N-glycosylation at the receptor binding site drives differences in receptor binding specificity between influenza B virus lineages.

Receptor specificity plays a critical role in influenza virus host tropism and pathogenesis. Influenza A and influenza B viruses (FLUAVs and FLUBVs, respectively) utilize N-glycans with terminal sialic acids on glycoproteins decorating the surface of mucosal epithelial cells as receptors for the virus hemagglutinin (HA) protein. For FLUAVs, the specificity of HA binding to distinct sialic acid linkages on host glycans is a major determinant of species specificity. Amino acid motifs and N-linked glycosylation sites influencing FLUAV HA receptor specificity are well defined. In contrast, considerably less is known regarding determinants of FLUBV receptor specificity, despite its significant contribution to the global influenza disease burden and unique restriction for human hosts. To address this knowledge gap, we utilize microarrays populated with glycans resembling structures found in the respiratory tract to comprehensively define the receptor binding profiles of FLUBVs from different decades and lineages and confirm these results with complementary virus-glycan binding assays. Using wild-type and reverse genetics FLUBVs having singular mutations in the HA receptor binding site (RBS), as well as structural models, we identify an N-glycosylation site at amino acid 196 within the RBS that determines the breadth of HA binding to terminal sialic acids. The definition of the presence of an N-linked glycan as the primary determinant for FLUBV receptor specificity provides a clear mechanism for lineage-specific differences in HA receptor binding. This may help explain the distinct tropism observed between Victoria and Yamagata lineage FLUBVs and provide insights into the disappearance of the Yamagata lineage.IMPORTANCEInfluenza B viruses (FLUBVs) are a major cause of human respiratory disease, but the molecular determinants influencing receptor specificity for the hemagglutinin protein remain largely undefined. We defined the receptor specificity of a panel of Early, Victoria, and Yamagata lineage viruses spanning over 50 years and showed that Victoria lineage viruses can have expanded receptor specificity, compared to Yamagata lineage viruses. We identified a critical N-glycosylation site within the hemagglutinin that regulates hemagglutinin binding to sialic acid receptors on host cells. Recent successful subclades of the Victoria lineage viruses lost this glycosylation site, enabling binding to both human-type and avian-type sialic acid receptors, which may influence respiratory tract tropism. These viruses also showed increased endemic activity. The expanded receptor tropism of Victoria lineage viruses may have fitness benefits, helping to explain epidemiologic features of the lineage, perhaps contributing to the lineage's recent success, while the Yamagata lineage appears to have become extinct.