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Molecular Biology

, Volume 52, Issue 6, pp 891–898 | Cite as

Three Mutations in the Stalk Region of Hemagglutinin Affect the pH of Fusion and Pathogenicity of H5N1 Influenza Virus

  • N. F. LomakinaEmail author
  • G. K. Sadykova
  • T. A. Timofeeva
  • I. A. Rudneva
  • E. Yu. Boravleva
  • P. A. Ivanov
  • A. G. Prilipov
  • A. S. Gambaryan
MOLECULAR CELL BIOLOGY

Abstract

Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4–5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70–5.80 to 5.25–5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.

Keywords:

influenza A virus H5N1 hemagglutinin amino acid substitutions reverse genetics reverse selection attenuation 

Notes

REFERENCES

  1. 1.
    Goodsell, D.S. 2006. Molecule of the month: Hemagglutinin. RCSB PDB Molecule of the month. http:// pdb101.rcsb.org/motm/76. doi 10.2210/rcsb_pdb/ mom_2006_410.2210/rcsb_pdb/mom_2006_4Google Scholar
  2. 2.
    Hamilton B.S., Whittaker G.R., Daniel S. 2012. Influenza virus-mediated membrane fusion: Determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion. Viruses. 4 (7), 1144–1168. doi 10.3390/v4071144CrossRefGoogle Scholar
  3. 3.
    Influenza A cleavage sites. 2016. Version 6. http://www. offlu.net/fileadmin/home/en/resource-centre/pdf/ Influenza_A_Cleavage_Sites.pdf.Google Scholar
  4. 4.
    Reed M.L., Bridges O.A., Seiler P., Kim J.-K., Yen H.-L., Salomon R., Govorkova E.A., Webster R.G., Russell C.J. 2010. The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity and transmissibility in ducks. J. Virol. 84, 1527–1535.CrossRefGoogle Scholar
  5. 5.
    Hanson A., Imai M., Hatta M., McBride R., Imai H., Taft A., Zhong G., Watanabe T., Suzuki Y., Neumann G., Paulson J.C., Kawaoka Y. 2015. Identification of stabilizing mutations in an H5 hemagglutinin influenza virus protein. J. Virol. 90 (6), 2981–2992. doi 10.1128/JVI.02790-15CrossRefGoogle Scholar
  6. 6.
    Galloway S.E., Reed M.L., Russell C.J., Steinhauer D.A. 2013. Influenza HA subtypes demonstrate divergent phenotypes for cleavage activation and pH of fusion: implications for host range and adaptation. PLoS Pathog. 9, e1003151.CrossRefGoogle Scholar
  7. 7.
    Daidoji T., Watanabe Y., Ibrahim M.S., Yasugi M., Maruyama H., Masuda T., Arai F., Ohba T., Honda A., Ikuta K., Nakaya T. 2015. Avian influenza virus infection of immortalized human respiratory epithelial cells depends upon a delicate balance between hemagglutinin acid stability and endosomal pH. J. Biol. Chem. 290 (17), 10627–10642. doi 10.1074/jbc.M114.611327CrossRefGoogle Scholar
  8. 8.
    Webster R.G. 1997. Influenza virus: Transmission between species and relevance to emergence of the next human pandemic. Arch. Virol. Suppl. 13, 105–113.Google Scholar
  9. 9.
    Baumann J., Kouassi N.M, Foni E., Klenk H.D., Matrosovich M. 2015. H1N1 swine influenza viruses differ from avian precursors by a higher pH optimum of membrane fusion. J. Virol. 90 (3), 1569–1577. doi 10.1128/JVI.02332-15CrossRefGoogle Scholar
  10. 10.
    DuBois R.M., Zaraket H., Reddivari M., Heath R.J., White S.W., Russell, C.J. 2011. Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity. PLoS Pathog. 7, e1002398CrossRefGoogle Scholar
  11. 11.
    Gerlach T., Hensen L., Matrosovich T., Bergmann J., Winkler M., Peteranderl C., Klenk H.D., Weber F., Herold S., Pöhlmann S., Matrosovich M. 2017. pH optimum of hemagglutinin-mediated membrane fusion determines sensitivity of influenza A viruses to the interferon-induced antiviral state and IFITMs. J. Virol. 91 (11), e00246-17. doi 10.1128/JVI.00246-17CrossRefGoogle Scholar
  12. 12.
    Krenn B.M., Egorov A., Romanovskaya-Romanko E., Wolschek M., Nakowitsch S., Ruthsatz T., Kiefmann B., Morokutti A., Humer J., Geiler J., Cinatl J., Michaelis M., Wressnigg N., Sturlan S., Ferko B., et al. 2011. Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1. PLoS One. 6 (4), e18577.Google Scholar
