Fig. 4: Identified natural compounds inhibit early stages of SARS-CoV-2 infection.
From: Fluorogenic reporter enables identification of compounds that inhibit SARS-CoV-2
a, Schematic of the staging experiment. SARS-CoV-2 (MOI = 5) was added to Vero-E6 cells, then natural compounds (1 μM) were added at various time points as indicated. b,c, Samples were characterized by plaque assay (b) and RT–qPCR (c). Total RNAs were extracted from Vero cells with Trizol reagent. vRNA genomes were measured by RT–qPCR with primers targeting the nucleocapsid gene (N) of SARS-CoV-2, normalized to internal control HRT1. d, SARS-CoV-2 binding assay with immunofluorescence staining. Vero cells were preincubated with natural compounds (2 μM), followed by the addition of SARS-CoV-2 (MOI = 100) for adsorption at 4 °C for 1 h. Left, experimental procedures; middle, immunofluorescence images; right, quantification of immunofluorescence against N protein. b–d, Data presented as mean ± s.d. (n = 3). P values, two-sided nonpaired t-test between compound- and DMSO-treated groups. *P < 0.05 considered significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values <0.0001 (b,c); starting from anisomycin: 0.00020, 0.00021, 0.00020 (d). Final concentration of natural compounds was 2 μM for the above experiments. d, Scale bars, 40 μm. e, Schematic of the rVSV-eGFP-SARS-CoV-2 pseudovirus infection assay. The rVSV-eGFP-SARS-CoV-2 pseudovirus comprises replication-competent rVSVs encoding the spike protein of SARS-CoV-2 in place of the original G glycoprotein. A549 cells stably expressing hACE2 were preincubated with natural compounds for 3 h, followed by incubation with rVSV-eGFP-SARS-CoV-2 (MOI = 1) at 37 °C for 1 h and washing with fresh medium containing compounds. Cell eGFP fluorescence was imaged at 16 h.p.i. f, Dose–response curves of inhibitors against rVSV-eGFP-SARS-CoV-2 pseudovirus infection assay. Relative infection was calculated based on eGFP fluorescence. Data presented as mean ± s.d. (n = 3).
