In silico, in vitro and in cellula models for monitoring SARS-CoV-2 spike/human ACE2 complex, viral entry and cell fusion

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 on viral protein complexes to the community. The Severe Acute Respiratory Syndrome-coronavirus 2, SARS-CoV-2, is the etiologic agent responsible for the recent coronavirus disease 2019 (Covid-19) pandemic. The productive SARS-CoV-2 infection relies on the virus entry into cells expressing the 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 and it has been consequently considered an attractive therapeutic target. In order to characterize further this interaction and the cellular processes engaged in the entry step of the viruswehave set up various in silico, in vitro and in cellula systems allowing to specifically monitor the S/ACE2 association based on the expertise of our team in virus/host interactions. We perform computational modelization of the SARS-CoV-2 S/ACE2 complex as well as its biochemical and biophysic monitoring using pull down, alphaLISA and bilayer interferometry (BLI). 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 cellula transduction assays using SARS-CoV-2 pseudotyped lentiviral vector and HEK293TACE2cell lines generated in collaboration with theVect'UB platform. This led us to recapitulate the early replication stage of the infection mediated by S/ACE2 interaction as well as to detect cell fusion induced by the interaction. Finally, a cell imaging system has been setup to directly monitor the S/ACE2 interaction in a cellular context and a flux cytometry assay has been developed to quantify this association at the surface of the cells. All these different approaches are now available for both fundamental and therapeutic purpose aiming to characterize the entry step of the initial SARS-CoV-2 strain and its variants. They could also be used for investigating the possible chemical modulation of this interaction and, thus, identifying newantiviral agent as well as newchemical tools for dissecting this entry step.