The study of the established interactions between viral glycoproteins and their host receptors is of vital importance for a better understanding of the entry of the virus into cells. Entry of the novel SARS-CoV-2 coronavirus into host cells is mediated by its peak glycoprotein (glycoprotein S), and angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which glycoprotein S binds to the ACE2 receptor.

We demonstrate, both on model surfaces and in living cells, that the receptor-binding domain (RBD) serves as a binding interface within the S-glycoprotein with the ACE2 receptor, and we extract the kinetic and thermodynamic properties of this binding pocket. Taken together, these results provide a picture of the established interaction in living cells. Finally, we tested several binding inhibitor peptides that target the early stages of virus binding, offering new insights in the treatment of SARS-CoV-2 infection.

In December 2019, a novel coronavirus (CoV) was determined to be responsible for an outbreak of life-threatening atypical pneumonia, finally defined as a coronavirus-19 disease (COVID-19), in Wuhan, China. This new CoV, termed severe acute respiratory syndrome (SARS) -CoV-2, was found to share similarities with the SARS-CoV that was responsible for the SARS pandemic that occurred in 2002. The resulting outbreak of COVID-19 has emerged as a severe pandemic. The SARS-CoV-2 genome shares approximately 80% identity with that of SARS-CoV and is approximately 96% identical to that of the bat coronavirus BatCoV RaTG13.

The entry of CoV into host cells is mediated by its peak transmembrane glycoprotein (S) that forms homotrimers that protrude from the viral surface2 (Fig. 1a).

Glycoprotein S comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit that includes the receptor-binding domain (RBD)) or for the fusion of viral and cellular membranes (S2 subunit). Recent studies affirmed that the angiotensin-converting enzyme 2 (ACE2), previously identified as the cellular receptor for SARS-CoV, also acts as a receptor for the new coronavirus (SARS-CoV-2) 3 (Fig. 1b). In the case of SARS-CoV, the S-glycoprotein on the surface of the virion mediates receptor recognition (Fig. 1c) and membrane fusion4,5. Recently, the high-resolution cryoelectron microscopy structure obtained on full-length human ACE2 in the presence of the SARS-CoV-2 S-glycoprotein RBD suggests the simultaneous binding of two S-glycoprotein trimers to an ACE23 dimer.

The S2 subunit is further cleaved by host proteases located immediately upstream of the fusion peptide6, leading to activation of the glycoprotein that undergoes extensive and irreversible conformational changes that facilitate the membrane fusion process. Taken together, the information obtained so far highlights the fact that CoV entry into susceptible cells is a complex process that requires concerted receptor binding and proteolytic activation of glycoprotein S on the host cell surface. to finally promote virus-cell membrane fusion. However, to date, direct evidence is lacking on the dynamics of S1- binding to the ACE2 receptor at the single-molecule level.

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