1887

Abstract

There are three conserved -linked glycosites, namely, Asn10, Asn23 and Asn286, in the stem region of haemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/Huadong/S/2005. The results indicated that these three -glycans were dispensable for the generation of replication-competent influenza viruses. However, when -glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF cells in comparison with that of the WT virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the -glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role in HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.

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2015-06-01
2024-03-28
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References

  1. Bommakanti G., Citron M. P., Hepler R. W., Callahan C., Heidecker G. J., Najar T. A., Lu X., Joyce J. G., Shiver J. W. et al. 2010; Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci U S A 107:13701–13706 [View Article][PubMed]
    [Google Scholar]
  2. Brown J. D., Swayne D. E., Cooper R. J., Burns R. E., Stallknecht D. E. 2007; Persistence of H5 and H7 avian influenza viruses in water. Avian Dis 51:(Suppl.)285–289 [View Article][PubMed]
    [Google Scholar]
  3. Brown J. D., Goekjian G., Poulson R., Valeika S., Stallknecht D. E. 2009; Avian influenza virus in water: infectivity is dependent on pH, salinity and temperature. Vet Microbiol 136:20–26 [View Article][PubMed]
    [Google Scholar]
  4. Chen H., Deng G., Li Z., Tian G., Li Y., Jiao P., Zhang L., Liu Z., Webster R. G., Yu K. 2004; The evolution of H5N1 influenza viruses in ducks in southern China. Proc Natl Acad Sci U S A 101:10452–10457 [View Article][PubMed]
    [Google Scholar]
  5. Chen W., Zhong Y., Qin Y., Sun S., Li Z. 2012; The evolutionary pattern of glycosylation sites in influenza virus (H5N1) hemagglutinin and neuraminidase. PLoS ONE 7:e49224 [View Article][PubMed]
    [Google Scholar]
  6. Claas E. C., Osterhaus A. D., van Beek R., De Jong J. C., Rimmelzwaan G. F., Senne D. A., Krauss S., Shortridge K. F., Webster R. G. 1998; Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351:472–477 [View Article][PubMed]
    [Google Scholar]
  7. Deshpande K. L., Fried V. A., Ando M., Webster R. G. 1987; Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc Natl Acad Sci U S A 84:36–40 [View Article][PubMed]
    [Google Scholar]
  8. Ekiert D. C., Bhabha G., Elsliger M. A., Friesen R. H., Jongeneelen M., Throsby M., Goudsmit J., Wilson I. A. 2009; Antibody recognition of a highly conserved influenza virus epitope. Science 324:246–251 [View Article][PubMed]
    [Google Scholar]
  9. Gao Y., Zhang Y., Shinya K., Deng G., Jiang Y., Li Z., Guan Y., Tian G., Li Y. 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 [View Article][PubMed]
    [Google Scholar]
  10. Gu M., Zhao G., Zhao K., Zhong L., Huang J., Wan H., Wang X., Liu W., Liu H. et al. 2013; Novel variants of clade 2.3.4 highly pathogenic avian influenza A(H5N1) viruses, China. Emerg Infect Dis 19:2021–2024 [View Article][PubMed]
    [Google Scholar]
  11. Hartshorn K. L., Webby R., White M. R., Tecle T., Pan C., Boucher S., Moreland R. J., Crouch E. C., Scheule R. K. 2008; Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D. Respir Res 9:65 [View Article][PubMed]
    [Google Scholar]
  12. Hoffmann E., Neumann G., Kawaoka Y., Hobom G., Webster R. G. 2000; A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97:6108–6113 [View Article][PubMed]
    [Google Scholar]
  13. Kawaoka Y., Webster R. G. 1989; Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin. J Virol 63:3296–3300[PubMed]
    [Google Scholar]
  14. Killian M. L. 2008; Hemagglutination assay for the avian influenza virus. Methods Mol Biol 436:47–52[PubMed]
