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

Figure 5.

Figure 5

Apra S4 inhibits formation of double-membrane vesicles and SARS-CoV-2 replication. (A) Transmission electron microscopy demonstrates block of the formation of viral replication organelles. Vero CCL81 cells were pretreated with DMSO (top) or 1 μM Apra S4 (bottom) for 2 h before cells were infected with SARS-CoV-2 BetaCoV/Germany/BavPat1/2020 (MOI = 2) in the presence of DMSO or Apra S4. At 16 h postinfection cells were fixed, dehydrated, and embedded. Ultrathin sections were stained with lead citrate and analyzed by transmission electron microscopy. Arrows indicate budding viruses. Scale bars indicate 1 μm. (B) Negative-stranded viral RNA synthesis. Vero E6 cells were electroporated with in vitro transcribed viral RNA. Two hours after seeding, the cells were treated with Apra S4 at the indicated concentration. Negative-stranded RNA was then quantified at 12 h postelectroporation. Error bars represent SEM for n = 3 independent experiments. (C) dsRNA expression. Vero CCL81 cells were pretreated with DMSO or 1 μM Apra S4 for 2 h. Cells were infected with SARS-CoV-2 BetaCoV/Germany/BavPat1/2020 (MOI = 2) in the presence of DMSO or Apra S4. At 16 h postinfection, cells were fixed and stained for dsRNA (green). Nuclei are stained in blue. Scale bars indicate 8 and 5 μm for the merged and merged zoom images, respectively. (D) Quantification of dsRNA. The mean fluorescence intensity of dsRNA-expressing cells from (C) was quantified using ImageJ software. Shown is mean fluorescence intensity (MFI) of two independent experiments (RI/RII). Error bars indicate standard deviation. A Mann–Whitney U test was performed to test for statistical analyses. *P < 0.05; ****P < 0.0001 (E) Vero CCL81 cells were treated with DMSO or 1 μM Apra S4 for 18 h and cell viability was determined using the CellTiter-Glo kit. Mean values from three independent experiments are shown relative to the DMSO control. Error bars depict standard deviation. (F) Vero CCL81 cells were pretreated with DMSO or 1 μM Apra S4 for 2 h before cells were infected with SARS-CoV-2 BetaCoV/Germany/BavPat1/2020 (MOI = 2) in the presence of DMSO or Apra S4. At 16 h postinfection virus titers in the supernatants were determined. Mean values from three independent experiments are shown. Error bars depict the standard deviation. Dotted line represents the limit of detection. An unpaired t test on log-transformed data was performed to test for statistical significance. **P < 0.01.