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J Virol. 2018 Oct 24. pii: JVI.01413-18. doi: 10.1128/JVI.01413-18. [Epub ahead of print]
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 JJS1, Abente EJ2, Obadan AO1, Thompson AJ3, Ferreri L1, Geiger G1, Gonzalez-Reiche AS1,4, Lewis NS5, Burke DF6, Raj?o DS1, Paulson JC3, Vincent AL2, Perez DR7.
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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 (aa) substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of aa 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 aa 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 major impact on virus replication in vitro All substitutions retained receptor binding specificity, but 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 aa 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 virus' hemagglutinin (HA) of the H3N2 IAVs, the aa substitution N145K causes significant antigenic changes. We show that aa 145 displays remarkable amino acid plasticity in vitro tolerating multiple aa substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment on virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145K substitution in antibody immunodominance. We provide a better understanding of the functional effects of aa substitutions implicated in antigenic drift and its consequences on receptor binding and antigenicity. The mutation analyses presented in this report represent a significant dataset to aid and test computational approaches' ability to predict binding of glycans and in antigenic cartography analyses.
PMID: 30355680 DOI: 10.1128/JVI.01413-18
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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 JJS1, Abente EJ2, Obadan AO1, Thompson AJ3, Ferreri L1, Geiger G1, Gonzalez-Reiche AS1,4, Lewis NS5, Burke DF6, Raj?o DS1, Paulson JC3, Vincent AL2, Perez DR7.
Author information
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 (aa) substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of aa 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 aa 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 major impact on virus replication in vitro All substitutions retained receptor binding specificity, but 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 aa 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 virus' hemagglutinin (HA) of the H3N2 IAVs, the aa substitution N145K causes significant antigenic changes. We show that aa 145 displays remarkable amino acid plasticity in vitro tolerating multiple aa substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment on virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145K substitution in antibody immunodominance. We provide a better understanding of the functional effects of aa substitutions implicated in antigenic drift and its consequences on receptor binding and antigenicity. The mutation analyses presented in this report represent a significant dataset to aid and test computational approaches' ability to predict binding of glycans and in antigenic cartography analyses.
PMID: 30355680 DOI: 10.1128/JVI.01413-18
Free full text