The Spatial Location of Single Amino Acid Substitutions in Proteins of Cold-Adapted Influenza B Viruses and Their Impact upon Cold Adaptation

  • E. A. StepanovaEmail author
  • E. V. Krutikova
  • I. V. Kiseleva
  • L. G. Rudenko


This review summarizes the locations of unique amino acid substitutions in proteins of cold-adapted influenza B viruses, master donor viruses of live attenuated influenza vaccines: B/Ann Arbor/1/66ca, B/USSR/60/69, B/Leningrad/14/17/55, B/Victoria/2/63/87, and B/Vienna/1/99ca. The analysis is based on data on the viral sequences and information on the functional organization of internal proteins of the influenza B virus. A description is given of the main features of the structure of internal proteins from influenza B virus and a recent understanding of the functioning of their domains. We describe 35 substitutions in proteins of the ribonucleoprotein complex [PB1 (3), PB2 (10), PA (11), NP (11)] of cold-adapted influenza B viruses. In total, there are 44 amino acid positions that are considered to be associated with cold adaptation. Protein domains involved in protein–protein interactions are the most susceptible to changes. In the polymerase complex, the largest number of replacements were found in C-terminal domains of PA and PB2 proteins, forming the external parts of the polymerase complex structure and providing interaction among subunits, with cellular factors, and with the nucleoprotein (NP). Substitutions in the NP are located in the areas responsible for its oligomerization (flexible N-terminal fragment), as well as for interaction with the polymerase complex (the outer surface of the NP head and body domains). All the cold-adapted viruses had changes in the seventh segment of the genome, leading to amino acid substitutions in the matrix protein or BM2 proton channel. An analysis of the location of amino acid substitutions allows it to be theorized that an important role is played by the internal gene constellation in attenuation of cold-adapted viruses.


