Only Two Residues Are Responsible for the Dramatic Difference in Receptor Binding between Swine and New Pandemic H1 Hemagglutinin

Abstract

In view of its critical role in influenza A virus (IAV) tropism and pathogenesis, we evaluated the receptor binding properties of HA proteins of the closely related swine and new pandemic human IAVs. We generated recombinant soluble trimeric H1 ectodomains of several IAVs and analyzed their sialic acid binding properties using fetuin-binding and glycan array analysis. The results show that closely related swine and new pandemic H1 proteins differ dramatically in their ability to bind these receptors. Although new pandemic H1 protein exhibited hardly any binding, swine H1 bound efficiently to a number of ␣2-6-linked sialyl glycans. The responsible amino acids were identified by analyzing chimeric H1 proteins and by performing systematic site-directed mutagenesis of swine and new pandemic human H1 proteins. The difference was found to map to residues at positions 200 and 227. Although substitution of either residue significantly affected the binding phenotype, substitution of both was found to act synergistically and reverse the phenotype almost completely. Modeling of the T200A and E227A substitutions into the crystal structure of the new pandemic human H1 protein revealed the loss of potential hydrogen bond formation with Gln 191 , which is part of the 190-loop of the receptor binding site, and with the penultimate galactose, respectively. Thus, a residue not belonging to the receptor binding site may affect the interaction of HA with its receptor. Interestingly, whereas alanine at position 200 is found in most new pandemic human viruses, the residue at position 227 in these viruses is invariably a glutamic acid. All influenza A virus (IAV)2 pandemics known so far originated from avian or swine IAV strains that managed to cross the species barrier to humans and acquire the capacity of human-to-human transmission. The most recent example of this was the new H1N1 swine origin IAV that emerged in 2009 and rapidly spread around the world (1, 2). The specificity of the interaction of HA with sialic acid (SIA), the cellular receptor, largely explains the host range of IAVs (3). Thus, viruses that infect humans bind preferentially to SIA linked to the penultimate galactose in an ␣2-6 configuration, whereas avian viruses prefer binding to SIA with ␣2-3 linkages (4). However, the adaptations in HA required for swine IAVs to become infectious for humans and to establish themselves in the human population are much less characterized.The HA receptor binding site (RBS) is formed by three structural elements at the tip of the HA molecule, an ␣-helix composed by residues 190 -198 (the 190-helix) and two loop structures formed by residues 133-138 (the 130-loop) and 220 -229 (the 220-loop). Four conserved residues, comprising Tyr 98 , Trp 153 , His 183 , and Tyr 195 , form the base of the RBS (5). The amino acid residues in the RBS that are critical for the recognition of either avian or human receptors have been well characterized (4, 6, 7). For H1, glutamic acid and glycine residues at positions 190 and 225, respectively, resu...

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

“…Our crystal structures confirm the relatively low affinity for ␣2-3-linked receptor analogs, as evidenced by the relatively disordered electron densities and low occupancy of ligands in these structures. Such small differences in individual HA/glycan affinity are amplified by multivalent binding into distinguishable binding properties (40,48), as also suggested by our glycan-binding experiments as well as by others (7,10,15,29,49).…”

“…is not among the HA residues directly interacting with glycan receptors, the T200A substitution in CA04 was shown to improve the receptor binding of recombinant HA on the glycan array (15). The lower avidity of the CA04 HA relative to those of the other pandemic strains also was evident in a plate-based ELISA.…”

Substitutions T200A and E227A in the Hemagglutinin of Pandemic 2009 Influenza A Virus Increase Lethality but Decrease Transmission

“…Residues 200 and 227 were recently reported to be responsible for the difference in the binding affinity for the sialic acid receptors between the HAs of swine and A(H1N1)pdm09 influenza virus isolates (6). Furthermore, recent publications have shed light on the role of HA residue 227 in cell tropism and transmission, confirming its role in sialic acid recognition (7).…”

“…Our results suggest that substitutions T200A and E227A of Cal/09 influenza virus HA affect viral replication, most likely because of their involvement in receptor binding affinity (6), thus affecting pathogenicity, virulence, and transmission. It is interesting that these mutations allow the virus to replicate to higher levels in MDCK cells and mice, while they delay transmission in ferrets.…”

“…In fetuin binding and glycan array assays, it was observed that swine virus-derived HA showed stronger binding to ␣2,6-linked sialic acids. When substitutions T200A and E227A were introduced into A(H1N1)pdm09 HA, its receptor binding increased to the same level as its swine HA counterpart (6).…”