Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses
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摘要
Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.
Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.
Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.
引文
Fouchier,R.,Schneeberger,P.,Rozendaal,F.,Broekman,J.,Kemink,S.,Munster,V.,Kuiken,T.,Rimmelzwaan,G.,Schutten,M.,Van Doornum,G.,et al.(2004).Avian influenza A virus(H7N7)associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome.Proc Natl Acad Sci USA 101,1356-1361.
Gao,Y.,Zhang,Y.,Shinya,K.,Deng,G.,Jiang,Y.,Li,Z.,Guan,Y.,Tian,G.,Li,Y.,Shi,J.,et al.(2009).Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host.PLoS Pathog 5,e1000709.
Herfst,S.,Schrauwen,E.,Linster,M.,Chutinimitkul,S.,de Wit,E.,Munster,V.,Sorrell,E.,Bestebroer,T.,Burke,D.,Smith,D.,et al.(2012).Airborne transmission of influenza A/H5N1 virus between ferrets.Science 336,1534-1541.
Imai,M.,Watanabe,T.,Hatta,M.,Das,S.,Ozawa,M.,Shinya,K.,Zhong,G.,Hanson,A.,Katsura,H.,Watanabe,S.,et al.(2012).Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.Nature486,420-428.
Jiang,Y.,Yu,K.,Zhang,H.,Zhang,P.,Li,C.,Tian,G.,Li,Y.,Wang,X.,Ge,J.,Bu,Z.,et al.(2007).Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector.Antiviral Res 75,234-241.
Kawaoka,Y.,Naeve,C.,and Webster,R.(1984).Is virulence of H5N2influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin?Virology 139,303-316.
Kimble,J.,Sorrell,E.,Shao,H.,Martin,P.,and Perez,D.(2011).Compatibility of H9N2 avian influenza surface genes and 2009pandemic H1N1 internal genes for transmission in the ferret model.Proc Natl Acad Sci USA 108,12084-12088.
K?hler,G.,and Milstein,C.(1976).Derivation of specific antibodyproducing tissue culture and tumor lines by cell fusion.Eur J Immunol6,511-519.
Li,C.,Bu,Z.,and Chen,H.(2014a).Avian influenza vaccines against H5N1‘bird flu’.Trends Biotech 32,147-156.
Li,X.,Shi,J.,Guo,J.,Deng,G.,Zhang,Q.,Wang,J.,He,X.,Wang,K.,Chen,J.,Li,Y.,et al.(2014b).Genetics,receptor binding property,and transmissibility in mammals of naturally isolated H9N2 Avian Influenza viruses.PLoS Pathog 10,e1004508.
Li,Y.,Shi,J.,Zhong,G.,Deng,G.,Tian,G.,Ge,J.,Zeng,X.,Song,J.,Zhao,D.,Liu,L.,et al.(2010).Continued evolution of H5N1 influenza viruses in wild birds,domestic poultry,and humans in China from 2004to 2009.J Virol 84,8389-8397.
Li,Z.,Chen,H.,Jiao,P.,Deng,G.,Tian,G.,Li,Y.,Hoffmann,E.,Webster,R.G.,Matsuoka,Y.,and Yu,K.(2005).Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model.J Virol 79,12058-12064.
Liu,L.,Zeng,X.,Chen,P.,Deng,G.,Li,Y.,Shi,J.,Gu,C.,Kong,H.,Suzuki,Y.,Jiang,Y.,et al.(2016).Characterization of clade 7.2 H5avian influenza viruses that continue to circulate in chickens in China.JVirol 90,9797-9805.
Neumann,G.,and Kawaoka,Y.(2006).Host range restriction and pathogenicity in the context of influenza pandemic.Emerg Infect Dis12,881-886.
Ping,J.,Li,C.,Deng,G.,Jiang,Y.,Tian,G.,Zhang,S.,Bu,Z.,and Chen,H.(2008).Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody.Biochem BioPhys Res Commun 371,168-171.
Senne,D.,Panigrahy,B.,Kawaoka,Y.,Pearson,J.,Suss,J.,Lipkind,M.,Kida,H.,and Webster,R.(1996).Survey of the hemagglutinin(HA)cleavage site sequence of H5 and H7 avian influenza viruses:amino acid sequence at the HA cleavage site as a marker of pathogenicity potential.Avian Dis 40,425-437.
