Fig. 2.

Effect of Bergamottin on SARS-CoV-2 life cycles. (A) Time-of-addition assay. SARS-CoV-2 infected Caco-2 cells were incubated with bergamottin at indicated time points as the diagram shows. Infection at 8h post-infection was quantified by immunostaining for NP. Data represents mean ± SD for n = 3 biological replicates. (B) Bergamottin inhibited VSV-based SARS-CoV-2 pv infection. Caco-2 cells were pretreated with the indicated concentration of bergamottin and infected with a pseudovirus. Luciferase activity was measured 24 h later. (C) Bergamottin inhibited SARS-CoV-2 S protein-mediated membrane fusion. Vero E6 cells co-transfected with SARS-CoV-2 spike and EGFP plasmids. After 4 h post-transfection, the medium was replaced by bergamottin and the images were acquired 24 h later using fluorescent microcopy. Scale bar: 1000 μm. Representative images selected from three independent experiments are shown. (D) Dual-split-protein (DSP)-based cell-cell fusion assay. The effector HEK 293T cells were co-transfected with S protein and a DSP_1-7 expressing plasmid, the target cells were transfected with DSP_8-11 plasmid. Before the target cells were transferred to the effector cells, bergamottin was added to the effector cells and incubated for 1h. The relative luminescence units were calculated and representative images were captured 24 h later (data not shown). Three independent replicates are shown. (E) Effect of bergamottin on the binding between SARS-CoV-2 RBD and ACE2. Gradient diluted bergamottin or vehicle was added to compete with ACE2-His to combine with immobilized SARS-CoV-2 S Protein RBD. The OD_450nm was measured using a microplate reader. Data represents mean ± SD for n = 2 biological replicates. (F) Impacts of bergamottin on ACE2 and TMPRSS2 expression. Caco-2 cells were incubated with bergamottin at indicated concentrations for 24 h. Cell lysates were then subjected to a Western Blot analysis. The experiment was repeated twice. (G) The effects of bergamottin on viral RNA synthesis. The BHK-21 cells were electroporated with SARS-CoV-2 wild-type (WT) replicon, and then treated with bergamottin or vehicle, as indicated. The relative luciferase activity was measured 24h later. Data represent mean ± SD for n = 3 biological replicates. (H) Bergamottin shows negligible inhibition of 3CL^pro protease activity. The activity of purified SARS-CoV-2 3CL^pro enzymes was measured after adding substrates. Enzyme activity in the absence and presence of bergamottin was calculated. The data shown represent two biological replicates.