review influenza B virus cold adaptation temperature sensitivity attenuation 



  1. 1.
    Massin, P., van der Werf, S., and Naffakh, N., Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses, J. Virol., 2001, vol. 75, no. 11, pp. 5398–5404.CrossRefGoogle Scholar
  2. 2.
    Isakova-Sivak, I. and Rudenko, L., Safety, immunogenicity and infectivity of new live attenuated influenza vaccines, Expert Rev. Vaccines, 2015, vol. 14, no. 10, pp. 1313–1329.CrossRefGoogle Scholar
  3. 3.
    Belshe, R.B., Coelingh, K., Ambrose, C.S., et al., Efficacy of live attenuated influenza vaccine in children against influenza B viruses by lineage and antigenic similarity, Vaccine, 2010, vol. 28, no. 9, pp. 2149–2156.CrossRefGoogle Scholar
  4. 4.
    Chen, Z., Aspelund, A., Kemble, G., and Jin, H., Genetic mapping of the cold-adapted phenotype of B/Ann Arbor/1/66, the master donor virus for live attenuated influenza vaccines (FluMist), Virology, 2006, vol. 345, no. 2, pp. 416–423.CrossRefGoogle Scholar
  5. 5.
    Kiseleva, I.V., Voeten, J.T.M., Teley, L.C.P., et al., PB2 and PA genes control the expression of the temperature-sensitive phenotype of cold-adapted B/USSR/60/69 influenza master donor virus, J. Gen. Virol., 2010, vol. 91, part 4, pp. 931–937.CrossRefGoogle Scholar
  6. 6.
    Hoffmann, E., Mahmood, K., Chen, Z., et al., Multiple gene segments control the temperature sensitivity and attenuation phenotypes of ca B/Ann Arbor/1/66, J. Virol., 2005, vol. 79, no. 17, pp. 11014–11021.CrossRefGoogle Scholar
  7. 7.
    Aleksandrova, G.I., RF Patent 2068271, 1996.Google Scholar
  8. 8.
    Gendon, Yu.Z., Markushin, S.G., Tsfasman, T.M., et al., New cold-adapted donor strains for live influenza vaccine, Vopr. Virusol., 2013, vol. 58, no. 1, pp. 11–17.Google Scholar
  9. 9.
    Katinger, H., Egorov, A., Ferko, B., et al., Int. Patent WO 2002024876 A2, 2002. patents/WO2002024876A2. Accessed July 5, 2017.Google Scholar
  10. 10.
    Seo, S.-U., Byun, Y.-H., Lee, E.-Y., et al., Development and characterization of a live attenuated influenza B virus vaccine candidate, Vaccine, 2008, vol. 26, no. 7, pp. 874–881.CrossRefGoogle Scholar
  11. 11.
    Lee, E.-Y., Lee, K.-H., Jung, E.-J., et al., Genotyping and screening of reassortant live-attenuated influenza B vaccine strain, J. Virol. Methods, 2010, vol. 165, no. 2, pp. 133–138.CrossRefGoogle Scholar
  12. 12.
    Jang, Y.H., Lee, E.-Y., Byun, Y.H., et al., Protective efficacy in mice of monovalent and trivalent live attenuated influenza vaccines in the background of cold-adapted A/X-31 and B/Lee/40 donor strains, Vaccine, 2014 vol. 32, no. 5, pp. 535–543.CrossRefGoogle Scholar
  13. 13.
    Maassab, H.F. and DeBorde, D.C., Development and characterization of cold-adapted viruses for use as live virus vaccines, Vaccine, 1985, vol. 3, no. 5, pp. 355–369.CrossRefGoogle Scholar
  14. 14.
    Donabedian, A.M., DeBorde, D.C., and Maassab, H.F., Genetics of A/X-31 and B/Lee/40 donor strains, Vaccine, 2014, vol. 32, no. 5, pp. 535–543.CrossRefGoogle Scholar
  15. 15.
    Rudenko, L. and Alexandrova, G., Current strategies for the prevention of influenza by the Russian cold-adapted live influenza vaccine among different populations, Int. Congr. Ser., 2001, vol. 1219, pp. 945–950.CrossRefGoogle Scholar
  16. 16.
    Alexandrova, G.I., Maassab, H.F., Kendal, A.P., et al., Laboratory properties of cold-adapted influenza B live vaccine strains developed in the US and USSR, and their B/Ann Arbor/1/86 cold-adapted reassortant vaccine candidates, Vaccine, 1990, vol. 8, no. 1, pp. 61–64.CrossRefGoogle Scholar
  17. 17.
    Resa-Infante, P., Jorba, N., Coloma, R., and Ortín, J., The influenza RNA synthesis machine, RNA Biol., 2011, vol. 8, no. 2, pp. 207–215.CrossRefGoogle Scholar
  18. 18.
    Liu, Y., Yang, Y., Fan, J., et al., The crystal structure of the PB2 cap-binding domain of influenza B virus reveals a novel cap recognition mechanism, J. Biol. Chem., 2015, vol. 290, no. 14, pp. 9141–9149.CrossRefGoogle Scholar
  19. 19.
