P[5]型猪C组轮状病毒Por2011 VP8*蛋白的表达及糖结合特征研究
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摘要
轮状病毒(Rotaviruses,RVs)是引起人和动物病毒性腹泻的重要病原,本文旨在研究猪C组轮状病毒(Group C rotaviruses,RVCs)VP8*蛋白的受体结合特征。本研究合成一株P[5]型猪C组轮状病毒(Por2011)的VP8*基因,经体外原核表达,采用亲和层析纯化获得VP8*目的蛋白,利用糖点阵实验、寡糖结合实验研究其寡糖结合特征,再通过序列比对和突变分析确定其潜在受体结合位点。结果显示猪C组轮状病毒Por2011 VP8*蛋白与P1抗原特异性结合,但第108位氨基酸突变后的蛋白则不能与P1抗原结合。本研究表明猪C组轮状病毒的某些型别可能以P1抗原为潜在受体,该受体结合位点与人C组轮状病毒的受体结合位点较为接近,这为猪C组轮状病毒感染机制的研究提供了一定的依据和基础。
Rotaviruses(RVs)are one of the most common pathogens causing viral gastroenteritis. In order to explore the glycan-binding specificity of the VP8* protein of porcine group C rotaviruses(RVCs),the VP8*protein of a P[5] porcine group C rotavirus strain(Por2011)was expressed and purified. The glycan-binding specificity of Por2011 VP8* was analyzed by glycans microarray and ELISA-based oligosaccharide-binding assays. The potential glycan binding sites of Por2011 VP8* were explored via the amino-acid mutation assay.The Por2011 VP8* protein showed significant binding to P1 antigen,whereas the VP8* protein with an Y108 L mutation did not show significant binding to P1 antigen. These data suggest that P1 antigen may be a potential attachment factor for the porcine Por2011 rotavirus. According to sequencing analyses,the glycan binding site of por2011 may be similar to that in human group C rotaviruses. This study provides a basis for understanding the infection mechanism of group C rotaviruses and is helpful for their surveillance.
Rotaviruses(RVs)are one of the most common pathogens causing viral gastroenteritis. In order to explore the glycan-binding specificity of the VP8* protein of porcine group C rotaviruses(RVCs),the VP8*protein of a P[5] porcine group C rotavirus strain(Por2011)was expressed and purified. The glycan-binding specificity of Por2011 VP8* was analyzed by glycans microarray and ELISA-based oligosaccharide-binding assays. The potential glycan binding sites of Por2011 VP8* were explored via the amino-acid mutation assay.The Por2011 VP8* protein showed significant binding to P1 antigen,whereas the VP8* protein with an Y108 L mutation did not show significant binding to P1 antigen. These data suggest that P1 antigen may be a potential attachment factor for the porcine Por2011 rotavirus. According to sequencing analyses,the glycan binding site of por2011 may be similar to that in human group C rotaviruses. This study provides a basis for understanding the infection mechanism of group C rotaviruses and is helpful for their surveillance.
引文
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[11]Suzuki T,Hasebe A.A provisional complete genomebased genotyping system for rotavirus species C from terrestrial mammals[J].J Gen Virol,2017,6(10):2647-2662.
[12]Amimo J O,Vlasova A N,Saif L J.Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio,USA and identification of a potential new VP4 genotype[J].Vet Microbiol,2013,164(1-2):27-38.
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[14]Kuga K,Miyazaki A,Suzuki T,Takagi M,Hattori N,Katsuda K,Mase M,Sugiyama M,Tsunemitsu H.Genetic diversity and classification of the outer capsid glycoprotein VP7 of porcine group B rotaviruses[J].Arch Virol,2009,154(11):1785-1795.
[15]Kattoor J J,Saurabh S,Malik Y S,Sircar S,Dhama K,Ghosh S,Bányai K,Kobayashi N,Singh R K.Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene[J].Vet Q,2017,37(12):252-261.
[16]Soma J,Tsunemitsu H,Miyamoto T,Suzuki G,Sasaki T,Suzuki T.Whole-genome analysis of two bovine rotavirus C strains:Shintoku and Toyama[J].Gen Virol,2013,94(1):128-135.
[17]Suzuki T,Hasebe A,Miyazaki A,Tsunemitsu H.Analysis of genetic divergence among strains of porcine rotavirus C,with focus on VP4 and VP7 genotypes in Japan[J].Virus Res,2015,197(2):26-34.
[18]Chang K O,Nielsen P R,Ward L A,Saif L J.Dual infection of gnotobiotic calves with bovine strains of group A and porcine-like group C rotaviruses influences pathogenesis of the group C rotavirus[J].J Virol,1999,73(11):9284-9293.
[19]Gabbay Y B,Borges A A,Oliveira D S,Linhares AC,Mascarenhas J D,Barardi C R,Sim?es C M,Wang Y,Glass R I,Jiang B.Evidence for zoonotic transmission of group C rotaviruses among children in Belem,Brazil[J].J Med Virol,2008,80(9):1666-1674.
