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Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties

Nemanichvili, Nikoloz, Tomris, Ilhan, Turner, Hannah L., McBride, Ryan, Grant, Oliver C., van der Woude, Roosmarijn, Aldosari, Mohammed H., Pieters, Roland J., Woods, Robert J., Paulson, James C., Boons, Geert-Jan, Ward, Andrew B., Verheije, Monique H., de Vries, Robert P.
Journal of molecular biology 2019 v.431 no.4 pp. 842-856
Influenza A virus, animal tissues, antibodies, binding properties, binding sites, fluorescence, green fluorescent protein, hemagglutinins, polysaccharides, staining, viruses
Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA–receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA–sfGFP proteins was studied using glycan arrays and tissue staining. The HA–sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA–sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.