    Reich, S., Guilligay, D., Pflug, A., et al., Structural insight into cap-snatching and RNA synthesis by influenza polymerase, Nature, 2014, vol. 516, no. 7531, pp. 361–366.Google Scholar
  20. 20.
    Pflug, A., Guilligay, D., Reich, S., and Cusack, S., Structure of influenza A polymerase bound to the viral RNA promoter, Nature, 2014, vol. 516, no. 7531, pp. 355–360.CrossRefGoogle Scholar
  21. 21.
    Massin, P., van der Werf, S., and Naffakh, N., Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses, J. Virol., 2001, vol. 75, no. 11, pp. 5398–5404.CrossRefGoogle Scholar
  22. 22.
    Subbarao, E.K., London, W., and Murphy, B.R., A single amino acid in the PB2 gene of influenza A virus is a determinant of host range, J. Virol., 1993, vol. 67, no. 4, pp. 1761–1764.Google Scholar
  23. 23.
    Boivin, S., Cusack, S., Ruigrok, R.W.H., and Hart, D.J., Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms, J. Biol. Chem., 2010, vol. 285, no. 37, pp. 28411–28417.CrossRefGoogle Scholar
  24. 24.
    He, X., Zhou, J., Bartlam, M., et al., Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza H5N1 virus, Nature, 2008, vol. 454, no. 7208, pp. 1123–1126.CrossRefGoogle Scholar
  25. 25.
    Obayashi, E., Yoshida, H., Kawai, F., et al., The structural basis for an essential subunit interaction in influenza virus RNA polymerase, Nature, 2008, vol. 454, no. 7208, pp. 1127–1131.CrossRefGoogle Scholar
  26. 26.
    Ng, A.K.-L., Lam M.K.-H., Zhang H., et al., Structural basis for RNA binding and homo-oligomer formation by influenza B virus nucleoprotein, J. Virol., 2012, vol. 86, no. 12, pp. 6758–6767.CrossRefGoogle Scholar
  27. 27.
    Liu, M, Lam, M.K.-H., Zhang, Q., et al., The functional study of the N-terminal region of influenza B virus nucleoprotein, PLoS One, 2015, vol. 10, no. 9, p. e0137802.CrossRefGoogle Scholar
  28. 28.
    Wanitchang, A., Narkpuk, J., and Jongkaewwattana, A., Nuclear import of influenza B virus nucleoprotein: involvement of an N-terminal nuclear localization signal and a cleavage-protection motif, Virology, 2013, vol. 443, no. 1, pp. 59–68.CrossRefGoogle Scholar
  29. 29.
    Hatta, M., Kohlmeier, C.K., Hatta, Y., et al., Region required for protein expression from the stop-start pentanucleotide in the M gene of influenza B virus, J. Virol., 2009, vol. 83, no. 11, pp. 5939–5942.CrossRefGoogle Scholar
  30. 30.
    Safo, M.K., Musayev, F.N., Mosier, P.D., et al., Crystal structures of influenza A virus matrix protein M1: Variations on a theme, PLoS One, 2014, vol. 9, no. 10, p. e109510.CrossRefGoogle Scholar
  31. 31.
    Cao, S., Jiang, J., Li, J., et al., Characterization of the nucleocytoplasmic shuttle of the matrix protein of influenza B virus, J. Virol., 2014, vol. 88, no. 13, pp. 7464–7473.CrossRefGoogle Scholar
  32. 32.
    Biasini, M., Bienert, S., Waterhouse, A., et al., SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information, Nucleic Acids Res., 2014, vol. 42, no. 1, pp. 252–258.CrossRefGoogle Scholar
  33. 33.
    Wang, J., Pielak, R.M., McClintock, M.A., and Chou, J.J., Solution structure and functional analysis of the influenza B proton channel, Nat. Struct. Mol. Biol., 2009, vol. 16, no. 12, pp. 1267–1271.CrossRefGoogle Scholar
  34. 34.
    Wakefield, L. and Brownlee, G.G., RNA-binding properties of influenza A virus matrix protein M1, Nucleic Acids Res., 1989, vol. 17, no. 21, pp. 8569–8580.CrossRefGoogle Scholar
  35. 35.
    Sha, B. and Luo, M., Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1, Nat. Struct. Biol., 1997, vol. 4, no. 3, pp. 239–244.CrossRefGoogle Scholar
  36. 36.
    Arzt, S., Baudin, F., Barge, A., et al., Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer, Virology, 2001, vol. 279, no. 2, pp. 439–446.CrossRefGoogle Scholar
  37. 37.
    Shtykova, E.V., Baratova, L.A., Fedorova, N.V., et al., Structural analysis of Influenza A virus matrix protein M1 and its self-assemblies at low pH, PLoS One, 2013, vol. 8, no. 12, p. e82431.CrossRefGoogle Scholar
  38. 38.