[20]Sun X,Wang L,Qi J,Li D,Wang M,Cong X,Peng R,Chai W,Zhang Q,Wang H,Wen H,Gao G F,Tan M,Duan Z.Human group C rotavirus VP8*s recognize type A histoblood group antigens as ligand[J/OL].J Virol,2018,92(11):e00442-18.
[21]Petrice F S,Steven L S.The P blood group system:biochemical,serological,and clinical aspects[J].Transfus Med Rev,1995,9(2):110-122.
[22]Liu Z Y,Lu Y Q,Zhang J B,Pardee K,Wang P G.P1 trisaccharide(Gal1,4Gal-1,4GlcNAc)synthesis by enzyme glycosylation eeactions using recombinant escherichia coli[J].Appl Environ Microbiol,2003,4(4):2110-2115.
[23]David K C,Gugen K,Rafael O.Oligosaccharides and discordant xenotransplantation[J].Immunol Rev,1994,141(10):31-58.
[24]Iwamura K,Furukawa K,Uchikawa M,Sojka B N,Kojima Y,Wiels J,Shiku H,Urano T,Furukawa K.The blood group P1 synthase gene is identical to the Gb3/CD77 synthase gene[J].J Biol Chem,2003,278(45):44429-44438.
[25]Maria D,Grazyna K A,Elena Y K,Nicolai V B,Stanislawa B,Elwira L.Anti-α-galactosyl antibodies recognizing epitopes terminating with a1,4-linked galactose:human natural and mouse monoclonal antiNOR and anti-P1 antibodies[J].Glycobiology,2005,15(9):109-118.
[26]Jacob F,Anugraham M,Pochechueva T,Tse B W,Alam S,Guertler R,Bovin N V,Fedier A,Hacker NF,Huflejt M E,Packer N,Heinzelmann S V.The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration[J].Br JCancer,2014,111(8):1634-1645.
[27]Kaczmarek R,Katarzyna S K,Bere?nicka A,Miko?ajczyk K,Duk M,Majorczyk E,Krop W A,Klausa E,Skowrońska J,Michalewska B,Brojer E,Czerwinski M.Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77synthase and underlie the P1PK blood group system[J/OL].PLoS One,2018,13(4):e0196627
[28]Motswaledi M S,Kasvosve I,Oguntibeju O O.Blood group antigens C,Lub P1 may have a role in HIVinfection in Africans[J/OL].PLoS One,2016,11(2):e0149883.
[2]Jiang B,Tsunemitsu H,Dennehy P H,Oishi I,Brown D,Schnagl R D,Oseto M,Fang Z Y,Avendano L F,Saif L J,Glass R I.Sequence conservation and expression of the gene encoding the outer capsid glycoprotein among human group C rotaviruses of global distribution[J].Arch Virol,1996,141(2):381-390.
[3]Padilla-Noriega L,Dunn S J,Lopez S,Greenberg HB,Arias C F.Identification of two independent neutralization domains on the VP4 trypsin cleavage products VP5*and VP8*of human rotavirus ST3[J].Virology,1995,206(1):148-154.
[4]Jelle M,Max C,Sarah M,McDonald,Houssam A,Krisztián B,Brister J R,Javier B,Mathew D E,Estes M K,Gentsch J R,Miren I G,Reimar J,Carl D K,Vito M,Mertens P C,Osamu N,Viviana P,Mustafizur R,Franco M R,Linda J S,Norma S,Andrej S,Koki T,John T P,Ulrich D,Marc V R.Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group(RCWG)[J].Arch Virol,2011,156(8):1397-1413.
[5]Ciarlet M,Ludert J E,Iturriza-Gomara M,Liprandi F,Gray J J,Desselberger U,Estes M K.Initial interaction of rotavirus strains with N-acetylneuraminic(sialic)acid residues on the cell surface correlates with VP4genotype,not species of origin[J].J Virol,2002,76(8):4087-4095.
[6]Liu Y,Ramelot T A,Huang P,Liu Y,Li Z,Feizi T,Zhong W,Wu F T,Tan M,Kennedy M A,Jiang X.Glycan specificity of P[19]rotavirus and comparison with those of related P genotypes[J].J Virol,2016,90(21):9983-9996.
[7]Jiang X,Liu Y,Tan M.Histo-blood group antigens as receptors for rotavirus,new understanding on rotavirus epidemiology and vaccine strategy[J/OL].Emerging Microbes Infections,2017,6(4):e22.
[8]Bányai K,Kemenesi G,Budinski I,F?ldes F,Zana B,Marton S,Varga K R,Oldal M,Kurucz K,Jakab F.Candidate new rotavirus species in Schreiber′s bats,Serbia[J].Infect Genet Evol,2017,3(48):19-26.
[9]Saif L J,Bohl E H,Theil K W,Cross R F,House J A.Rotavirus-like,calicivirus-like,and 23-nm virus-like particles associated with diarrhea in young pigs[J].JClin Microbiol,1980,12(1):105-111.
