Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin

Abstract

The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA-receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.

Fig. 1.

Biolayer-interferometry binding curves of influenza viruses (100 pM) to the human receptor analog α2,6-sialyl lactosamine bound to the sensor chip show a large decline in avidity of the virus for receptor over the period of 2001–2010. Viruses used are indicated as follows: 1968, X31 (a high growth reassortant virus carrying the HA and NA genes of A/Aichi/2/68); 2001 A/Toulouse/878/2001; 2003, A/Trieste/2/2003; 2004, A/Finland/486/2004; 2004 (S193F) reverse genetic virus with the HA and NA genes of A/Finland/486/2004 carrying the substitution Ser-193→Phe in HA1; 2004 (D225N), reverse genetic virus with the HA and NA genes of A/Finland/486/2004 with the substitution Asp-225→Asn in HA1; 2005, A/Hong Kong/4443/2005; 2010a, A/Hong Kong/3615/2010; 2010b, A/Esfahan/6117/2010.

Fig. 2.

Crystal structures of H3 HA trimers from 1968 (A) and 2004 (B) viruses shown in a surface electrostatics representation. Negative potential is colored red and positive in blue, and the bound sialic acid moieties from receptor complexes are colored in yellow. Additional potential glycosylation sites present in the 2004 HA, but not 1968 HA, are marked in green and labeled on the structure of the 2004 HA.

Fig. 3.

The structure of the receptor binding site of H3 HAs in complex with human receptor analogs. (A) Overlap of apo (gray) and human receptor complex of 2004 HA (protein in green; receptor in yellow) showing the hydrogen bond interactions between Asp-225 and the 3-hydroxyl of galactose-2 made possible by the altered conformation of the 220-loop in the receptor complex. (B) Similar overlap to A of the apo (gray) and human receptor complex of 2005 HA (protein in blue; receptor in yellow), illustrating their similarity. In the 2005 complex, only the sialic acid moiety of the receptor can be built. (C) Overlap of the human receptor complexes of the 1968 HA (colored magenta) and 2004 HA (colored green). Key differences between the two, including the greater distance between the hydrophobic Ile-226 and the bridging carbon of the receptor in the 2004 complex (4.5 versus 3.8 Å) and the presence of Glu-190 in the 1968 structure versus Asp-190 in the 2004 structure, are indicated. Selected interactions are shown that are additional to the highly conserved hydrogen bonds formed between the sialic acid carboxylate and the OH of Ser-136 and main-chain amide 137, and between main-chain carbonyl 135 and the N of the acetamido substituent. The selected interactions shown are as follows: (A) Ser-137 OH indirect through H₂O to sialic acid carboxylate, Ser-137 OH direct to sialic acid carboxylate, and Thr-135 OH indirect through H₂O to sialic acid 4-OH. (B) Ser-136 OH indirect through H₂O to sialic acid carboxylate. Thr-135 OH indirect through H₂O to sialic acid 4-OH.