Glycosylation Focuses Sequence Variation in the Influenza A Virus H1 Hemagglutinin Globular Domain

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Abstract
Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the “flow index” (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation. Influenza A virus is highly susceptible to neutralizing antibodies specific for the viral hemagglutinin glycoprotein (HA), and is easily controlled by standard vaccines. Influenza A virus remains an important human pathogen, however, due to its ability to rapidly evade antibody responses. This process, termed antigenic drift, is due to the accumulation of amino acid substitutions that modify HA antigenic sites recognized by neutralizing antibodies. In this study, we perform bioinformatic analysis on thousands of influenza A virus isolates to better understand the influence of N-linked glycosylation on antigenic drift. HA from human IAV isolates can accommodate up to 6 oligosaccharides in its globular domain. We show that for H1, H2, and to a somewhat less extent H3, HAs, the number of glycosylation sites in the globular domain does not greatly modify the total degree of variation in antigenic sites, but rather focuses variation on sites whose access to antibodies is unaffected by glycosylation. Our findings imply that glycosylation protects HA from antibody neutralization, but functional impairment limits the number of oligosaccharides that HA can accommodate.