Influenza A virus membrane fusion is regulated by the balance between receptor binding and cleavage
Interactions between influenza A virus (IAV) and its host receptor, sialic acid, influence multiple stages of infection through the opposing activities of hemagglutinin (HA) and neuraminidase (NA). Multivalent HA binding to low-affinity receptors generates high avidity and attachment specificity, while NA-mediated receptor cleavage promotes penetration through sialylated respiratory mucus and progeny virion release. The role of receptor interactions in post-attachment entry, particularly during genome delivery through HA-mediated endosomal membrane fusion, remains unresolved due to conflicting data, limited control of receptor conditions, and difficulty separating attachment from fusion at low efficiency. Here, we address this question using a flow-cytometry based assay that quantifies time-resolved lipid mixing in hundreds of individual virion-membrane pairs per second, combined with programmable control of receptor density and chemistry on target membranes. We show that HA-receptor interactions regulate membrane fusion efficiency by promoting productive fusion-peptide insertion by HA. This effect depends on receptor context: within a defined regime, lipid-mixing efficiency increases with receptor density whereas NA activity reduces it by depleting receptors. Receptor type and HA-receptor binding avidity further modulate lipid-mixing outcomes, establishing a direct link between HA-receptor interactions and fusion efficiency. Together, these results identify receptor binding as an active regulator of membrane fusion and provide a framework that reconciles prior conflicting observations. More broadly, they extend the functional interplay between HA and NA to the level of membrane fusion, with implications for viral adaptation and host specificity. Significance StatementWe resolve a long-standing question by showing that host receptor binding by HA and cleavage by NA regulate influenza A virus membrane fusion, the entry step delivering the viral genome into the cell. HA-receptor engagement promotes productive HA membrane insertion, leading to fusion. Receptor density, HA-receptor affinity, and NA catalytic activity tune fusion outcomes. These results were enabled by two methodological advances: precise control of receptor presentation on target membranes and sensitive measurement of attachment and membrane fusion at the single-virion level. By defining how the opposing activities of HA and NA regulate fusion, this study extends their functional interplay to a key step in entry and provides new insight into how these proteins coevolve during viral adaptation.