[10]Chang K O,Nielsen P R,Ward L A,Saif L J.Dual infection of gnotobiotic calves with bovine strains of group A and porcine-like group C rotaviruses influences pathogenesis of the group C rotavirus[J].J Virol,1999,73(11):9284-9293.
[11]Suzuki T,Hasebe A.A provisional complete genomebased genotyping system for rotavirus species C from terrestrial mammals[J].J Gen Virol,2017,6(10):2647-2662.
[12]Amimo J O,Vlasova A N,Saif L J.Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio,USA and identification of a potential new VP4 genotype[J].Vet Microbiol,2013,164(1-2):27-38.
[13]Vlasova A N,Amimo J O,Saif L J.Porcine RotavirusesEpidemiology,Immune responses and control strategies[J/OL].Viruses,2017,9(3):E48.
[14]Kuga K,Miyazaki A,Suzuki T,Takagi M,Hattori N,Katsuda K,Mase M,Sugiyama M,Tsunemitsu H.Genetic diversity and classification of the outer capsid glycoprotein VP7 of porcine group B rotaviruses[J].Arch Virol,2009,154(11):1785-1795.
[15]Kattoor J J,Saurabh S,Malik Y S,Sircar S,Dhama K,Ghosh S,Bányai K,Kobayashi N,Singh R K.Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene[J].Vet Q,2017,37(12):252-261.
[16]Soma J,Tsunemitsu H,Miyamoto T,Suzuki G,Sasaki T,Suzuki T.Whole-genome analysis of two bovine rotavirus C strains:Shintoku and Toyama[J].Gen Virol,2013,94(1):128-135.
[17]Suzuki T,Hasebe A,Miyazaki A,Tsunemitsu H.Analysis of genetic divergence among strains of porcine rotavirus C,with focus on VP4 and VP7 genotypes in Japan[J].Virus Res,2015,197(2):26-34.
[18]Chang K O,Nielsen P R,Ward L A,Saif L J.Dual infection of gnotobiotic calves with bovine strains of group A and porcine-like group C rotaviruses influences pathogenesis of the group C rotavirus[J].J Virol,1999,73(11):9284-9293.
[19]Gabbay Y B,Borges A A,Oliveira D S,Linhares AC,Mascarenhas J D,Barardi C R,Sim?es C M,Wang Y,Glass R I,Jiang B.Evidence for zoonotic transmission of group C rotaviruses among children in Belem,Brazil[J].J Med Virol,2008,80(9):1666-1674.
[20]Sun X,Wang L,Qi J,Li D,Wang M,Cong X,Peng R,Chai W,Zhang Q,Wang H,Wen H,Gao G F,Tan M,Duan Z.Human group C rotavirus VP8*s recognize type A histoblood group antigens as ligand[J/OL].J Virol,2018,92(11):e00442-18.
[21]Petrice F S,Steven L S.The P blood group system:biochemical,serological,and clinical aspects[J].Transfus Med Rev,1995,9(2):110-122.
[22]Liu Z Y,Lu Y Q,Zhang J B,Pardee K,Wang P G.P1 trisaccharide(Gal1,4Gal-1,4GlcNAc)synthesis by enzyme glycosylation eeactions using recombinant escherichia coli[J].Appl Environ Microbiol,2003,4(4):2110-2115.
[23]David K C,Gugen K,Rafael O.Oligosaccharides and discordant xenotransplantation[J].Immunol Rev,1994,141(10):31-58.
[24]Iwamura K,Furukawa K,Uchikawa M,Sojka B N,Kojima Y,Wiels J,Shiku H,Urano T,Furukawa K.The blood group P1 synthase gene is identical to the Gb3/CD77 synthase gene[J].J Biol Chem,2003,278(45):44429-44438.
[25]Maria D,Grazyna K A,Elena Y K,Nicolai V B,Stanislawa B,Elwira L.Anti-α-galactosyl antibodies recognizing epitopes terminating with a1,4-linked galactose:human natural and mouse monoclonal antiNOR and anti-P1 antibodies[J].Glycobiology,2005,15(9):109-118.
[26]Jacob F,Anugraham M,Pochechueva T,Tse B W,Alam S,Guertler R,Bovin N V,Fedier A,Hacker NF,Huflejt M E,Packer N,Heinzelmann S V.The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration[J].Br JCancer,2014,111(8):1634-1645.
[27]Kaczmarek R,Katarzyna S K,Bere?nicka A,Miko?ajczyk K,Duk M,Majorczyk E,Krop W A,Klausa E,Skowrońska J,Michalewska B,Brojer E,Czerwinski M.Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77synthase and underlie the P1PK blood group system[J/OL].PLoS One,2018,13(4):e0196627
[28]Motswaledi M S,Kasvosve I,Oguntibeju O O.Blood group antigens C,Lub P1 may have a role in HIVinfection in Africans[J/OL].PLoS One,2016,11(2):e0149883.