Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants

Abstract

We used a panel of monoclonal antibodies to H9 hemagglutinin to select 18 escape mutants of mouse-adapted influenza A/Swine/Hong Kong/9/98 (H9N2) virus. Cross-reactions of the mutants with the antibodies and the sequencing of hemagglutinin genes revealed two minimally overlapping epitopes. We mapped the amino acid changes to two areas of the recently reported three-dimensional structure of A/Swine/Hong Kong/9/98 hemagglutinin. The grouping of the antigenically relevant amino acid positions in H9 hemagglutinin differs from the pattern observed in H3 and H5 hemagglutinins. Several positions in site B of H3 hemagglutinin are distributed in two sites of H9 hemagglutinin. Unlike any subtype analyzed so far, H9 hemagglutinin does not contain an antigenic site corresponding to site A in H3 hemagglutinin. Positions 145 and 193 (H3 numbering), which in H3 hemagglutinin belong to sites A and B, respectively, are within one site in H9 hemagglutinin. This finding is consistent with the peculiarity of the three-dimensional structure of the H9 molecule, that is, the absence from H9 hemagglutinin of the lateral loop that forms site A in H3 and the equivalent site in H5 hemagglutinins. The escape mutants analyzed displayed phenotypic variations, including decreased virulence for mice and changes in affinity for sialyl substrates. Our results demonstrate a correlation between intersubtype differences in three-dimensional structure and variations among subtypes in the distribution of antigenic areas. Our findings also suggest that covariation and pleiotropic effects of antibody-selected mutations may be important in the evolution of H9 influenza virus, a possible causative agent of a future pandemic.

FIG. 1.

Locations of the amino acid changes in the HA of H9 escape mutants mapped on the three-dimensional map of A/Swine/Hong Kong/9/98 HA (8). Amino acids were mapped on the three-dimensional structure with the RasMol 2.6 program (available at http://www.umass.edu/microbio/rasmol/getras.htm). Positions of amino acid changes detected by the binding of MAbs are shown in red, and the position of the change resulting from mouse adaptation is shown in blue. Numbering corresponds to the HA of subtype H3. H9 numbering (8) is presented in red in parentheses.

FIG. 2.

Antigenic sites on the globular head of HA of the H3 (A), H5 (B), and H9 (C) subtypes mapped on the three-dimensional structure of the molecule (top view). Images were created with RasMol 2.6, and HA structures are from the Protein Data Bank (PDB accession numbers 1HGF, 1JSM, and 1JSD, respectively). Antigenic sites in H3 (A) are marked in red, green, and yellow, corresponding to sites A, B,and D, respectively, described by Skehel and Wiley (23). Antigenic sites at the top of the H5 HA (B) and H9 HA (C) are shown in blue and green. Residue numbering corresponds to the HA of subtype H3. Residues in H9 contributing to the overlap of epitopes are marked by asterisks. H5 antigenic sites correspond to those given in our previous paper (10).