Delving into the interaction between the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and heparin using atomic force microscopy: a single-molecule approach

At the dawn of 2020, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) took the world by storm with a still ongoing COVID-19 pandemic. In the fight against the spread of the novel virus, numerous vaccines and therapeutics have been conceived with remarkable alacrity. Most of the developed treatments are based on the virus's spike (S) glycoprotein. Like any virus, SARS-CoV-2 initiates its infection by attaching and entering into a host cell. These first steps are mediated by the S glycoprotein, which comprises two main subunits: S1, in charge of binding, and S2, responsible for cell-virus fusion. A comprehensive understanding of the virus's entry mechanisms is necessary to find efficient ways of blocking the virus before it gets to enter cells and propagate. In the quest to infection, SARS-CoV-2's S1 domain interacts with two types of host-cell factors: receptors and attachment factors. Virions make use of attachment factors to anchor themselves to the cells and then bind to a receptor that actively promotes their entry. While there are many binding candidates for SARS-CoV-2, we focused on the entry mediated by the recognized functional receptor, Angiotensin-Converting Enzyme 2 (ACE2), and heparan sulfate (HS). More precisely, we performed an in vitro study of the interaction between the S1 subunit and heparan sulfate with heparin as a model surface. To probe the interaction of the S1-heparin complex, we used single-molecule force spectroscopy in atomic force microscopy (AFM). The method enables the extraction of thermodynamic and kinetic parameters of an interaction in near-physiological conditions with a high spatial and temporal resolution. We coupled nanoscale tips with the S1 domain and monitored its interaction with heparin-coated surfaces immersed in a phosphate buffer. Through control experiments, we confirmed the binding specificity between S1 and heparin. We found that removing the S1 from the biosensor had the same effect as the injection of free heparin into the medium, extending the potential inhibitory properties of heparin. We conducted dynamic force spectroscopy experiments to extract the kinetic and thermodynamic parameters of the S1-heparin interaction. We managed to extract the width of the free-energy barrier crossed by the complex when it unbinds, as well as the binding and dissociation rates. More particularly, we obtained a dissociation constant in the µM range, indicating a moderate binding affinity between S1 and heparin and thus heparan sulfate. The results were in agreement with other experiments probing similar systems. The kinetic and thermodynamic parameters were further used for multivalency assessment. Our data suggested the presence of uncorrelated double bonds but did not support the existence of triple bonds. As we achieved the characterization of the S1-heparin system using only S1 and functionalized surfaces, a future investigation of the binding could be carried out with cells and virions for physiological relevance. In addition, using mutated S proteins or the RBD instead of the whole S1 domain for measurements would help distinguish which binding sites on the S glycoprotein are involved in SARS-CoV-2 attachment.