Glycosylation of the hemagglutinin protein of H9N2 subtype avian influenza virus influences its replication and virulence in mice
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摘要
N-Linked glycosylation of hemagglutinin(HA) has been demonstrated to regulate the virulence and receptor-binding specificity of avian influenza virus(AIV). In this study, we characterized the variation trend of naturally isolated H9 N2 viruses for the potential N-linked glycosylation sites in HA proteins, and explored any important role of some glycosylation sites. HA genes of 19 H9 N2 subtype AIV strains since 2001 were sequenced and analyzed for the potential glycosylation sites. The results showed that the viruses varied by losing one potential glycosylation site at residues 200 to 202, and having an additional one at residues 295 to 297 over the past few years. Further molecular and single mutation analysis revealed that the N200 Q mutation lost an N-linked glycosylation at positions 200 to 202 of the HA protein and affected the human-derived receptor affinity. We further found that this N-linked glycosylation increased viral productivity in the lung of the infected mice. These findings provide a novel insight on understanding the determinants of host adaption and virulence of H9 N2 viruses in mammals.
N-Linked glycosylation of hemagglutinin(HA) has been demonstrated to regulate the virulence and receptor-binding specificity of avian influenza virus(AIV). In this study, we characterized the variation trend of naturally isolated H9 N2 viruses for the potential N-linked glycosylation sites in HA proteins, and explored any important role of some glycosylation sites. HA genes of 19 H9 N2 subtype AIV strains since 2001 were sequenced and analyzed for the potential glycosylation sites. The results showed that the viruses varied by losing one potential glycosylation site at residues 200 to 202, and having an additional one at residues 295 to 297 over the past few years. Further molecular and single mutation analysis revealed that the N200 Q mutation lost an N-linked glycosylation at positions 200 to 202 of the HA protein and affected the human-derived receptor affinity. We further found that this N-linked glycosylation increased viral productivity in the lung of the infected mice. These findings provide a novel insight on understanding the determinants of host adaption and virulence of H9 N2 viruses in mammals.
N-Linked glycosylation of hemagglutinin(HA) has been demonstrated to regulate the virulence and receptor-binding specificity of avian influenza virus(AIV). In this study, we characterized the variation trend of naturally isolated H9 N2 viruses for the potential N-linked glycosylation sites in HA proteins, and explored any important role of some glycosylation sites. HA genes of 19 H9 N2 subtype AIV strains since 2001 were sequenced and analyzed for the potential glycosylation sites. The results showed that the viruses varied by losing one potential glycosylation site at residues 200 to 202, and having an additional one at residues 295 to 297 over the past few years. Further molecular and single mutation analysis revealed that the N200 Q mutation lost an N-linked glycosylation at positions 200 to 202 of the HA protein and affected the human-derived receptor affinity. We further found that this N-linked glycosylation increased viral productivity in the lung of the infected mice. These findings provide a novel insight on understanding the determinants of host adaption and virulence of H9 N2 viruses in mammals.
引文
Butt K M,Smith G J,Chen H,Zhang L J,Leung Y H,Xu K M,Lim W,Webster R G,Yuen K Y,Peiris J S,Guan Y.2005.Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003.Journal of Clinical Microbiology,43,5760-5767.
Chen R A,Lai H Z,Li L,Liu Y P,Pan W L,Zhang W Y,Xu J H,He D S,Tang Z X.2013.Genetic variation and phylogenetic analysis of hemagglutinin genes of H9 avian influenza viruses isolated in China during 2010-2012.Veterinary Microbiology,165,312-318.
Crecelius D M,Deom C M,Schulze I T.1984.Biological properties of a hemagglutinin mutant of influenza virus selected by host cells.Virology,139,164-177.
Deshpande K L,Fried V A,Ando M,Webster R G.1988.Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.Proceedings of the National Academy of Sciences of the United States of America,84,36-40.
Gallagher P,Henneberry J,Wilson I,Sambrook J,Gething MJ.1988.Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding,transport,and activity of the molecule.The Journal of Cell Biology,107,2059-2073.
Gao Y,Zhang Y,Shinya K,Deng G,Jiang Y,Li Z,Guan Y,Tian G,Li Y,Shi J,Liu L,Zeng X,Bu Z,Xia X,Kawaoka Y,Chen H.2009.Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host.PLoS Pathogens,5,e1000709.
