Plasticity of Amino Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A Viruses and Its Impact on Receptor Binding and Antibody Recognition

Santos, J J S and Abente, E J and Obadan, A O and Thompson, A J and Ferreri, L and Geiger, G and Gonzalez-Reiche, A S and Lewis, N S and Burke, D F and Rajão, D S and Paulson, J C and Vincent, A L and Perez, D R (2019) Plasticity of Amino Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A Viruses and Its Impact on Receptor Binding and Antibody Recognition. JOURNAL OF VIROLOGY, 93 (2). e01413-18.

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Abstract

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication in vitro. All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift.