New models for studying SARS CoV-2 S/ACE2 interaction available in MobileVIR team

04 February 2021 by Delphine LAPAILLERIE

https://doi.org/10.1101/2021.02.03.429555

From the beginning of the pandemic our team has been deeply involved in the research about SARS-CoV-2 with the only purpose to provide additional tools and approaches based on our expertise to the community.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we developed a strong collaboration network between our MobileVIR team, MFP lab, Vect’UB (V. Guyonnet-Dupeyrat), IMPACT platform in Nantes (C. Charlier and P. Weigel), IINS (A. Favereaux) and BioSIM, University of Porto. This collaborative work led us to set up various in silico, in vitro and in cellulo approaches that allow to specifically monitor the S/ACE2 association. We report especially a novel computational model of the SARS-CoV-2 S/ACE2 complex as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context, and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step as currently ongoing in the team.

 

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