Research Article

Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus

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Science  21 Apr 2006:
Vol. 312, Issue 5772, pp. 404-410
DOI: 10.1126/science.1124513
  1. Fig. 1.

    Crystal structure of Viet04 HA and comparison with 1918 human H1, duck H5, and 1968 human H3 HAs. (A) Overview of the Viet04 trimer, represented as a ribbon diagram. For clarity, each monomer has been colored differently. Carbohydrates observed in the electron-density maps are colored orange, and all the asparagines that make up a glycosylation site are labeled. Only Glu20, Glu289, and Phe154 are not labeled, as these are on the back of the molecule. The location of the receptor binding, cleavage, and basic patch sites are highlighted only on one monomer. All the figures were generated and rendered with the use of MacPymol (66). (B) Structural comparison of the Viet04 monomer (olive) with duck H5 (orange) and 1918 H1 (red) HAs. Structures were first superimposed on the HA2 domain of Viet04 through the following residues: Viet04, Gly1 to Pro160; 1918 H1 (PDB: 1rd8), Gly1 to Pro160; H3(PDB:2hmg), Gly1 to Pro160; H5 (PDB: 1jsm), Gly1 to Pro160. Orientation of the overlay approximates to the blue monomer in (A). (C) Superimposition of the two long α-helices of HA2 for 1918 H1 (PDB: 1rd8), avian H5 (PDB: 1jsm), human H3 (PDB: 2hmg), and Viet04 reveal that the extended interhelical loop of Viet04 is more similar to the 1918 H1 than to the existing avian H5 structure. The side chain of Phe63 is illustrated as an example of the close proximity of the two structures.

  2. Fig. 2.

    Antigenic variation in recent H5N1 viruses mapped onto the Viet04 structure. (Left) Side view of the Viet04 structure in which natural mutations identified by comparison of 2005 with 2004 isolates (23) are colored yellow; escape mutants (24, 25) are blue; and those that overlap in both analyses are green. All of the 2004 and 2005 strains have a new potential glycosylation site at position 158 in the HA1 chain (orange). The receptor binding site is highlighted with a red oval. (Right) Top view looking down onto the globular membrane distal end of the trimer around the RBD showing that the mutations mainly cluster around the RBD.

  3. Fig. 3.

    Analysis of Viet04 receptor binding site. (A) The Viet04 receptor-binding domain (RBD) with the side chains of key residues for receptor binding labeled. The binding site comprises three structural elements: an α-helix (190-helix) and two loops (130-loop and 220-loop). Residues mutated in this study are labeled red. (B) Overlay of the RBDs of Viet04 with Sing97 structure (PDB: 1jsm) reveals a similar RBD. The most divergent part of the pocket is the loop made up of residues 210 to 221, in which the Viet04 loop is displaced ∼1 Å farther away from the binding pocket compared with the 1997 avian H5. Only two residues, at position 216 and 221, differ in these two RBDs.

  4. Fig. 4.

    Glycan microarray analyses of (A) Viet04, (B) Dk97, and (C) an avian H1, Dk76. The Dk97 HA sequence is identical to that in the published structure of duck virus Sing97, so a direct structural comparison can be made. Binding to different types of glycans on the array are highlighted where orange represents glycoproteins; yellow, α2-3 ligands; green, α2-6 ligands; blue, α2-8 ligands; and purple, other ligands such as β-linkages, modified sialic acid analogs or glycolylsialic acid glycans. Red bars indicate sulfated or additional negatively charged ligands. See table S4 for list and tabulated binding results. Because of continual glycan microarray development, a number of new ligands were printed between analyzing the Dk76 protein (C) and the remaining samples reported in this study. Binding to glycans nos. 37 to 44, 56, 58 to 60, 67, and 70 was not determined for Dk76 and its three mutants in Fig. 5.

  5. Fig. 5.

    Glycan microarray analysis of mutants of Viet04 and Dk76. Mutations of an avian H1, Dk76: (A) E190D, (B) G225D, and (C) E190D and G225D were generated and subjected to glycan microarray analysis. Both positions were reported to be important for conversion of α2-6 receptor specificity of the human 1918 virus HA to avian α2-3 specificity (35, 45). These mutations did indeed result in exclusive α2-6 specificity for this avian H1 HA. (D to F) Consequently, Viet04 mutations were generated at the same positions, but did not result in a switch of receptor specificity, except to 6′-sialyllactose, although they did result in decreased α2-3 binding, particularly to nonsulfated glycans (compare Fig. 4A). (G to I) Viet04 was mutated at positions 226 and 228, known to be important for H3 HA α2-6 receptor adaptation. Again, no clear switch in receptor specificity was observed, although binding to biantennary α2-6 moieties was observed, as well as reduced α2-3 binding in the double and single (Q226L) mutant. Graphs are generated as described in the legend for Fig. 4 and labels to the introduced mutations.