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Structural restrictions for influenza neuraminidase activity promote adaptation and diversification

Nature Microbiology volume 4, pages 2565–2577 (2019)Cite this article

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An Author Correction to this article was published on 16 June 2020

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Abstract

Influenza neuraminidase (NA) is a sialidase that contributes to viral mobility by removing the extracellular receptors for the haemagglutinin (HA) glycoprotein. However, it remains unclear why influenza NAs evolved to function as Ca2+-dependent tetramers that display variable stability. Here, we show that the Ca2+ ion located at the centre of the NA tetramer is a major stability determinant, as this Ca2+ ion is required for catalysis and its binding affinity varies between NAs. By examining NAs from 2009 pandemic-like H1N1 viruses, we traced the affinity variation to local substitutions that cause residues in the central Ca2+-binding pocket to reposition. A temporal analysis revealed that these local substitutions predictably alter the stability of the 2009 pandemic-like NAs and contribute to the tendency for the stability to vary up and down over time. In addition to the changes in stability, the structural plasticity of NA was also shown to support the formation of heterotetramers, which creates a mechanism for NA to obtain hybrid properties and propagate suboptimal mutants. Together, these results demonstrate how the structural restrictions for activity provide influenza NA with several mechanisms for adaptation and diversification.

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Fig. 1: Influenza NAs display distinct Ca2+ sensitive thermostability profiles.
Fig. 2: NA stability is affected by substitutions near the axis of symmetry.
Fig. 3: Optimal NA thermostability and catalysis require the central Ca2+ ion.
Fig. 4: Affinity for the NA central Ca2+ ion defines the Ca2+ level required for optimal replication.
Fig. 5: NA from 2009 pandemic-like H1N1 IAVs shows temporal variation in the central Ca2+-binding site.
Fig. 6: NA tetramer plasticity provides a mechanism to increase heterogeneity and rescue deficient mutants.
Fig. 7: Structural mapping of N1 and N2 sequence conservation.

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Data availability

All of the raw data for the figures presented in this study are available in the Supplementary Information.

Change history

  • 16 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. Harris, A. et al. Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc. Natl Acad. Sci. USA 103, 19123–19127 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dou, D., Revol, R., Östbye, H., Wang, H. & Daniels, R. Influenza A virus cell entry, replication, virion assembly and movement. Front. Immunol. 9, 1581 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Weis, W. et al. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature 333, 426–431 (1988).

    Article  CAS  PubMed  Google Scholar 

  4. Hamilton, B. S., Whittaker, G. R. & Daniel, S. Influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion. Viruses 4, 1144–1168 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Burnet, F. M. Mucins and mucoids in relation to influenza virus action; inhibition of virus haemagglutination by glandular mucins. Aust. J. Exp. Biol. Med. Sci. 26, 371–380 (1948).

    Article  CAS  PubMed  Google Scholar 

  6. Matrosovich, M. N., Matrosovich, T. Y., Gray, T., Roberts, N. A. & Klenk, H. D. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J. Virol. 78, 12665–12667 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cohen, M. et al. Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase. Virol. J. 10, 321 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Yang, X. et al. A beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus. PLoS ONE 9, e110026 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Burnet, F. M., Mc, C. J. & Anderson, S. G. Mucin as substrate of enzyme action by viruses of the mumps influenza group. Nature 160, 404–405 (1947).

    Article  CAS  PubMed  Google Scholar 

  10. Burnet, F. M. Mucins and mucoids in relation to influenza virus action; the destruction of Francis inhibitor activity in a purified mucoid by virus action. Aust. J. Exp. Biol. Med. Sci. 26, 389–402 (1948).

    Article  CAS  PubMed  Google Scholar 

  11. Gottschalk, A. Neuraminidase: the specific enzyme of influenza virus and vibrio cholerae. Biochim. Biophys. Acta 23, 645–646 (1957).

    Article  CAS  PubMed  Google Scholar 

  12. Sakai, T., Nishimura, S. I., Naito, T. & Saito, M. Influenza A virus hemagglutinin and neuraminidase act as novel motile machinery. Sci. Rep. 7, 45043 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Webster, R. G. & Laver, W. G. Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. J. Immunol. 99, 49–55 (1967).

    CAS  PubMed  Google Scholar 

  14. Seto, J. T. & Rott, R. Functional significance of sialidose during influenza virus multiplication. Virology 30, 731–737 (1966).

    Article  CAS  PubMed  Google Scholar 

  15. Kilbourne, E. D., Laver, W. G., Schulman, J. L. & Webster, R. G. Antiviral activity of antiserum specific for an influenza virus neuraminidase. J. Virol. 2, 281–288 (1968).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Compans, R. W., Dimmock, N. J. & Meier-Ewert, H. Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus. J. Virol. 4, 528–534 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Palese, P. & Compans, R. W. Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action. J. Gen. Virol. 33, 159–163 (1976).

