Development of a fusion-entry system based on GFP complementation: an innovative approach for the screening of SARS-CoV-2 therapeutics

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a Betacoronavirus that causes COVID-19 disease. The mechanism of viral entry into human host cells consists in the binding of the Spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. Molecules targeting SARS-CoV-2 entry mechanisms are a promising therapeutic approach since they inhibit the first step of infection. This experimental study provides an innovative assay to screen new antiviral drugs with a fusion-entry system based on Green Fluorescent Protein (GFP) complementation. The system development consists in the production of stable ACE2 or Spike-expressing cell lines, to allow the fusion between membranes. Each cell line also carries a construct encoding a part of the split GFP (GFP1-10 or GFP11); upon membrane fusion, complementation occurs reconstituting GFP. The expression of each component of the system is monitored through flow cytometry allowing the sorting of cells. Pilot cell-cell fusion assays are then performed, and fusion is monitored through fluorescent microscopy, flow cytometry and fluorescence plate reader. The assay, once optimized, will be used to screen new antiviral drugs that target SARS-COV-2 Spike - human ACE2 mediated entry-fusion.