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

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.Surveillance of influenza viruses is essential for updating vaccines, for tracking the emergence of drug resistant viruses, and for monitoring zoonotic infections. It also gives important insights into the mechanisms of virus evolution. This is particularly the case for interpreting the correlation between antigenic differences and changes in the sialic acid receptor binding properties of the HA glycoprotein. The correlation in these two properties arises because of the close proximity on HA of binding sites for antibodies that neutralize virus infectivity and the sialic acid receptor binding pocket (1), and accounts for the observations that mutations that prevent antibody binding can also result in changes in receptor binding (2–7). Reduction in affinity of human H3N2 viruses for avian receptors since the beginning of the pandemic in 1968 has meant that by the 1990s viruses with reduced ability to agglutinate chicken erythrocytes had emerged (8, 9). Moreover, viruses isolated after 1999 were shown to have reduced affinity for both human and avian receptors, a feature that correlated with their poor growth properties in eggs and different cells in culture (9–14). The evolution of the HA has resulted in at least three key changes that influence receptor binding. Two sequential substitutions occurred at residue 225: in 2001–2002, a substitution Gly-225→Asp was accompanied by a Trp-222→Arg substitution, and in 2004–2005, an Asp-225→Asn substitution was accompanied by the substitution Ser-193→Phe (while maintaining arginine at position 222). Residue 226, a key amino acid in determining receptor specificity (15), also changed twice: before 2001, Leu-226→Val, and in 2004, Val-226→Ile (Fig. S1).To correlate these amino acid substitutions with the biological properties of the viruses, we have analyzed the receptor binding characteristics of H3N2 viruses isolated between 2001 and 2010, examined changes in their ability to infect cells in culture, and determined the structures of two HAs of virus isolates from 2004 and 2005 in the absence of receptor and complexed with a human receptor analog. The data show that the progressive decrease in binding of these viruses to human receptors from 2000 onward correlates with changes in the efficiencies of infection of cultured cells. Comparison of structural data for HAs of viruses from 1968, 2004, and 2005 explain how particular mutations that affect the conformation of the HA1 220-loop component of the receptor binding site define the receptor binding phenotype of recent H3N2 human influenza viruses.