Shi,J.,Deng,G.,Kong,H.,Gu,C.,Ma,S.,Yin,X.,Zeng,X.,Cui,P.,Chen,Y.,Yang,H.,et al.(2017).H7N9 virulent mutants detected in chickens in China pose an increased threat to humans.Cell Res 27,1409-1421.
Shi,J.,Deng,G.,Ma,S.,Zeng,X.,Yin,X.,Li,M.,Zhang,B.,Cui,P.,Chen,Y.,Yang,H.,et al.(2018).Rapid evolution of H7N9 highly pathogenic viruses that emerged in China in 2017.Cell Host Microbe24,558-568.e7.
Skehel,J.,Stevens,D.,Daniels,R.,Douglas,A.,Knossow,M.,Wilson,I.,and Wiley,D.(1984).A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody..Proc Natl Acad Sci USA 81,1779-1783.
Smith,D.,Lapedes,A.,de Jong,J.,Bestebroer,T.,Rimmelzwaan,G.,Osterhaus,A.,and Fouchier,R.(2004).Mapping the antigenic and genetic evolution of influenza virus.Science 305,371-376.
Sorrell,E.,Wan,H.,Araya,Y.,Song,H.,and Perez,D.(2009).Minimal molecular constraints for respiratory droplet transmission of an avianhuman H9N2 influenza A virus.Proc Natl Acad Sci USA 106,7565-7570.
Swayne,D.(2012).Impact of vaccines and vaccination on global control of avian influenza.Avian Dis 56,818-828.
Wang,J.,Zeng,Y.,Xu,S.,Yang,J.,Wang,W.,Zhong,B.,Ge,J.,Yin,L.,Bu,Z.,Shu,H.,et al.(2018).A naturally occurring deletion in the effector domain of H5N1 swine influenza virus nonstructural protein 1regulates viral fitness and host innate immunity.J Virol 92,pii:e00149-18.
Waterhouse,A.,Bertoni,M.,Bienert,S.,Studer,G.,Tauriello,G.,Gumienny,R.,Heer,F.,de Beer,T.,Rempfer,C.,Bordoli,L.,et al.(2018).SWISS-MODEL:homology modelling of protein structures and complexes.Nucleic Acids Res 46,W296-W303.
Wei,X.,and Cui,J.(2018).Why were so few people infected with H7N9influenza A viruses in China from late 2017 to 2018?Sci China Life Sci61,1442-1444.
WHO-OIE-FAO H5 evolution working group.(2008).Toward a unified nomenclature system for highly pathogenic avian influenza virus(H5N1).Emerg Infect Dis 14,e1.
World Health Organization.(2014).Influenza virus infections in humans(February 2014).http://www.who.int/influenza/human_animal_interface/virology_laboratories_and_vaccines/influenza_virus_infections_humans_feb14.pdf?ua=1.
World Health Organization.(2018a).Influenza at the human-animal interface.http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_02_03_2018.pdf?ua=2011.
World Health Organization.(2018b).Cumulative number of confirmed human cases of avian influenza A(H5N1)reported to WHO.http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/.
Zeng,X.,Tian,G.,Shi,J.,Deng,G.,Li,C.,and Chen,H.(2018).Vaccination of poultry successfully eliminated human infection with H7N9 virus in China.Sci China Life Sci 61,doi:https://doi.org/10.1007/s11427-018-9420-1.
Zhang,Q.,Shi,J.,Deng,G.,Guo,J.,Zeng,X.,He,X.,Kong,H.,Gu,C.,Li,X.,Liu,J.,et al.(2013a).H7N9 influenza viruses are transmissible in ferrets by respiratory droplet.Science 341,410-414.
Zhang,Y.,Zhang,Q.,Kong,H.,Jiang,Y.,Gao,Y.,Deng,G.,Shi,J.,Tian,G.,Liu,L.,Liu,J.,et al.(2013b).H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet.Science 340,1459-1463.
Zhao,D.,Liang,L.,Wang,S.,Nakao,T.,Li,Y.,Liu,L.,Guan,Y.,Fukuyama,S.,Bu,Z.,Kawaoka,Y.,et al.(2017).Glycosylation of the hemagglutinin protein of H5N1 influenza virus increases its virulence in mice by exacerbating the host immune response.J Virol 91,pii:e02215-16.
