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. 2022 Jun 29;8(7):1265–1279. doi: 10.1021/acsinfecdis.2c00008

Figure 4.

Figure 4

Effect of Apra S4 on SARS-CoV-2 multicycle growth and entry. (A) SARS-CoV-2 growth curve. Left panel: Vero CCL81 cells were treated with Apra S4, Remdesivir, or DMSO (0.1%) at the indicated concentrations 2 h before, during, and after infection with SARS-CoV-2 BetaCoV/Germany/BavPat1/2020 (MOI = 0.01). Of note, compounds were not replenished during incubation. Cell supernatants were harvested 0, 16, 24, 48, and 72 h postinfection and virus titers were determined by plaque assay. Shown are mean virus titers of three independent experiments performed in duplicates. Error bars indicate standard deviation. LoD: limit of detection was at 100 PFU/mL. Of note, titers for 1 μM and 0.1 μM Apra S4 lie below the LoD. Right panel: Cell viability of Vero CCL81 after treatment similar to left panel, but left uninfected. At the indicated time points cell viability was determined using the CellTiter-Glo kit. (B) Time-of-Addition assay: Vero CCL81 cells were treated with Apra S4 (1 μM), Remdesivir (10 μM), or DMSO (0.1%) starting either 2 h before, during, or 2 h after infection with SARS-CoV-2 BetaCoV/Germany/BavPat1/2020 (MOI = 0.5). Cell supernatants were harvested 16 h postinfection and virus titers were determined by plaque assay. Shown are mean virus titers of three independent experiments performed in duplicates. Error bars indicate standard deviation. For both (A) and (B), an unpaired t test on log-transformed data was performed to test for statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. LoD: limit of detection was at 100 PFU/mL. (C) VSV-based pseudotyped viral particles assay. Vero E6 cells were pretreated with Apra S4 for 2 h and then infected with SARS-CoV-1 S/SARS-CoV-2 S pseudotyped particles harboring firefly luciferase for additional 1 h. Luciferase signal was quantified at 24 h postinoculation (left). The cell viability was measured at 24 h postinfection using WST-8 assay and normalized to the DMSO-treated samples (right). Error bars represent standard error of the mean (SEM).