Gu M,Li Q H,Gao R Y,He D C,Xu Y P,Xu H X,Xu L J,Wang X Q,Hu J,Liu X W,Hu S L,Peng D X,Jiao X A,Liu X F.2017.The T160A hemagglutinin substitution affects not only receptor binding property but also transmissibility of H5N1clade 2.3.4 avian influenza virus in guinea pigs.Veterinary Research,48,7.
Gupta R,Brunak S.2002.Prediction of glycosylation across the human proteome and the correlation to protein function.Pacific Symposium Biocomputing,7,312-322.
Herfst S,Imai M,Kawaoka Y,Fouchier R A.2014.Avian influenza virus transmission to mammals.Current Topics in Microbiology and Immunology,385,137-155.
Hoffmann E,Stech J,Guan Y,Webster R G,Perez D R.2001.Universal primer set for the full-length amplification of all influenza A viruses.Archives of Virology,146,2275-2289.
Horimoto T,Kawaoka Y.2001.Pandemic threat posed by avian influenza A viruses.Clinical Microbiology Reviews,14,129-149.
Imai M,Watanabe T,Hatta M,Das S C,Ozawa M,Shinya K,Zhong G,Hanson A,Katsura H,Watanabe S,Li C,Kawakami E,Yamada S,Kiso M,Suzuki Y,Maher E A,Neumann G,Kawaoka Y.2012.Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.Nature,486,420-428.
Kim J II,Park M S.2012.N-linked glycosylation in the hemagglutinin of influenza A viruses.Yonsei Medical Journal,53,886-893.
Li Z,Chen H,Jiao P,Deng G,Tian G,Li Y,Hoffmann E,Webster R G,Matsuoka Y,Yu K.2005.Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model.Journal of Virology,79,12058-12064.
Li Z,Watanabe T,Hatta M,Watanabe S,Nanbo A,Ozawa M,Kakugawa S,Shimojima M,Yamada S,Neumann G,Kawaoka Y.2009.Mutational analysis of conserved amino acids in the influenza A virus nucleoprotein.Journal of Virology,83,4153-4162.
Lin Y P,Shaw M,Gregory V,Cameron K,Lim W,Klimov A,Subbarao K,Guan Y,Krauss S,Shortridge K,Webster R,Cox N,Hay A.2002.Avian-to-human transmission of H9N2subtype influenza A viruses:Relationship between H9N2and H5N1 human isolates.Proceedings of the National Academy of Sciences of the United States of America,97,9654-9658.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method.Methods,25,402-408.
O’Donnell C D,Vogel L,Wright A,Das S R,Wrammert J,Li G M,McCausland M,Zheng N Y,Yewdell J W,Ahmed R,Wilson P C,Subbarao K.2012.Antibody pressure by a human monoclonal antibody targeting the 2009 pandemic H1N1 virus hemagglutinin drives the emergence of A virus with increased virulence in mice.mB io,3,e00120-e00131.
Reed L J,Muench H.1938.Simple method of estimating fifty percent end point.The American Journal of Hygiene,27,493-497.
Schulze I T.1997.Effects of glycosylation on the properties and functions of influenza virus hemagglutinin.Journal of Infectious Diseases,176,S24-S28.
Shen H Q,Yan Z Q,Zeng F G,Liao C T,Zhou Q F,Qin J P,Xie Q M,Bi Y Z,Chen F.2015.Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013.Journal of Veterinary Science,16,317-324.
Sun S,Wang Q,Zhao F,Chen W,Li Z.2012.Prediction of biological functions on glycosylation site migrations in human influenza H1N1 viruses.PLoS ONE,7,e32119.
Sun X,Xu X,Liu Q,Liang D,Li C,He Q,Jiang J,Cui Y,Li J,Zheng L,Guo J,Xiong Y,Yan J.2013.Evidence of avianlike H9N2 influenza A virus among dogs in Guangxi,China.Infection,Genetics and Evolution:Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases,20,471-475.
Sun Y P,Liu J H.2015.H9N2 influenza virus in China:A cause of concern.Protein Cell,6,18-25
Teng Q,Xu D,Shen W,Liu Q,Rong G,Li X,Yan L,Yang J,Chen H,Yu H,Ma W,Li Z.2016.A single mutation at position 190 in hemagglutinin enhances binding affinity for human type sialic acid receptor and replication of H9N2avian influenza virus in mice.Journal of Virology,90,9806-9825.
