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

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. IMPORTANCE The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity in vitro, tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses.

Variability of nonpathogenic influenza virus H5N3 under immune pressure

“…A mutation in this position may lead to conformational changes and may affect receptor specificity of those mutated influenza viruses. Indeed, the mutation S145P resulted in increased affinity towards sialyl receptors, as was shown earlier (Ilyushina et al, 2004, Santos et al, 2019. Resistance to high temperatures and increased affinity for cell receptors can give viruses an advantage of more successful environmental spread.…”

Genetic Characterization of Influenza A Viruses in Japanese Swine in 2015 to 2019

“…Single mutations at positions 145, 155, 156, 158, 159, 189, and 193 that are adjacent to the receptor binding site of the human H3 HA protein greatly reduced reactivity against serum obtained from ferrets infected with a wild-type strain (54). Of these 7 mutations, the single mutation at position 145 on H3 HA protein among IAVs-S also reduced reactivity against serum obtained from pigs infected with a wild-type strain (55). Likewise, in the present study, the single mutation at position 155 on H1 HA protein, which corresponds to 158 on H3 HA protein, might be critical for the altered antigenicity, enabling the variant to evade immunity against the previous strain on the farm.…”

Machine learning prediction and experimental validation of antigenic drift in H3 influenza A viruses in swine

“…Given the raw antigenic distances calculated from the pair of titers were 6 and 7 for the two serum samples, the real distance is likely somewhere between the two values. Consequently, our approach that was developed using a small IAV in swine empirical dataset made predictions that in the majority of cases are useful in biological applications Literature reports suggested that the conservation of biochemical properties of the amino acid mutation may also have some effect on the observed antigenic change (15,19). Unequal weighting of mutations in the model suggests antigenic distance may help improve vaccine antigen selection when compared to HA sequence comparison alone, as this approach captures not only sequence homology but how amino acid can influence antigen-antibody interactions.…”

“…Structural changes in the HA may alter the interaction with antibodies targeting the virus, and these changes are generally correlated with the number of accumulated amino acid mutations in the HA protein (19). Empirical data has also shown that certain amino acid mutations have a disproportionate effect on antigenic change based on the location of the amino acid in the protein structure (13,15). Though there are relatively few antigenically characterized swine IAV HA genes (9,13), this empirical data may be used to establish antigenic distances between multiple IAV in swine, and be used to gain insight on the contribution of site-specific amino acid mutations.…”