Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity

Fig. 1. Schematic diagram of the cell-based reporter assay system. (a) Reporter vector was engineered to measure the activity of SARS-CoV-2 RdRp by regulating FLuc under the control of the 5′ UTR. This reporter plasmid incorporates the FLuc gene between the 5′ UTR and 3′ UTR of SARS-CoV-2 in the reverse orientation, following the CMV promoter. (b) The RNA transcript, driven by the CMV promoter, undergoes ribozyme-mediated cleavage at both ends, producing the 5′ UTR-FLuc-3′ UTR RNA transcript. RdRp recognizes the 5′ UTR, initiates RNA polymerization, and transcribes FLuc in the positive direction. The level of FLuc transcribed by RdRp is quantified and expressed as the reporter activity value. The ribozyme is denoted as Rbz in both diagrams.

Fig. 2. Confirmation of reporter activity through expression of SARS-CoV-2 RdRp. (a) HEK 293 cells were co-transfected with our constructed reporter vector and RdRp at various ratios (1:1, 1:2, and 1:4). Reporter activity was evaluated after 48 h of incubation. (b) Reporter activity was compared between the single expression of RdRp and the co-expression of Nsp7, Nsp8, and RdRp in reporter-transfected HEK 293 cells. (c) The expression of the reporter vector was assessed at various time intervals following the transfection of HEK 293 cells with RdRp. The reporter activity value of the reporter vector and RdRp co-expression group was divided by the reporter activity value of the reporter vector expression group and expressed as a ratio. (d) Reporter activity was measured following the mutation of essential residues Lys73 and Asp218 (depicted as K73 and D218) in the NiRAN domain of RdRp, or the mutation of key residues Asp618, Asp760, and Asp761 (depicted as D618, D760, and D761) in the RdRp domain of RdRp. Each key residue that regulates the activity of SARS-CoV-2 was replaced with alanine, indicated as ‘A’ in the graph. All data on reporter activity are expressed as a percentage compared…

Fig. 3. Evaluation of a cell-based reporter assay system for HTS using Z-factor analysis. The experimental groups included a negative control group (reporter vector expression group; (●), a positive control group (reporter vector and RdRp co-expression group; (▲), and an RdRp inhibitor treatment group (positive group treated with remdesivir; 5 µM for 24 h; (■) in HEK 293 cells expressing the reporter. Positive and negative controls were treated with DMSO instead of remdesivir. Z- and Z′-factors were calculated using Zhang’s formula for each group: negative, positive, and inhibitor.

Fig. 4. Validation of reporter assay using inhibitors of SARS-CoV-2 RdRp activity. HEK 293 cells and A549 cells were transiently transfected with the reporter vector and RdRp. After 24 h, the cells were treated with various concentrations of (a) remdesivir, (b) molnupiravir, (c) tenofovir, and (d) sofosbuvir. These compounds were added to the reporter vector and RdRp co-expressed cells, followed by an additional 24 h incubation to measure reporter activity (solid line) and cell viability (dotted line). The data represent the results of three independent experiments. Reporter activity was expressed as a percentage relative to the reporter and RdRp co-expressed group. Statistical significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the reporter vector and RdRp co-expressed group.

Fig. 5. Screening of compounds inhibiting SARS-CoV-2 RdRp activity using our constructed reporter vector. (a) A heatmap illustrating the regulation of reporter activity following treatment with 2,704 kinase inhibitors. HEK 293 cells co-transfected with Reporter and RdRp were treated with DMSO or 10 µM of test compounds for 24 h. Luminescence was measured using a reporter assay kit. Reporter values were compared to the positive control group (the reporter vector and RdRp co-expressed group) and expressed as a percentage of reporter activity. (b) Chemical structure of MePT-S-N-PMe. (c) HEK 293 cells expressing reporter vector and RdRp were treated with MePT-S-N-PMe for 24 h. The inhibition rate of RdRp activity by MePT-S-N-PMe at various concentrations was determined using a reporter assay kit, while cytotoxicity was assessed using EZ-Cytox. Reporter activity and cell viability are represented as a solid line and a dotted line, respectively. Results are presented as percentages relative to the reporter vector and RdRp co-expressed group, based on three independent experiments conducted in triplicate. (d) The primer extension reaction was initiated by treating the active SARS-CoV-2 RdRp…

Fig. 6. Inhibition of viral replication by MePT-S-N-PMe in Vero cells. (a) Vero cells were treated with varying concentrations of MePT-S-N-PMe and then inoculated with the SARS-CoV-2 virus. After 24 h, cells were fixed with 4% paraformaldehyde and visualized by immunostaining for nuclei and nucleocapsid (N) protein of SARS-CoV-2. (b) The inhibition of SARS-CoV-2 infection (blue) and cell viability (red) were calculated as percentages compared to the untreated group and expressed as graphs. Mean ± standard deviation was calculated from duplicate experiments.