Rastering the Influenza Virus Surface with Molecular Rulers and Nanoparticles to Design Optimal Multivalent Inhibitors

Multivalency is a widespread phenomenon in many biological systems, and inevitable for specific surface-surface interactions. The influenza virus provides a suitable system for studying multivalent interactions as it is covered by a densely packed layer of hemagglutinin (HA), which provides the virus high affinity to the host cells glycocalyx by multivalent interactions. To prevent such interactions and thus, infection, one promising strategy is the use of inhibitors presenting multiple HA receptors. For efficient inhibition, we investigated the optimal spatial arrangement, and amount of ligands of multivalent structures, among others multivalent polyglycerols and DNA-PNA heteroduplexes. Natural receptors such as sialic acid or peptide fragments from HA antibodies, form the basis of our multivalent constructs. In order to determine exact distances between HA binding sites, we introduced bivalent DNA-PNA heteroduplexes, presenting sialic acid with distinct distances, and thus serving as molecular rulers. Inhibition of fluorescent virions to red blood cells by heteroduplexes was assessed by flow cytometry measurement. By this strategy, we determined the spatial arrangement of sialic acid for optimal binding to virus at angstrom resolution. Microscale thermophoresis (MST) technology served as an independent and complementary approach to assess binding. Here, we detected nanomolar dissociation constants to full virus particles free in solution. In some instances, we observed multiphasic binding kinetics of multivalent structures to influenza virions. These studies are not only highly relevant for inhibition of virus infection, but also to provide new insights on the fundamentals of multivalent interactions.