Hooking on Viral Glycoproteins with Single Molecule Force Spectroscopy to Study Single and Multiple Bond Formations

Abstract

The influenza A virus (IAV) interacts with sialic acid (SA) receptors on host cell membranes upon multivalent interactions of its major spike glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA mediates the viral attachment to cells, while NA enables the release of newly formed virus particles by destroying SA receptors. Since both proteins target the same receptor, but have antagonistic functions, virus infection depends on a properly tuned balance of the kinetics of HA and NA activities for viral spread. Here dynamic single molecule force spectroscopy (SMFS) was employed to determine bond specific kinetics, characterized by the off-rate and on-rate of virus binding, the rupture length as well as the related free energy barrier ΔG. Measurements were conducted on two relevant virus systems, differing in the HA/NA type, and by using a synthetic SA-displaying receptor attached to the SFM cantilever. The bond strength was tested to immobilized HA and NA of the IAV strains California/H1N1, and Aichi/H3N2 as well as intact virus particles of the latter. SMSF at variable force loading rates revealed most probable rupture forces of the protein-SA bond being in the range of 10-100 pN. Measurements of the binding probability at increasing contact time between the SFM force probe, and the surface allowed an estimation of KD, which suggests a stronger interaction for NA-SA than for HA-SA. A systematic study of multiple bonds connected in parallel demonstrated a rapid formation of multivalent interactions of both proteins increasing strongly the bond life-time (msec-sec). In conclusion, we obtained new insights for the orchestrated role of HA and NA in the viral binding and release process. In particular, our results provide strong evidence for involvement of NA into viral attachment to the host cell.