    Article  CAS  PubMed  Google Scholar 

  18. Mitnaul, L. J. et al. Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. J. Virol. 74, 6015–6020 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Xu, R. et al. Functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 H1N1 influenza pandemic. J. Virol. 86, 9221–9232 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Varghese, J. N., Laver, W. G. & Colman, P. M. Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 Å resolution. Nature 303, 35–40 (1983).

    Article  CAS  PubMed  Google Scholar 

  21. Dou, D., da Silva, D. V., Nordholm, J., Wang, H. & Daniels, R. Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion. Mol. Biol. Cell 25, 3363–3374 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Nordholm, J. et al. Translational regulation of viral secretory proteins by the 5′ coding regions and a viral RNA-binding protein. J. Cell Biol. 216, 2283–2293 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang, N., Glidden, E. J., Murphy, S. R., Pearse, B. R. & Hebert, D. N. The cotranslational maturation program for the type II membrane glycoprotein influenza neuraminidase. J. Biol. Chem. 283, 33826–33837 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. da Silva, D. V., Nordholm, J., Madjo, U., Pfeiffer, A. & Daniels, R. Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains. J. Biol. Chem. 288, 644–653 (2013).

    Article  PubMed  CAS  Google Scholar 

  25. Nordholm, J., da Silva, D. V., Damjanovic, J., Dou, D. & Daniels, R. Polar residues and their positional context dictate the transmembrane domain interactions of influenza a neuraminidases. J. Biol. Chem. 288, 10652–10660 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. da Silva, D. V. et al. The influenza virus neuraminidase protein transmembrane and head domains have coevolved. J. Virol. 89, 1094–1104 (2015).

    Article  PubMed  CAS  Google Scholar 

  27. Air, G. M. Influenza neuraminidase. Influenza Other Respir. Virus. 6, 245–256 (2012).

    Article  CAS  Google Scholar 

  28. Li, Q. et al. The 2009 pandemic H1N1 neuraminidase N1 lacks the 150-cavity in its active site. Nat. Struct. Mol. Biol. 17, 1266–1268 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Xu, X., Zhu, X., Dwek, R. A., Stevens, J. & Wilson, I. A. Structural characterization of the 1918 influenza virus H1N1 neuraminidase. J. Virol. 82, 10493–10501 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Colman, P. M., Varghese, J. N. & Laver, W. G. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 303, 41–44 (1983).

    Article  CAS  PubMed  Google Scholar 

  31. Crennell, S. J., Garman, E. F., Laver, W. G., Vimr, E. R. & Taylor, G. L. Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. Proc. Natl Acad. Sci. USA 90, 9852–9856 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Taylor, G. Sialidases: structures, biological significance and therapeutic potential. Curr. Opin. Struct. Biol. 6, 830–837 (1996).

    Article  CAS  PubMed  Google Scholar 

  33. Paterson, R. G. & Lamb, R. A. Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms. J. Cell Biol. 110, 999–1011 (1990).

    Article  CAS  PubMed  Google Scholar 

  34. Carroll, S. M. & Paulson, J. C. Complete metal ion requirement of influenza virus N1 neuraminidases. Arch. Virol. 71, 273–277 (1982).

    Article  CAS  PubMed  Google Scholar 

  35. Burmeister, W. P., Cusack, S. & Ruigrok, R. W. Calcium is needed for the thermostability of influenza B virus neuraminidase. J. Gen. Virol. 75, 381–388 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Sultana, I. et al. Stability of neuraminidase in inactivated influenza vaccines. Vaccine 32, 2225–2230 (2014).

    Article  CAS  PubMed  Google Scholar 

  37. Francis, T. Dissociation of hemagglutinating and antibody-measuring capacities of influenza virus. J. Exp. Med. 85, 1–7 (1947).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Baker, N. J. & Gandhi, S. S. Effect of Ca++ on the stability of influenza virus neuraminidase. Arch. Virol. 52, 7–18 (1976).

    Article  CAS  PubMed  Google Scholar 

  39. Briody, D. A. Characterization of the enzymic action of influenza viruses on human red cells. J. Immunol. 59, 115–127 (1948).

    CAS  PubMed  Google Scholar 

  40. Dimmock, N. J. Dependence of the activity of an influenza virus neuraminidase upon Ca2+. J. Gen. Virol. 13, 481–483 (1971).

    Article  CAS  PubMed  Google Scholar 

  41. Chong, A. K., Pegg, M. S. & von Itzstein, M. Influenza virus sialidase: effect of calcium on steady-state kinetic parameters. Biochim. Biophys. Acta 1077, 65–71 (1991).

    Article  CAS  PubMed  Google Scholar 

  42. Zanin, M. et al. An amino acid in the stalk domain of N1 neuraminidase is critical for enzymatic activity. J. Virol. 91, e00868-16 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Dai, M. et al. Identification of residues that affect oligomerization and/or enzymatic activity of influenza virus H5N1 neuraminidase proteins. J. Virol. 90, 9457–9470 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Mok, C. K. et al. Evolutionarily conserved residues at an oligomerization interface of the influenza A virus neuraminidase are essential for viral survival. Virology 447, 32–44 (2013).

