Article Figures & Data
Figures
- Fig 1
Sequence variation in the HA protein of pH1N1. Amino acid substitutions at every position of HA of pH1N1 (using all 7,132 HA sequences) were counted per half-year flu season (season I, 1 April 2009 to 30 September 2009, etc.). The most frequent substitutions were plotted (y axis, percent occurrence) for each season. The data for seasons IV to VI were grouped together because of the low number of sequences available for seasons V and VI. The locations of the signal peptide (SP), HA stem, and HA head domains are indicated at the bottom (the drawing is not to scale). The most frequent mutations in the head domain of HA in season III that still prevailed in seasons IV to VI are shaded in black.
- Fig 2
Receptor-binding avidity of HA proteins with single amino acid substitutions. The effect of the most frequently occurring amino acid substitutions on the binding avidity of soluble HA trimers of pH1N1 was determined by solid-phase binding assay (40 μg/ml HA on fetuin) (OD450, optical density at 450 nm) (A) and hemagglutination (human erythrocytes) (B). HA from strain A/California/07/2009 (Cal07) was used as the template for the introduction of the substitutions indicated on the x axis. (C) The effect of a single amino acid substitution (T200A) on the binding avidity of virus particles was determined by hemagglutination using strain A/California/04/2009 (Cal04) and a recombinant virus of this strain carrying substitution T200A in HA. Equal numbers of viral particles were used on the basis of quantification by quantitative PCR. The average result of four experiments (each performed in triplicate) with independently grown viral stocks is shown. The hemagglutination of the two viruses was significantly different (P < 0.05) (B). Error bars show standard deviations.
- Fig 3
Phylogenetic tree based on HA of pH1N1. A phylogenetic tree (obtained by neighbor joining) was constructed using full-length viral HA sequences (GenBank accession numbers are listed in Table S2 in the supplemental material) identical to the signature sequence of the cluster (see Materials and Methods) to which it belongs. Cluster numbers describe three properties. The first number (1 to 4) indicates the flu season (seasons I, II, III, and IV to VI, respectively) in which the cluster was the most prominent (as a percentage of the total number of sequences in a season). The decimal number ranks the clusters in order of size. The clusters dominant in season III (given a prefix of 3) and seasons IV to VI (given a prefix of 4) are colored red and blue, respectively. The number after the underscore indicates the number of sequences present in a cluster (all 7,132 sequences were assigned to a cluster). Substitutions defining the branches are indicated. The substitutions for the Cal04 and Cal07 reference strains are indicated in red and were analyzed for their effect on receptor binding.
- Fig 4
Distribution of HA pH1N1 clusters per flu season. Results for clusters that contained more than 5% of the total number of sequences in any of the seasons are plotted (the y axis indicates the percentage of sequences in a cluster relative to the total number of sequences in a season; e.g., cluster 1.1 harbors 47% of the total number of sequences in season I [green bar], 31% of the sequences in season II [red bar], but only 2% of the sequences in season III [yellow bar]). Clusters are sorted by cluster number on the x axis (numbering is according to that used for the phylogenetic tree and is explained in the legend to Fig. 3). Clusters belonging to clade A or B (Fig. 3) are indicated.
- Fig 5
Receptor-binding avidity of HA proteins of clades A and B with multiple amino acid substitutions. The effect of amino acid substitutions consecutively occurring during the evolution of clades A (A, C) and B (B, D) on the binding avidity of soluble HA trimers of pH1N1 was determined by a solid-phase binding assay (40 μg/ml HA on fetuin) (A, B) and hemagglutination (human erythrocytes) (C, D). HA from strain A/California/07/2009 (Cal07) was used as the template for the introduction of the substitutions indicated on the x axis.
- Fig 6
Receptor-binding specificity of HA proteins of clades A and B with multiple amino acid substitutions. Precomplexed HA trimers were applied to a custom-made glycan array. The effect of amino acid substitutions that were introduced into wild-type (Cal07) HA in a stepwise reconstruction of clades A and B was examined. The structures of glycans (arranged in separate panels for N-linked glycans, O-linked glycans, and single-chain glycans attached by an ethyl linker to the array) that were bound (relative fluorescence units are indicated on the y axes) are shown below the panels. Binding was strictly specific for glycans carrying α2-6-linked sialic acids (indicated by purple diamonds in the glycan structure) attached to a β1-4-linked galactose (yellow circles). Blue squares, N-acetylglucosamine; yellow squares, N-acetylgalactosamine; green circles, mannose.
- Fig 7
Structural model of the amino acid substitutions in clade A and B HA proteins. The two pairs of amino acid substitutions that were shown to compensate for the effects on HA binding of each other are circled in red (panels A and B show clade B mutations S188T/A200T; panels C and D show clade A mutations) in the wild type (A, C) and double-substitution mutants (B, D). The receptor trisaccharide NeuAca(2-6)-Galβ(1-4)-GlcNAc is colored in purple (SIA), yellow (Gal), and blue (GlcNAc). The three structural elements that interact with the receptor are indicated in ribbon presentation (white, 130 and 220 loops; purple, 190 helix). Important backbone and side chain structures are also displayed. Two water molecules involved in a hydrogen bond network (green dotted lines) between residue S186, the 220 loop, and the sialic acid are indicated in red. Substitution S186P disrupts this network by clashes (red dotted lines in panel D) between the hydrophobic proline backbone and both water molecules. Substitution A137T leads to the formation of a new hydrogen bond between the threonine side chain hydroxyl group and the sialic acid. Remarkably, formation of this new bond decreases binding avidity, whereas the disruption of the water-supported network by S186P increases binding.
Tables
- Table 1
Coevolution of substitution patterns in HA and NA
Clustera Substitution(s) in HA No. of sequences in each cluster No. (%) of sequences with the indicated mutation in NA N44S V241I N396K 3.8 S454N 14 1 (7) 4 (29) 3 (21) 4.15 S454N, E377K 7 0 (0) 1 (14) 2 (29) 4.7 S454N, E377K, S188T 39 2 (5) 36 (92) 37 (95) 4.2 S454N, E377K, S188T, D100N 77 0 (0) 74 (96) 75 (97) 3.2 S454N, E377K, S188T, A200T 55 49 (92) 50 (94) 51 (96) 4.9 S454N, E377K, S188T, A200T, I463T 19 19 (100) 19 (100) 19 (100) 4.1 S454N, E377K, S188T, A200T, S146G 86 82 (95) 84 (98) 84 (98) 4.5 S454N, E377K, S188T, A200T, S146G, L-7M 33 33 (100) 32 (97) 33 4.1 S454N, E377K, S188T, A200T, S146G, N263D 12 12 (100) 12 (100) 12 (100) ↵a The cluster number corresponds to the clusters shown in Fig. 3 and 4.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1 (Accession numbers of all HA protein sequences included in each cluster.)
XLS, 630K
- Supplemental file 2
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Table S2 (Accession number of the HA protein sequence from each cluster that was used for construction of the phylogenetic tree shown in Fig. 3.)
XLS, 20K
- Supplemental file 1
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