Deciphering the single molecule interaction between the DC-/L-SIGN and the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) S1 protein using Atomic Force Microscopy (AFM)

Details

Supervisors

Alsteens, David ; Köhler, Melanie

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) appeared for the first time in December 2019 in Wuhan city (China). Since then, this highly transmissible virus shattered the world by causing severe acute respiratory syndrome and became responsible for the global outbreak of coronavirus disease 2019 (COVID-19), with more than 6 million reported deaths. In this context, understanding the mechanism of viral infection is fundamental to develop therapeutic solutions and prevention. The SARS-CoV-2 virus must enter host cells to establish its infection and proliferation, which occurred by the interaction of its spike with cell surface molecules. This crucial step is mediated by the virus’s spike glycoprotein, composed of two subunits: S1, responsible for the host cell’s interaction and S2, in charge of the membrane fusion. But also, cells surface molecules: attachment factors that anchor the virus to the cells and promote the S1 interaction with its functional receptors, and the latter, which triggered the virus internalization process. Consequently, many studies focus on identifying the attachment factors and receptors involved in the SARS-CoV-2 infection process. This thesis is part of them, with the purpose to decipher the interaction between Dendritic Cell-Specific intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) and Liver/lymph node-Specific intercellular adhesion molecule-3-Grabbing Non-integrin (L-SIGN) with the S1 subunit of the SARS-CoV-2. To probe these interactions, we used atomic force microscopy (AFM) based on single-molecule force spectroscopy. This method allows us to establish the thermodynamic and kinetic parameters of the interaction ligand-receptors. To this end, we first performed an in vitro study by functionalizing nanoscale AFM tips with the SARS-CoV-2 S1 protein to study the interaction with either DC- or L-SIGN grafted on model surface. Then, dynamic force spectroscopy and contact time experiments were performed to extract the association and dissociation rate of the complexes by applying biophysical models (Bell-Evans and Williams-Evans model). These experiments also enable the determination of the width of the free-energy barrier, separating the complexes from the unbound state to the bound state. Secondly, we validated the in vitro results by in vivo experiments on living cells overexpressing the probed receptors. Finally, to fulfil the objective of deciphering the interaction of the established complexes, we additionally probed the interaction of the DC-/L-SIGN with the receptor-binding domain (RBD) of the S1 protein to determine its implication on the bond formation. Then, we checked if the DC-/L-SIGN interaction with SARS-CoV-2 S1 protein depend on the glycosylation present on the subunit, using a non-glycosylated S1 variant. This study found that both receptors bind specifically the SARS-CoV-2 S1 subunit with a high affinity and L-SIGN with a higher binding probability and affinity than DC-SIGN. Moreover, it was found that their binding occurs presumably in the RBD of the S1 protein, and that in all likelihood both receptors bind the S1 protein thanks to its glycosylation sites. Overall, this thesis has improved the knowledge of SARS-CoV-2 interaction with cells, through DC-/L-SIGN, by first identifying the thermodynamic and kinetic parameters of the interactions and secondly by investigating where and how the binding mechanisms take place. Together, the results of this thesis can be used for further research between SARS-CoV-2 and host cells.