    Article  CAS  PubMed  Google Scholar 

  45. Takahashi, T., Song, J., Suzuki, T. & Kawaoka, Y. Mutations in NA that induced low pH-stability and enhanced the replication of pandemic (H1N1) 2009 influenza a virus at an early stage of the pandemic. PLoS ONE 8, e64439 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Labadie, T., Batejat, C., Manuguerra, J. C. & Leclercq, I. Influenza virus segment composition influences viral stability in the environment. Front. Microbiol. 9, 1496 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Collins, P. J. et al. Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature 453, 1258–1261 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Xue, K. S., Hooper, K. A., Ollodart, A. R., Dingens, A. S. & Bloom, J. D. Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture. eLife 5, e13974 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Talley, K. & Alexov, E. On the pH-optimum of activity and stability of proteins. Proteins 78, 2699–2706 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. van der Vries, E. et al. H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase mutant explained by kinetic and structural analysis. PLoS Pathog. 8, e1002914 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Dou, D. et al. Analysis of IAV replication and co-infection dynamics by a versatile RNA viral genome labeling method. Cell Rep. 20, 251–263 (2017).

    Article  CAS  PubMed  Google Scholar 

  52. van der Vries, E. et al. Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets. PLoS Pathog. 7, e1002276 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Burmeister, W. P., Ruigrok, R. W. & Cusack, S. The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 11, 49–56 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Schmidt, P. M., Attwood, R. M., Mohr, P. G., Barrett, S. A. & McKimm-Breschkin, J. L. A generic system for the expression and purification of soluble and stable influenza neuraminidase. PLoS ONE 6, e16284 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Eichelberger, M. C. & Wan, H. Influenza neuraminidase as a vaccine antigen. Curr. Top. Microbiol. Immunol. 386, 275–299 (2015).

  56. Gao, J. et al. Antigenic drift of the influenza A(H1N1)pdm09 virus neuraminidase results in reduced effectiveness of A/California/7/2009 (H1N1pdm09)-specific antibodies. mBio 10, e00307-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Chen, Y. Q. et al. Influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies. Cell 173, 417–429 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Johansson, B. E. & Kilbourne, E. D. Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition. J. Virol. 67, 5721–5723 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Johansson, B. E., Matthews, J. T. & Kilbourne, E. D. Supplementation of conventional influenza A vaccine with purified viral neuraminidase results in a balanced and broadened immune response. Vaccine 16, 1009–1015 (1998).

    Article  CAS  PubMed  Google Scholar 

  60. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl Acad. Sci. USA 97, 6108–6113 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Mellroth, P. et al. LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan. J. Biol. Chem. 287, 11018–11029 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Harbury, P. B., Zhang, T., Kim, P. S. & Alber, T. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262, 1401–1407 (1993).

    Article  CAS  PubMed  Google Scholar 

  63. Chutinimitkul, S. et al. Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding. J. Virol. 84, 11802–11813 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Reed, L. J. & Muench, H. A simple method of estimating fifty per cent end points. Am. J. Epidemiol. 27, 493–497 (1938).

    Article  Google Scholar 

  65. Kawakami, E. et al. Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. J. Virol. Methods 173, 1–6 (2011).

    Article  CAS  PubMed  Google Scholar 

  66. Huang, Y., Niu, B., Gao, Y., Fu, L. & Li, W. CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics 26, 680–682 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Zhu, X. et al. Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic acid receptors. J. Virol. 86, 13371–13383 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank R. Fouchier (Erasmus Medical Center) and A. Pfeiffer for reading the manuscript and offering several suggestions, M. Oliveberg (Stockholm University), P. Ädelroth (Stockholm University) and J. Nordholm for discussions, and B. Fu for help generating PyMOL images. This work was supported in part by grants from the Swedish Research Council (K2015-57-21980-04-4) and the Carl Trygger Foundation (CTS17:111), as well as federal funds from the NIAID, National Institutes of Health, Department of Health and Human Services, under CEIRS contract number HHSN272201400005C.

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  1. Robert Daniels

    Present address: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    Hao Wang, Dan Dou, Henrik Östbye, Rebecca Revol & Robert Daniels

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Contributions

H.W. designed and performed the majority of the experiments and analysed most of the data. D.D. designed and performed most of the viral growth experiments under Ca2+-depletion conditions, some of the NA thermostability experiments and contributed to the statistical analysis. H.Ö. performed most of the NA temporal stability experiments, the bioinformatic analysis for the amino acid frequency at position 106 in NA and most of the co-transfection experiments. R.R. performed the amino acid conservation mapping for N1 and N2. R.D. conceived and supervised the study and wrote the manuscript with help from H.W.

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Correspondence to Robert Daniels.

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Supplementary Information

Supplementary Figs. 1–28, Supplementary Tables 1–5 and Supplementary Data.

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Supplementary Data 1

Excel file containing the raw NA activity measurements and normalizations.

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Wang, H., Dou, D., Östbye, H. et al. Structural restrictions for influenza neuraminidase activity promote adaptation and diversification. Nat Microbiol 4, 2565–2577 (2019). https://doi.org/10.1038/s41564-019-0537-z

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