    Shishkov, A., Bogacheva, E., Fedorova, N., et al., Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium, FEBS J., 2011, vol. 278, no. 24, pp. 4905–4916.CrossRefGoogle Scholar
  39. 39.
    Zhang, K., Wang, Z., Liu, X., et al., Dissection of influenza A virus M1 protein: pH-dependent oligomerization of N-terminal domain and dimerization of C‑terminal domain, PLoS One, 2012, vol. 7, no. 5, p. e37786.CrossRefGoogle Scholar
  40. 40.
    Zhang, K., Wang, Z., Fan, G.-Z., et al., Two polar residues within C-terminal domain of M1 are critical for the formation of influenza A virions, Cell. Microbiol., 2015, vol. 17, no. 11, pp. 1583–1593.CrossRefGoogle Scholar
  41. 41.
    Yin, C., Khan, J.A., Swapna, G.V.T., et al., Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses, J. Biol. Chem., 2007, vol. 282, no. 28, pp. 20584–20592.CrossRefGoogle Scholar
  42. 42.
    Wang, W. and Krug, R.M., The RNA-binding and effector domains of the viral NS1 protein are conserved to different extents among influenza A and B viruses, Virology, 1996, vol. 223, no. 1, pp. 41–50.CrossRefGoogle Scholar
  43. 43.
    Guan, R., Ma, L.-C., Leonard, P.G., et al., Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus, Proc. Natl. Acad. Sci. U. S. A., 2011, vol. 108, no. 33, pp. 13468–13473.CrossRefGoogle Scholar
  44. 44.
    Ma, L.-C., Guan, R., Hamilton, K., et al., A second RNA-binding site in the NS1 protein of influenza B virus, Structure, 2016, vol. 24, no. 9, pp. 1562–1572.CrossRefGoogle Scholar
  45. 45.
    Dauber, B., Schneider, J., and Wolff, T., Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon, J. Virol., 2006, vol. 80, no. 23, pp. 11667–11677.CrossRefGoogle Scholar
  46. 46.
    Imai, M., Watanabe, S., and Odagiri, T., Influenza B virus NS2, a nuclear export protein, directly associates with the viral ribonucleoprotein complex, Arch. Virol., 2003, vol. 148, no. 10, pp. 1873–1884.CrossRefGoogle Scholar
  47. 47.
    Paragas, J., Talon, J., O’Neill, R.E., et al., Influenza B and C virus NEP (NS2) proteins possess nuclear export activities, J. Virol., 2001, vol. 75, no. 16, pp. 7375–7383.CrossRefGoogle Scholar
  48. 48.
    Cha, T.-A., Kao, K., Zhao, J., et al., Genotypic stability of cold-adapted influenza virus vaccine in an efficacy clinical trial, J. Clin. Microbiol., 2000, vol. 38, no. 2, pp. 839–845.Google Scholar
  49. 49.
    Kiseleva, I.V., Klimov, A.I., Grigor’eva, E.P., et al., Genetic and phenotypic analysis of heterogeneous population of A/Leningrad/134/17/57 (H2N2) cold-adapted donor of the attenuation and donor-based reassortant influenza vaccine strains, Vopr. Virusol., 2005, vol. 50, no. 2, pp. 14–18.Google Scholar
  50. 50.
    Buonagurio, D.A., Bechert, T.M., Yang, C.-F., et al., Genetic stability of live, cold-adapted influenza virus components of the FluMist/CAIV-T vaccine throughout the manufacturing process, Vaccine, 2006, vol. 24, no. 12, pp. 2151–2160.CrossRefGoogle Scholar
  51. 51.
    Buonagurio, D.A., O’Neill, R.E., Shutyak, L., et al., Genetic and phenotypic stability of cold-adapted influenza viruses in a trivalent vaccine administered to children in a day care setting, Virology, 2006, vol. 347, no. 2, pp. 296–306.CrossRefGoogle Scholar
  52. 52.
    Murphy, B.R., Park, E.J., Gottlieb, P., and Subbarao, K., An influenza A live attenuated reassortant virus possessing three temperature-sensitive mutations in the PB2 polymerase gene rapidly loses temperature sensitivity following replication in hamsters, Vaccine, 1997, vol. 15, nos. 12–13, pp. 1372–1378.CrossRefGoogle Scholar
  53. 53.
    Treanor, J.J., Buja, R., and Murphy, B.R., Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus, J. Virol., 1991, vol. 65, no. 8, pp. 4204–4210.Google Scholar

Copyright information

© Allerton Press, Inc. 2018

Authors and Affiliations

  • E. A. Stepanova
    • 1
    Email author
  • E. V. Krutikova
    • 1
  • I. V. Kiseleva
    • 1
    • 2
  • L. G. Rudenko
    • 1
  1. 1.Institute of Experimental Medicine, St. PetersburgRussia
  2. 2.St. Petersburg State UniversitySt. PetersburgRussia

Personalised recommendations