Structural restrictions for influenza neuraminidase activity promote adaptation and diversification

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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.

Data availability

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


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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.

Author information

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.

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 (2019) doi:10.1038/s41564-019-0537-z

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