Vigerust D J,Shepherd V L.2007.Virus glycosylation:Role in virulence and immune interactions.Trends in Microbiology,15,211-218.
Wan H,Perez D R.2007.Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells.Journal of Virology,81,5181-5191.
Wang W,Lu B,Zhou H,Suguitan Jr A L,Cheng X,Subbarao K,Kemble G,Jin H.2010.Glycosylation at 158N of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets.Journal of Virology,84,6570-6577.
Xia J,Cui J Q,He X,Liu Y Y,Yao K C,Cao S J,Han X F,Huang Y.2017.Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China,2013-2016.PLoS ONE,12,e0171564.
Zhao D,Liang L,Wang S,Nakao T,Li Y,Liu L,Guan Y,Fukuyama S,Bu Z,Kawaoka Y,Chen H.2017.Glycosylation of the hemagglutinin protein of H5N1 influenza virus increases its virulence in mice by exacerbating the host immune response.Journal of Virology,91,e02215-e02230.
Zhao Y,Li S,Zhou Y,Song W,Tang Y,Pang Q,Miao Z.2015.Phylogenetic analysis of hemagglutinin genes of H9N2 avian influenza viruses isolated from chickens in Shandong,China,between 1998 and 2013.BioMed Research International,2015,267520.
Zhou P,Zhu W,Gu H,Fu X,Wang L,Zheng Y,He S,Ke C,Wang H,Yuan Z,Ning Z,Qi W,Li S,Zhang G.2014.Avian influenza H9N2 seroprevalence among swine farm residents in China.Journal of Medical Virology,86,597-600.
Chen R A,Lai H Z,Li L,Liu Y P,Pan W L,Zhang W Y,Xu J H,He D S,Tang Z X.2013.Genetic variation and phylogenetic analysis of hemagglutinin genes of H9 avian influenza viruses isolated in China during 2010-2012.Veterinary Microbiology,165,312-318.
Crecelius D M,Deom C M,Schulze I T.1984.Biological properties of a hemagglutinin mutant of influenza virus selected by host cells.Virology,139,164-177.
Deshpande K L,Fried V A,Ando M,Webster R G.1988.Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.Proceedings of the National Academy of Sciences of the United States of America,84,36-40.
Gallagher P,Henneberry J,Wilson I,Sambrook J,Gething MJ.1988.Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding,transport,and activity of the molecule.The Journal of Cell Biology,107,2059-2073.
Gao Y,Zhang Y,Shinya K,Deng G,Jiang Y,Li Z,Guan Y,Tian G,Li Y,Shi J,Liu L,Zeng X,Bu Z,Xia X,Kawaoka Y,Chen H.2009.Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host.PLoS Pathogens,5,e1000709.
Gu M,Li Q H,Gao R Y,He D C,Xu Y P,Xu H X,Xu L J,Wang X Q,Hu J,Liu X W,Hu S L,Peng D X,Jiao X A,Liu X F.2017.The T160A hemagglutinin substitution affects not only receptor binding property but also transmissibility of H5N1clade 2.3.4 avian influenza virus in guinea pigs.Veterinary Research,48,7.
Gupta R,Brunak S.2002.Prediction of glycosylation across the human proteome and the correlation to protein function.Pacific Symposium Biocomputing,7,312-322.
Herfst S,Imai M,Kawaoka Y,Fouchier R A.2014.Avian influenza virus transmission to mammals.Current Topics in Microbiology and Immunology,385,137-155.
Hoffmann E,Stech J,Guan Y,Webster R G,Perez D R.2001.Universal primer set for the full-length amplification of all influenza A viruses.Archives of Virology,146,2275-2289.
Horimoto T,Kawaoka Y.2001.Pandemic threat posed by avian influenza A viruses.Clinical Microbiology Reviews,14,129-149.
Imai M,Watanabe T,Hatta M,Das S C,Ozawa M,Shinya K,Zhong G,Hanson A,Katsura H,Watanabe S,Li C,Kawakami E,Yamada S,Kiso M,Suzuki Y,Maher E A,Neumann G,Kawaoka Y.2012.Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.Nature,486,420-428.
Kim J II,Park M S.2012.N-linked glycosylation in the hemagglutinin of influenza A viruses.Yonsei Medical Journal,53,886-893.
