Question

66. Describe the role of force fields in the approach to identify a protein that binds influenza hemagglutinin.

Answer 1

Influenza virus belongs to a wide range of enveloped viruses. The viral envelope contains two spike proteins, hemagglutinin (HA), and neuraminidase (NA), as well as the proton channel M2. The major spike protein hemagglutinin binds sialic acid slag of glycoproteins and glycolipids with segregation constants in the millimolar range.The crystal structure of the viral HA bound to its cellular receptor, sialic acid (SA), shows that the receptor-binding domain is located in the HA1 subunit.

• The first critical step of infection is marked by virus-host cell binding.Hence, forces involved in this process are essential.Optical tweezers (OT) and atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) provide powerful tools to measure these forces in biological systems. Using optical tweezers, binding forces were measured between viruses on beads and adherent cells. However, an assignment of forces to their underlying molecular interactions involved in these processes is difficult or cannot even be obtained by these techniques.
• We applied single-virus force spectroscopy (SVFS) to characterize the interacting forces between influenza A viruses (IAV) and living cells.The binding of individual Influenza A viruses (IAV) to living host cells is particularly observed by SMFS. In this type of analysis, Atomic Force Microscopy (AFM) cantilevers are covalently attached to intact influenza viruses, Which are then lowered on single living cells. While revealing kinetic and thermodynamic properties of the interactions cycles between cell binding and cantilever retraction, allow direct characterization of virus-cell binding. Thus, SVFS allows investigating virus-cell binding in an experimental system that closely mimics the natural situation.

• Single-Virus Force Measurements using AFM: For SVFS, already stated that intact viruses were covalently attached to AFM cantilevers. Binding to cells was measured in a dynamic range of increasing loading rates, i.e. pulling velocities to determine the dissociation rate at zero force k_off. To obtain the rupture force F as well as the effective spring constant k_eff, unbinding events were recorded and analyzed. We obtained the thermodynamic properties of the interaction, by fitting the force spectra to a single energy barrier model, and the separation of the receptor-bound state to the energy barrier. The dissociation rate k_off and its reciprocal, the bond lifetime ?_off, provide information about the stability of the underlying virus-cell interaction.