    [Google Scholar]
  15. Kim J. I., Park M. S. 2012; N-linked glycosylation in the hemagglutinin of influenza A viruses. Yonsei Med J 53:886–893 [View Article][PubMed]
    [Google Scholar]
  16. Kobayashi Y., Suzuki Y. 2012; Evidence for N-glycan shielding of antigenic sites during evolution of human influenza A virus hemagglutinin. J Virol 86:3446–3451 [View Article][PubMed]
    [Google Scholar]
  17. Li C., Chen H. 2014; Enhancement of influenza virus transmission by gene reassortment. Curr Top Microbiol Immunol 385:185–204[PubMed]
    [Google Scholar]
  18. Li Y., Shi J., Zhong G., Deng G., Tian G., Ge J., Zeng X., Song J., Zhao D. et al. 2010; Continued evolution of H5N1 influenza viruses in wild birds, domestic poultry, and humans in China from 2004 to 2009. J Virol 84:8389–8397 [View Article][PubMed]
    [Google Scholar]
  19. Liao H. Y., Hsu C. H., Wang S. C., Liang C. H., Yen H. Y., Su C. Y., Chen C. H., Jan J. T., Ren C. T. et al. 2010; Differential receptor binding affinities of influenza hemagglutinins on glycan arrays. J Am Chem Soc 132:14849–14856 [View Article][PubMed]
    [Google Scholar]
  20. Matrosovich M., Zhou N., Kawaoka Y., Webster R. 1999; The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73:1146–1155[PubMed]
    [Google Scholar]
  21. Negovetich N. J., Webster R. G. 2010; Thermostability of subpopulations of H2N3 influenza virus isolates from mallard ducks. J Virol 84:9369–9376 [View Article][PubMed]
    [Google Scholar]
  22. Ohuchi M., Ohuchi R., Feldmann A., Klenk H. D. 1997a). Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J Virol 71:8377–8384[PubMed]
    [Google Scholar]
  23. Ohuchi R., Ohuchi M., Garten W., Klenk H. D. 1997b). Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity. J Virol 71:3719–3725[PubMed]
    [Google Scholar]
  24. Proença-Módena J. L., Macedo I. S., Arruda E. 2007; H5N1 avian influenza virus: an overview. Braz J Infect Dis 11:125–133 [View Article][PubMed]
    [Google Scholar]
  25. Reading P. C., Pickett D. L., Tate M. D., Whitney P. G., Job E. R., Brooks A. G. 2009; Loss of a single N-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice. Respir Res 10:117 [View Article][PubMed]
    [Google Scholar]
  26. Roberts P. C., Garten W., Klenk H.-D. 1993; Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin. J Virol 67:3048–3060[PubMed]
    [Google Scholar]
  27. Rossman J. S., Lamb R. A. 2011; Influenza virus assembly and budding. Virology 411:229–236 [View Article][PubMed]
    [Google Scholar]
  28. Skehel J. J., Wiley D. C. 2000; Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531–569 [View Article][PubMed]
    [Google Scholar]
  29. Skehel J. J., Stevens D. J., Daniels R. S., Douglas A. R., Knossow M., Wilson I. A., Wiley D. C. 1984; A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci U S A 81:1779–1783 [View Article][PubMed]
    [Google Scholar]
  30. Song J., Feng H., Xu J., Zhao D., Shi J., Li Y., Deng G., Jiang Y., Li X. et al. 2011; The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks. J Virol 85:2180–2188 [View Article][PubMed]
    [Google Scholar]
  31. Subbarao K., Klimov A., Katz J., Regnery H., Lim W., Hall H., Perdue M., Swayne D., Bender C. et al. 1998; Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279:393–396 [View Article][PubMed]
    [Google Scholar]
  32. Sui J., Hwang W. C., Perez S., Wei G., Aird D., Chen L. M., Santelli E., Stec B., Cadwell G. et al. 2009; Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16:265–273 [View Article][PubMed]
    [Google Scholar]
  33. Takahashi T., Kurebayashi Y., Ikeya K., Mizuno T., Fukushima K., Kawamoto H., Kawaoka Y., Suzuki Y., Suzuki T. 2010; The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication. PLoS ONE 5:e15556 [View Article][PubMed]