Zhu,Q.,Yang,H.,Chen,W.,Cao,W.,Zhong,G.,Jiao,P.,Deng,G.,Yu,K.,Yang,C.,Bu,Z.,et al.(2008).A naturally occurring deletion in its NSgene contributes to the attenuation of an H5N1 swine influenza virus in chickens.J Virol 82,220-228.
Gao,Y.,Zhang,Y.,Shinya,K.,Deng,G.,Jiang,Y.,Li,Z.,Guan,Y.,Tian,G.,Li,Y.,Shi,J.,et al.(2009).Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host.PLoS Pathog 5,e1000709.
Herfst,S.,Schrauwen,E.,Linster,M.,Chutinimitkul,S.,de Wit,E.,Munster,V.,Sorrell,E.,Bestebroer,T.,Burke,D.,Smith,D.,et al.(2012).Airborne transmission of influenza A/H5N1 virus between ferrets.Science 336,1534-1541.
Imai,M.,Watanabe,T.,Hatta,M.,Das,S.,Ozawa,M.,Shinya,K.,Zhong,G.,Hanson,A.,Katsura,H.,Watanabe,S.,et al.(2012).Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.Nature486,420-428.
Jiang,Y.,Yu,K.,Zhang,H.,Zhang,P.,Li,C.,Tian,G.,Li,Y.,Wang,X.,Ge,J.,Bu,Z.,et al.(2007).Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector.Antiviral Res 75,234-241.
Kawaoka,Y.,Naeve,C.,and Webster,R.(1984).Is virulence of H5N2influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin?Virology 139,303-316.
Kimble,J.,Sorrell,E.,Shao,H.,Martin,P.,and Perez,D.(2011).Compatibility of H9N2 avian influenza surface genes and 2009pandemic H1N1 internal genes for transmission in the ferret model.Proc Natl Acad Sci USA 108,12084-12088.
K?hler,G.,and Milstein,C.(1976).Derivation of specific antibodyproducing tissue culture and tumor lines by cell fusion.Eur J Immunol6,511-519.
Li,C.,Bu,Z.,and Chen,H.(2014a).Avian influenza vaccines against H5N1‘bird flu’.Trends Biotech 32,147-156.
Li,X.,Shi,J.,Guo,J.,Deng,G.,Zhang,Q.,Wang,J.,He,X.,Wang,K.,Chen,J.,Li,Y.,et al.(2014b).Genetics,receptor binding property,and transmissibility in mammals of naturally isolated H9N2 Avian Influenza viruses.PLoS Pathog 10,e1004508.
Li,Y.,Shi,J.,Zhong,G.,Deng,G.,Tian,G.,Ge,J.,Zeng,X.,Song,J.,Zhao,D.,Liu,L.,et al.(2010).Continued evolution of H5N1 influenza viruses in wild birds,domestic poultry,and humans in China from 2004to 2009.J Virol 84,8389-8397.
Li,Z.,Chen,H.,Jiao,P.,Deng,G.,Tian,G.,Li,Y.,Hoffmann,E.,Webster,R.G.,Matsuoka,Y.,and Yu,K.(2005).Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model.J Virol 79,12058-12064.
Liu,L.,Zeng,X.,Chen,P.,Deng,G.,Li,Y.,Shi,J.,Gu,C.,Kong,H.,Suzuki,Y.,Jiang,Y.,et al.(2016).Characterization of clade 7.2 H5avian influenza viruses that continue to circulate in chickens in China.JVirol 90,9797-9805.
Neumann,G.,and Kawaoka,Y.(2006).Host range restriction and pathogenicity in the context of influenza pandemic.Emerg Infect Dis12,881-886.
Ping,J.,Li,C.,Deng,G.,Jiang,Y.,Tian,G.,Zhang,S.,Bu,Z.,and Chen,H.(2008).Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody.Biochem BioPhys Res Commun 371,168-171.
Senne,D.,Panigrahy,B.,Kawaoka,Y.,Pearson,J.,Suss,J.,Lipkind,M.,Kida,H.,and Webster,R.(1996).Survey of the hemagglutinin(HA)cleavage site sequence of H5 and H7 avian influenza viruses:amino acid sequence at the HA cleavage site as a marker of pathogenicity potential.Avian Dis 40,425-437.
Shi,J.,Deng,G.,Kong,H.,Gu,C.,Ma,S.,Yin,X.,Zeng,X.,Cui,P.,Chen,Y.,Yang,H.,et al.(2017).H7N9 virulent mutants detected in chickens in China pose an increased threat to humans.Cell Res 27,1409-1421.
