Structure, receptor binding, and antigenicity of influenza virus hemagglutinins from the 1957 H2N2 pandemic

Abstract

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 A resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

FIG. 1.

Crystal structure of H2 HA. (a) Chemical structures of α2,3- and α2,6-linked glycans, with the terminal sialic acid and galactose shown here. (b) Overview of the 1957 H2 trimer. One of the monomers is highlighted in green (HA1) and blue (HA2), respectively. Five potential glycosylation sites are found on each monomer (as labeled). Glycans in the density map are shown in orange. (c) Receptor binding site of H2. Residues involved in receptor binding, as suggested by the H3 structures, are shown in sticks. Aromatic residues comprising the base of the binding site are absolutely conserved in various HA subtypes. Residues from the 220 loop and position 190 are critical for the receptor specificity switch in H1, H2, and H3.

FIG. 2.

Switch of binding specificity in H2 HA. (a) Amino acids at positions 226 and 228 in selected H2 HAs of avian, human, and swine origins. (b to d) Residues at positions 226 and 228 dictate binding specificity in H2. Sialosides are grouped into α2,3 (residues 1 to 46)- and α2,6 (residues 47 to 73)-linked glycans for interrogation on the array. In each graph, binding to α2,3- and α2,6-linked glycans are shown as green and red bars, respectively. Glycans are also color-coded in subgroups along the x axis and in the glycan list in the supplemental material (orange, α2,3 sulfated sialosides; blue, α2,3 branched disialosides; green, α2,3 linear sialosides; red, α2,3 fucosylated sialosides; light blue, α2,3 internal sialosides; yellow, α2,6 sulfated sialosides; purple, α2,6 branched di-sialosides; magenta, α2,6 linear sialosides; brown, α2,6 internal sialosides). H2-human HA (d) reveals classic α2,6 specificity, while H2-avian HA (b) binds only α2,3-linked glycans. A single mutant H2-226L/228G (H2-LG) (c) shows relatively low binding to both α2,3 and α2,6 sialosides.

FIG. 3.

Structural comparison of H2 mutants in a stereo representation. H2-avian (orange), H2-human (cyan), and H2-226L/228G (gray). The binding pocket is extended by about 0.5 Å by mutations Q226L/G228S from H2-avian to H2-human. The 220 loop is shown in Cα trace for clarity.

FIG. 4.

Antigenic structure of H2. Positions of escape mutants are mapped on one monomer, in side view (a) and top view (b). Mutations that add a glycosylation site are colored in green, whereas other single residue mutations are colored in blue. A mutation on the stem region (Lys⁴⁰; shown in red) reveals a highly conserved epitope for antibody binding. (c) Sequence alignment of the conserved epitope within the stem domain (HA2, residues 38 to 57) from the four structures is shown. Residues conserved in all four structures are highlighted in red, with residues conserved only in the H2 clade colored in green. (d) Comparison of the conserved epitope from various HA subtypes. Helix 1 from H1 (1ru7) (gray), H3 (2hmg) (blue), and H5 (1jsm) (yellow) are superimposed on the same segment of H2 (green). Positions of escape mutants in this region, identified in two independent studies, are shown in sticks and labeled in red.