    [Google Scholar]
  34. Tang Y. H., Wu P. P., Sun Q., Peng D. X., Zhang W. J., Li Y. F., Wang W. B., Long J. X., Zhang P. H., Liu X. F. 2008; Role of amino acid residues at positions 322 and 329 of haemagglutinin in virulence of H5N1 avian influenza virus. Bing Du Xue Bao 24:340–344 (in Chinese) [PubMed]
    [Google Scholar]
  35. Tang Y., Wu P., Peng D., Wang X., Wan H., Zhang P., Long J., Zhang W., Li Y. et al. 2009; Characterization of duck H5N1 influenza viruses with differing pathogenicity in mallard (Anas platyrhynchos) ducks. Avian Pathol 38:457–467 [View Article][PubMed]
    [Google Scholar]
  36. Tate M. D., Job E. R., Brooks A. G., Reading P. C. 2011; Glycosylation of the hemagglutinin modulates the sensitivity of H3N2 influenza viruses to innate proteins in airway secretions and virulence in mice. Virology 413:84–92 [View Article][PubMed]
    [Google Scholar]
  37. Wagner R., Wolff T., Herwig A., Pleschka S., Klenk H. D. 2000; Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol 74:6316–6323 [View Article][PubMed]
    [Google Scholar]
  38. Wagner R., Heuer D., Wolff T., Herwig A., Klenk H.-D. 2002; N-Glycans attached to the stem domain of haemagglutinin efficiently regulate influenza A virus replication. J Gen Virol 83:601–609[PubMed] [CrossRef]
    [Google Scholar]
  39. Wang C. C., Chen J. R., Tseng Y. C., Hsu C. H., Hung Y. F., Chen S. W., Chen C. M., Khoo K. H., Cheng T. J. et al. 2009; Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc Natl Acad Sci U S A 106:18137–18142 [View Article][PubMed]
    [Google Scholar]
  40. Wang T. T., Tan G. S., Hai R., Pica N., Petersen E., Moran T. M., Palese P. 2010a). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog 6:e1000796 [View Article][PubMed]
    [Google Scholar]
  41. Wang W., Lu B., Zhou H., Suguitan A. L. Jr, Cheng X., Subbarao K., Kemble G., Jin H. 2010b). 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. J Virol 84:6570–6577 [View Article][PubMed]
    [Google Scholar]
  42. Wanzeck K., Boyd K. L., McCullers J. A. 2011; Glycan shielding of the influenza virus hemagglutinin contributes to immunopathology in mice. Am J Respir Crit Care Med 183:767–773 [View Article][PubMed]
    [Google Scholar]
  43. Wiley D. C., Skehel J. J. 1987; The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem 56:365–394 [View Article][PubMed]
    [Google Scholar]
  44. Yamanouchi K., Barrett T. 1994; Progress in the development of a heat-stable recombinant rinderpest vaccine using an attenuated vaccinia virus vector. Rev Sci Tech 13:721–735[PubMed]
    [Google Scholar]
  45. Yen H. L., Lipatov A. S., Ilyushina N. A., Govorkova E. A., Franks J., Yilmaz N., Douglas A., Hay A., Krauss S. et al. 2007; Inefficient transmission of H5N1 influenza viruses in a ferret contact model. J Virol 81:6890–6898 [View Article][PubMed]
    [Google Scholar]
  46. Yuen K. Y., Chan P. K., Peiris M., Tsang D. N., Que T. L., Shortridge K. F., Cheung P. T., To W. K., Ho E. T. et al. 1998; Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 351:467–471 [View Article][PubMed]
    [Google Scholar]
  47. Zhang X., Chen S., Jiang Y., Huang K., Huang J., Yang D., Zhu J., Zhu Y., Shi S. et al. 2015; Hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the H5N1 avian influenza virus. Vet Microbiol 175:244–256 [View Article][PubMed]
    [Google Scholar]
  48. Zhao K., Gu M., Zhong L., Duan Z., Zhang Y., Zhu Y., Zhao G., Zhao M., Chen Z. et al. 2013; Characterization of three H5N5 and one H5N8 highly pathogenic avian influenza viruses in China. Vet Microbiol 163:351–357 [View Article][PubMed]
    [Google Scholar]
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