  13. 13.
    Russier M., Yang G., Rehg J.E., Wong S.S., Mostafa H.H., Fabrizio T.P., Barman S., Krauss S., Webster R.G., Webby R.J., Russell C J. 2016. Molecular requirements for a pandemic influenza virus: an acid-stable hemagglutinin protein. Proc. Natl. Acad. Sci. U. S. A. 113 (6), 1636–1641, doi 10.1073/pnas.1524384113CrossRefGoogle Scholar
  14. 14.
    Zaraket H., Bridges O.A., Russell C.J. 2013. The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice. J. Virol. 87 (9), 4826–4834. doi 10.1128/JVI.03110-12CrossRefGoogle Scholar
  15. 15.
    Reed M.L., Yen H.L., DuBois R.M., Bridges O.A., Salomon R., Webster R.G., Russell C.J. 2009. Amino acid residues in the fusion peptide pocket regulate the pH of activation of the H5N1 influenza virus hemagglutinin protein. J. Virol. 83 (8), 3568–3580. doi 10.1128/JVI.02238-08CrossRefGoogle Scholar
  16. 16.
    Lomakina N.F., Boravleva E.Yu., Kropotkina E.A., Yamnikova S.S., Drygin V.V., Gambaryan A.S. 2011. Attenuation of A/Chicken/Kurgan/3/2005 (H5N1) influenza virus using selection in an environment simu-lating the life cycle of wild duck viruses. Mol. Genet. Microbiol. Virol. 26 (3), 132–136.CrossRefGoogle Scholar
  17. 17.
    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.CrossRefGoogle Scholar
  18. 18.
    World Organization for Animal Health. 2018. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. http://www.oie.int/standard-setting/terrestrial-manual/access-online/Google Scholar
  19. 19.
    Stech J., Stech O., Herwig A., Altmeppen H., Hundt J., Gohrbandt S., Kreibich A., Weber S., Klenk H.D., Mettenleiter T.C. 2008. Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification. Nucleic Acids Res. 36 (21), e139. doi 10.1093/nar/gkn646CrossRefGoogle Scholar
  20. 20.
    Kreibich A., Stech J., Mettenleiter T.C., Stech O. 2009. Simultaneous one-tube full-length amplification of the NA, NP, M, and NS genes of influenza A viruses for reverse genetics. J. Virol. Methods. 159 (2), 308–310. doi 10.1016/j.jviromet.2009.04.020CrossRefGoogle Scholar
  21. 21.
    World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO). H5N1 Evolution Working Group. 2014. Revised and updated nomenclature for highly pathogenic avian influenza A (H5N1) viruses. Influenza Other Respir. Viruses. 8 (3), 384–388.Google Scholar
  22. 22.
    Squires R.B., Noronha J., Hunt V., García-Sastre A., Macken C., Baumgarth N., Suarez D., Pickett B.E., Zhang Y., Larsen C.N., Ramsey A., Zhou L., Zaremba S., Kumar S., Deitrich J., et al. 2012. Influenza research database: An integrated bioinformatics resource for influenza research and surveillance. Influenza Other Respir. Viruses. 6, 404–416. doi 10.1111/j.1750-2659.2011.00331.xCrossRefGoogle Scholar
  23. 23.
    Stevens J., Blixt O., Tumpey T.M., Taubenberger J.K., Paulson J.C., Wilson I.A. 2006. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science. 312, 404–410. doi 10.1126/science.1124513CrossRefGoogle Scholar
  24. 24.
    Gambaryan A.S., Boravleva E.Y., Lomakina N.F., Kropotkina E.A., Gordeychuk I.V., Chvala I.A., Drygin V.V., Klenk H.D., Matrosovich M.N. 2016. Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus. Acta Virol. 60 (3), 316–327. doi 10.4149/ av_2016_03_316CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • N. F. Lomakina
    • 1
    • 2
    Email author
  • G. K. Sadykova
    • 2
  • T. A. Timofeeva
    • 2
  • I. A. Rudneva
    • 2
  • E. Yu. Boravleva
    • 1
  • P. A. Ivanov
    • 2
    • 3
  • A. G. Prilipov
    • 2
  • A. S. Gambaryan
    • 1
  1. 1.Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products, Russian Academy of SciencesMoscowRussia
  2. 2.N.F. Gamaleya National Center of Epidemiology and Microbiology, Ministry of Health of the Russian FederationMoscowRussia
  3. 3.The Mental Health Research CenterMoscowRussia

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