Li Z,Chen H,Jiao P,Deng G,Tian G,Li Y,Hoffmann E,Webster R G,Matsuoka Y,Yu K.2005.Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model.Journal of Virology,79,12058-12064.
Li Z,Watanabe T,Hatta M,Watanabe S,Nanbo A,Ozawa M,Kakugawa S,Shimojima M,Yamada S,Neumann G,Kawaoka Y.2009.Mutational analysis of conserved amino acids in the influenza A virus nucleoprotein.Journal of Virology,83,4153-4162.
Lin Y P,Shaw M,Gregory V,Cameron K,Lim W,Klimov A,Subbarao K,Guan Y,Krauss S,Shortridge K,Webster R,Cox N,Hay A.2002.Avian-to-human transmission of H9N2subtype influenza A viruses:Relationship between H9N2and H5N1 human isolates.Proceedings of the National Academy of Sciences of the United States of America,97,9654-9658.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method.Methods,25,402-408.
O’Donnell C D,Vogel L,Wright A,Das S R,Wrammert J,Li G M,McCausland M,Zheng N Y,Yewdell J W,Ahmed R,Wilson P C,Subbarao K.2012.Antibody pressure by a human monoclonal antibody targeting the 2009 pandemic H1N1 virus hemagglutinin drives the emergence of A virus with increased virulence in mice.mB io,3,e00120-e00131.
Reed L J,Muench H.1938.Simple method of estimating fifty percent end point.The American Journal of Hygiene,27,493-497.
Schulze I T.1997.Effects of glycosylation on the properties and functions of influenza virus hemagglutinin.Journal of Infectious Diseases,176,S24-S28.
Shen H Q,Yan Z Q,Zeng F G,Liao C T,Zhou Q F,Qin J P,Xie Q M,Bi Y Z,Chen F.2015.Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013.Journal of Veterinary Science,16,317-324.
Sun S,Wang Q,Zhao F,Chen W,Li Z.2012.Prediction of biological functions on glycosylation site migrations in human influenza H1N1 viruses.PLoS ONE,7,e32119.
Sun X,Xu X,Liu Q,Liang D,Li C,He Q,Jiang J,Cui Y,Li J,Zheng L,Guo J,Xiong Y,Yan J.2013.Evidence of avianlike H9N2 influenza A virus among dogs in Guangxi,China.Infection,Genetics and Evolution:Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases,20,471-475.
Sun Y P,Liu J H.2015.H9N2 influenza virus in China:A cause of concern.Protein Cell,6,18-25
Teng Q,Xu D,Shen W,Liu Q,Rong G,Li X,Yan L,Yang J,Chen H,Yu H,Ma W,Li Z.2016.A single mutation at position 190 in hemagglutinin enhances binding affinity for human type sialic acid receptor and replication of H9N2avian influenza virus in mice.Journal of Virology,90,9806-9825.
Vigerust D J,Shepherd V L.2007.Virus glycosylation:Role in virulence and immune interactions.Trends in Microbiology,15,211-218.
Wan H,Perez D R.2007.Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells.Journal of Virology,81,5181-5191.
Wang W,Lu B,Zhou H,Suguitan Jr A L,Cheng X,Subbarao K,Kemble G,Jin H.2010.Glycosylation at 158N of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets.Journal of Virology,84,6570-6577.
Xia J,Cui J Q,He X,Liu Y Y,Yao K C,Cao S J,Han X F,Huang Y.2017.Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China,2013-2016.PLoS ONE,12,e0171564.
Zhao D,Liang L,Wang S,Nakao T,Li Y,Liu L,Guan Y,Fukuyama S,Bu Z,Kawaoka Y,Chen H.2017.Glycosylation of the hemagglutinin protein of H5N1 influenza virus increases its virulence in mice by exacerbating the host immune response.Journal of Virology,91,e02215-e02230.
Zhao Y,Li S,Zhou Y,Song W,Tang Y,Pang Q,Miao Z.2015.Phylogenetic analysis of hemagglutinin genes of H9N2 avian influenza viruses isolated from chickens in Shandong,China,between 1998 and 2013.BioMed Research International,2015,267520.
Zhou P,Zhu W,Gu H,Fu X,Wang L,Zheng Y,He S,Ke C,Wang H,Yuan Z,Ning Z,Qi W,Li S,Zhang G.2014.Avian influenza H9N2 seroprevalence among swine farm residents in China.Journal of Medical Virology,86,597-600.