title: Glycosylation of the Hemagglutinin Protein of H5N1 Influenza Virus Increases Its Virulence in Mice by Exacerbating the Host Immune Response.
abstract: The highly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in nature and threaten public health. Although several viral determinants and host factors that influence the virulence of HPAI H5N1 viruses in mammals have been identified, the detailed molecular mechanism remains poorly defined and requires further clarification. In our previous studies, we characterized two naturally isolated HPAI H5N1 viruses that had similar viral genomes but differed substantially in their lethality in mice. In this study, we explored the molecular determinants and potential mechanism for this difference in virulence. By using reverse genetics, we found that a single amino acid at position 158 of the hemagglutinin (HA) protein substantially affected the systemic replication and pathogenicity of these H5N1 influenza viruses in mice. We further found that the G158N mutation introduced an N-linked glycosylation at positions 158 to 160 of the HA protein and that this N-linked glycosylation enhanced viral productivity in infected mammalian cells and induced stronger host immune and inflammatory responses to viral infection. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.IMPORTANCEHighly pathogenic avian influenza (HPAI) H5N1 viruses continue to evolve in nature and threaten human health. Key mutations in the virus hemagglutinin (HA) protein or reassortment with other pandemic viruses endow HPAI H5N1 viruses with the potential for aerosol transmissibility in mammals. A thorough understanding of the pathogenic mechanisms of these viruses will help us to develop more effective control strategies; however, such mechanisms and virulent determinants for H5N1 influenza viruses have not been fully elucidated. In this study, we identified glycosylation at positions 158 to 160 of the HA protein of two naturally occurring H5N1 viruses as an important virulence determinant. This glycosylation event enhanced viral productivity, exacerbated the host response, and thereby contributed to the high pathogenicity of H5N1 virus in mice.
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.|Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Dongming
authors.initials: D
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Libin
authors.initials: L
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.affiliation: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Nakao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Liling
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Guan
authors.affiliation: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fukuyama
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Zhigao
authors.initials: Z
authors.lastname: Bu
authors.affiliation: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.|Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawaoka
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China chenhualan@caas.cn.
authors.forename: Hualan
authors.initials: H
authors.lastname: Chen
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D049109:Cell Proliferation; D004285:Dogs; D005260:Female; D006031:Glycosylation; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007113:Immunity, Innate; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000472:pathogenicity*; D061985:Madin Darby Canine Kidney Cells; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000276:immunology / Q000821:virology*; D011499:Protein Processing, Post-Translational; D014774:Virulence; D014779:Virus Replication
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; C483468:hemagglutinin, human influenza A virus
keywords: H5N1 viruses; glycosylation; mouse; pathogenicity