Cell-based sialoglycan arrays for directly comparing influenza A virus receptor requirements for binding and infection

Influenza A viruses multivalently engages sialoglycan attachment factors. Synthetic glycan arrays provide meticulous insight into primary binding specificity but do not capture dynamic post-binding virus-receptor interactions leading to cell entry. Establishing an HEK293 cell-based array of genetically dissected sialoglycan assemblies enabled screening of the complete interaction cascade from binding to infection, at physiologically relevant low virus doses. Screening forty years of H3N2 receptor binding evolution showed that besides N-glycans, deemed as principal receptors for primary attachment, specific O-glycans or glycosphingolipids independently supported all steps from primary binding to entry. For all three glycoconjugate classes, receptor preferences gradually evolved toward utilization of human-type α2-6-linked sialic acid receptors, followed by regaining use of avian-type α2-3-linked receptors after 1995. The screen identified a lack of quantitative correlation between binding and infection efficiency, suggesting specific receptor requirements beyond attachment. Virus-glycan interactions and other sialoglycan-dependent interactions with cells can be functionally analyzed using this system.