Fig 3. Luminescent biosensors of authentic SARS-CoV-2 infection.

(A) Activation of luciferase-based reporters by recombinant SARS-CoV-2 protease expression. HEK293T cells were co-transfected with indicated 30F biosensors ± MPro, PLPro c.d., or empty pcDNA3.1. (B) Activation of luciferase-based reporters by SARS-CoV-2 infection. HEK293T-ACE2 cells were transfected with indicated 30F biosensors, incubated for 12 h, then mock-infected or infected with SARS-CoV-2 at MOI = 0.01. (C-E) Quantitation of infected cells. HEK293T-ACE2 cells were transfected with the 30F-PLP2 biosensor, incubated for 12 h, then mock-infected or infected with increasing doses of SARS-CoV-2. 1 μL of viral stock corresponds to MOI≈0.01. Cells were analysed in parallel 24 h post-infection by either epifluorescence microscopy for SARS-CoV-2 spike protein (C), or luminometry for Firefly and Renilla luciferase activities (D). Spike+ cells were enumerated by automated microscopy (Cellomics). Illustrative microscopy data (C) and mean values ± SEM (D) are shown for an experiment performed in duplicate (microscopy) or triplicate (luminometry). The correlation between the ratio of Firefly/Renilla luminescence and the proportions of spike+ cells is shown in E. Spike, red. DAPI, blue. DIC, differential interference contrast. R², Pearson’s correlation coefficient. Representative of 2 independent experiments. (F-G) Inhibition of SARS-CoV-2 replication by candidate antivirals. HEK293T-ACE2 cells were transfected with the 30F-PLP2 biosensor, incubated for 12 h, then infected with SARS-CoV-2 at MOI = 0.01 in the presence of DMSO or decreasing doses of candidate antivirals. Titration curves and IC50s are shown for remdesivir and GC376. Representative of 2 independent experiments. For all experiments, Firefly and Renilla luciferase activities were measured by luminometry 24 h post-transfection (A) or infection (B-G). Unless otherwise stated, mean values ± SEM are shown for experiments performed in triplicate, representative of at least 3 independent experiments. For F-G, Firefly/Renilla luminescence is shown as % luminescence in the DMSO condition. **** p<0.0001. MPro, recombinant SARS-CoV-2 Main Protease. PLPro c.d., catalytic domain of recombinant SARS-CoV-2 Papain-Like Protease.