Influenza A Virus Binding to α,2–3- and α,2–8-Linked Sialo-Gangliosides Reconstituted in Phase-Separated Vesicles

We investigated the binding of influenza A viruses (IAVs) to gangliosides containing either α,2-3- or α,2-8-linked sialic acid residues, or a combination of both. The respective gangliosides were reconstituted into phase-separated giant unilamellar vesicles to mimic the cellular membrane environment. Dual-color fluorescence imaging of influenza particles interacting with these vesicles revealed that the order of virus binding density and the lateral mobility of bound particles does not correlate with (i) the intrinsic affinity of viral hemagglutinin (HA) for specific sialic acid conformations or (ii) the number of sialic acid residues present in the gangliosides. Instead, we observe that the extent of viral attachment closely follows the surface charge of the ganglioside-rich vesicles. Because influenza particles carry a net negative charge, they experience electrostatic repulsion when approaching negatively charged membranes. Consequently, electrostatic interactions modulate the ability of viruses to establish multivalent contacts with membrane-bound sialic acids. Taken together, our findings demonstrate that electrostatic repulsion, glycosidic linkage, and lipid packing collectively govern influenza binding to phase-separated membranes. These results suggest that the commonly discussed preferential binding of influenza to specific sialic acid linkages and its association with viral species tropism should be reconsidered in the broader context of membrane surface charge and lipid organization.