Shi,J.,Deng,G.,Ma,S.,Zeng,X.,Yin,X.,Li,M.,Zhang,B.,Cui,P.,Chen,Y.,Yang,H.,et al.(2018).Rapid evolution of H7N9 highly pathogenic viruses that emerged in China in 2017.Cell Host Microbe24,558-568.e7.
Skehel,J.,Stevens,D.,Daniels,R.,Douglas,A.,Knossow,M.,Wilson,I.,and Wiley,D.(1984).A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody..Proc Natl Acad Sci USA 81,1779-1783.
Smith,D.,Lapedes,A.,de Jong,J.,Bestebroer,T.,Rimmelzwaan,G.,Osterhaus,A.,and Fouchier,R.(2004).Mapping the antigenic and genetic evolution of influenza virus.Science 305,371-376.
Sorrell,E.,Wan,H.,Araya,Y.,Song,H.,and Perez,D.(2009).Minimal molecular constraints for respiratory droplet transmission of an avianhuman H9N2 influenza A virus.Proc Natl Acad Sci USA 106,7565-7570.
Swayne,D.(2012).Impact of vaccines and vaccination on global control of avian influenza.Avian Dis 56,818-828.
Wang,J.,Zeng,Y.,Xu,S.,Yang,J.,Wang,W.,Zhong,B.,Ge,J.,Yin,L.,Bu,Z.,Shu,H.,et al.(2018).A naturally occurring deletion in the effector domain of H5N1 swine influenza virus nonstructural protein 1regulates viral fitness and host innate immunity.J Virol 92,pii:e00149-18.
Waterhouse,A.,Bertoni,M.,Bienert,S.,Studer,G.,Tauriello,G.,Gumienny,R.,Heer,F.,de Beer,T.,Rempfer,C.,Bordoli,L.,et al.(2018).SWISS-MODEL:homology modelling of protein structures and complexes.Nucleic Acids Res 46,W296-W303.
Wei,X.,and Cui,J.(2018).Why were so few people infected with H7N9influenza A viruses in China from late 2017 to 2018?Sci China Life Sci61,1442-1444.
WHO-OIE-FAO H5 evolution working group.(2008).Toward a unified nomenclature system for highly pathogenic avian influenza virus(H5N1).Emerg Infect Dis 14,e1.
World Health Organization.(2014).Influenza virus infections in humans(February 2014).http://www.who.int/influenza/human_animal_interface/virology_laboratories_and_vaccines/influenza_virus_infections_humans_feb14.pdf?ua=1.
World Health Organization.(2018a).Influenza at the human-animal interface.http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_02_03_2018.pdf?ua=2011.
World Health Organization.(2018b).Cumulative number of confirmed human cases of avian influenza A(H5N1)reported to WHO.http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/.
Zeng,X.,Tian,G.,Shi,J.,Deng,G.,Li,C.,and Chen,H.(2018).Vaccination of poultry successfully eliminated human infection with H7N9 virus in China.Sci China Life Sci 61,doi:https://doi.org/10.1007/s11427-018-9420-1.
Zhang,Q.,Shi,J.,Deng,G.,Guo,J.,Zeng,X.,He,X.,Kong,H.,Gu,C.,Li,X.,Liu,J.,et al.(2013a).H7N9 influenza viruses are transmissible in ferrets by respiratory droplet.Science 341,410-414.
Zhang,Y.,Zhang,Q.,Kong,H.,Jiang,Y.,Gao,Y.,Deng,G.,Shi,J.,Tian,G.,Liu,L.,Liu,J.,et al.(2013b).H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet.Science 340,1459-1463.
Zhao,D.,Liang,L.,Wang,S.,Nakao,T.,Li,Y.,Liu,L.,Guan,Y.,Fukuyama,S.,Bu,Z.,Kawaoka,Y.,et al.(2017).Glycosylation of the hemagglutinin protein of H5N1 influenza virus increases its virulence in mice by exacerbating the host immune response.J Virol 91,pii:e02215-16.
Zhu,Q.,Yang,H.,Chen,W.,Cao,W.,Zhong,G.,Jiao,P.,Deng,G.,Yu,K.,Yang,C.,Bu,Z.,et al.(2008).A naturally occurring deletion in its NSgene contributes to the attenuation of an H5N1 swine influenza virus in chickens.J Virol 82,220-228.