue. This study adds to theories about the mechanism of competition between WSSV and IHHNV for receptor sites on the cell membrane of prawn gill tissue, and lays a foundation for further studies of protein interactions in the context of WSSV and prawn culture.
引文
[1]Walker P J,Gudkovs N,Mohan C V,et al.Longitudinal disease studies in small-holder black tiger shrimp(Penaeus monodon)ponds in Andhra Pradesh,India.II.Multiple WSSV genotypes associated with disease outbreaks in ponds seeded with uninfected postlarvae[J].Aquaculture,2011,319(1–2):18–24.
[2]Durand S,Lightner D V,Redman R M,et al.Ultrastructure and morphogenesis of white spot syndrome baculovirus(WSSV)[J].Dis Aquat Organ,1997,29(3):205–211.
[3]Walker P J,Mohan C V.Viral disease emergence in shrimp aquaculture:origins,impact and the effectiveness of health management strategies[J].Rev Aquac,2009,1(2):125–154.
[4]van Hulten M C W V,Vlak J M.Identification and phylogeny of a protein kinase gene of white spot syndrome virus[J].Virus Genes,2001,22(2):201–207.
[5]Rajan P R,Ramasamy P,Purushothaman V,et al.White spot baculovirus syndrome virus in the Indian shrimp Penaeus Monodon and P.Indicus[J].Aquaculture,2000,184(1–2):31–44.
[6]Wongteerasupaya C,Vickers J E,Sriurairatana S,et al.A non-occluded systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon[J].Dis Aquat Organ,1995,21(1):69–77.
[7]Nadala E C,Loh P C.A comparative study of three different isolates of white spot virus[J].Dis Aquat Organ,1998,33(3):231–234.
[8]Chang Y S,Liu W J,Chou T L,et al.Characterization of white spot syndrome virus envelope protein VP51A and its interaction with viral tegument protein VP26[J].J Virol,2008,82(24):12555–12564.
[9]Tang K F J,Durand S V,White B L,et al.Induced resistance to white spot syndrome virus infection in Penaeus stylirostristhrough pre-infection with infectious hypodermal and hematopoietic necrosis virus—a preliminary study[J].Aquaculture,2003,216(1):19–29.
[10]Melena J,Bayot B,Betancourt I,et al.Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus(WSSV)confers protection against WSSV in Penaeus vannamei(Boone)post-larvae[J].J Fish Dis,2006,29(10):589–600.
[11]Steeg P S,Bevilacqua G,Kopper L,et al.Evidence for a novel gene associated with low tumor metastatic potential2[J].J Natl Cancer Inst,1988,80(3):200–204.
[12]Xu L,Feng R F,Ma Z R.Progress on virus receptors[J].Progress in Veterinary Medicine,2015,36(2):88–92.[徐雷,冯若飞,马忠仁.病毒受体研究进展[J].动物医学进展,2015,36(2):88–92.]
[13]Fan Z,Beresford P J,Oh D Y,et al.Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTLmediated apoptosis,and the nucleosome assembly protein SET is its inhibitor[J].Cell,2003,112(5):659–672.
[14]Postel E H.Multiple biochemical activities of NM23/NDP kinase in gene regulation[J].J Bioenerg Biomembr,2003,35(1):31–40.
[15]Song E J,Kim Y S,Chung J Y,et al.Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J].Biochemistry,2000,39(33):10090–10097.
[16]Pinon V P,Millot G,Munier A,et al.Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines:specific nuclear localization of the B subunit[J].Exp Cell Res,1999,246(2):355–367.
[17]Kaetzel D M,Zhang Q,Yang M,et al.Potential roles of 3′-5′exonuclease activity of NM23-H1 in DNA repair and malignant progression[J].J Bioenerg Biomembr,2006,38(3–4):163–167.
[18]Van Etten J L.Preface to lesser known large ds DNA viruses[J].Curr Topics Microbiol Immunol,2009,328:5–7.
[19]Li L,Lin S,Yang F.Characterization of an envelope protein(VP110)of white spot syndrome virus[J].J Gen Virol,2006,87(7):1909–1915.
[2]Durand S,Lightner D V,Redman R M,et al.Ultrastructure and morphogenesis of white spot syndrome baculovirus(WSSV)[J].Dis Aquat Organ,1997,29(3):205–211.
[3]Walker P J,Mohan C V.Viral disease emergence in shrimp aquaculture:origins,impact and the effectiveness of health management strategies[J].Rev Aquac,2009,1(2):125–154.
[4]van Hulten M C W V,Vlak J M.Identification and phylogeny of a protein kinase gene of white spot syndrome virus[J].Virus Genes,2001,22(2):201–207.
[5]Rajan P R,Ramasamy P,Purushothaman V,et al.White spot baculovirus syndrome virus in the Indian shrimp Penaeus Monodon and P.Indicus[J].Aquaculture,2000,184(1–2):31–44.
[6]Wongteerasupaya C,Vickers J E,Sriurairatana S,et al.A non-occluded systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon[J].Dis Aquat Organ,1995,21(1):69–77.
[7]Nadala E C,Loh P C.A comparative study of three different isolates of white spot virus[J].Dis Aquat Organ,1998,33(3):231–234.
[8]Chang Y S,Liu W J,Chou T L,et al.Characterization of white spot syndrome virus envelope protein VP51A and its interaction with viral tegument protein VP26[J].J Virol,2008,82(24):12555–12564.
[9]Tang K F J,Durand S V,White B L,et al.Induced resistance to white spot syndrome virus infection in Penaeus stylirostristhrough pre-infection with infectious hypodermal and hematopoietic necrosis virus—a preliminary study[J].Aquaculture,2003,216(1):19–29.
[10]Melena J,Bayot B,Betancourt I,et al.Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus(WSSV)confers protection against WSSV in Penaeus vannamei(Boone)post-larvae[J].J Fish Dis,2006,29(10):589–600.
[11]Steeg P S,Bevilacqua G,Kopper L,et al.Evidence for a novel gene associated with low tumor metastatic potential2[J].J Natl Cancer Inst,1988,80(3):200–204.
[12]Xu L,Feng R F,Ma Z R.Progress on virus receptors[J].Progress in Veterinary Medicine,2015,36(2):88–92.[徐雷,冯若飞,马忠仁.病毒受体研究进展[J].动物医学进展,2015,36(2):88–92.]
[13]Fan Z,Beresford P J,Oh D Y,et al.Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTLmediated apoptosis,and the nucleosome assembly protein SET is its inhibitor[J].Cell,2003,112(5):659–672.
[14]Postel E H.Multiple biochemical activities of NM23/NDP kinase in gene regulation[J].J Bioenerg Biomembr,2003,35(1):31–40.
[15]Song E J,Kim Y S,Chung J Y,et al.Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J].Biochemistry,2000,39(33):10090–10097.
[16]Pinon V P,Millot G,Munier A,et al.Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines:specific nuclear localization of the B subunit[J].Exp Cell Res,1999,246(2):355–367.
[17]Kaetzel D M,Zhang Q,Yang M,et al.Potential roles of 3′-5′exonuclease activity of NM23-H1 in DNA repair and malignant progression[J].J Bioenerg Biomembr,2006,38(3–4):163–167.
[18]Van Etten J L.Preface to lesser known large ds DNA viruses[J].Curr Topics Microbiol Immunol,2009,328:5–7.
[19]Li L,Lin S,Yang F.Characterization of an envelope protein(VP110)of white spot syndrome virus[J].J Gen Virol,2006,87(7):1909–1915.