Engineering a Reliable and Convenient SARS-CoV-2 Replicon System for Analysis of Viral RNA Synthesis and Screening of Antiviral Inhibitors

Abstract

The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.

Keywords: COVID-19; SARS-CoV-2; safety replicon.

FIG 1

Design of the SARS-CoV-2 replicon system. (A) (Top) The genomic organization of SARS-CoV-2; (bottom) schematic structure of the replicon system consisting of ps2V, ps2AN, ps2AC, and ps2B. (B) The replicases and transcriptases were expressed from ps2AN, ps2AC, and ps2B plasmids. ps2V plasmid expressed the replicon RNA. Replicase and transcriptase mediated replicon replication and the subgenomic luciferase RNA synthesis. The luciferase protein was expressed from subgenomic luciferase RNA.

FIG 2

Characterization of the SARS-CoV-2 replicon system. ps2V (0.1 μg), ps2AN (0.05 μg), ps2AC (0.4 μg), and ps2B (0.4 μg) were cotransfected into 293T cells (cell density: 6.5 × 10⁴/cm²) seeded in a 12-well plate. (A, B, and C) RNA was isolated 48 h after transfection following treatment with DNase (Promega) before reverse transcription. The RNA was reverse transcribed by specific forward primer for minus RNA or oligo(dT) primer for plus RNA. The minus and plus sgRNA expression was detected through qPCR with specific primers. Human GAPDH mRNA was measured as endogenous controls. The sequence information of primers is listed in Materials and Methods. The PCR products were inserted into T-vector and followed by DNA sequence. (D) The luciferase activity was detected by the luciferase reporter assay system at different time points (hours). (E) The construction expressing renilla luciferase (40 ng) was cotransfected. The luciferase activity was detected after 54 h of transfection by the dual luciferase reporter assay system. FL, firefly luciferase; RL, Renilla luciferase. (F) The mentioned plasmid combinations (consistency of plasmid amount for each group has been ensured via adding empty vector) were transfected into 293T cells. The luciferase activity was detected by the luciferase reporter assay system. (G to K) After 24 to 30 h of transfection, the indicated drugs were added into the cell medium. The luciferase activity was detected after drug treatment for 24 h. IC₅₀ of drugs was measured using 9 to 10 different concentrations. The inhibition (%) was calculated by the formula (1 − Luc_{each concentration}/Luc_DMSO) × 100%. (L) The ps2V plasmid was transfected into 293T cells (cell density: 6.5 × 10⁴/cm²). After transfection for 12 h, the indicated drugs (10 μM) were added into the cell medium for treatment for 24 h. DMSO treatment served as a negative control. The fluorescence-field micrograph image was obtained and processed in the same way. Scale bar, 100 μm. Images were quantified by Image J software. Graph shows relative fluorescence signal of GFP after different treatments. All experimental data were analyzed using GraphPad Prism. All data are representative of at least three experiments.

FIG 3

Analysis of SARS-CoV-2 protein function based on the SARS-CoV-2 replicon system. (A, B, E, F, and H) ps2V (0.1 μg), ps2AN (0.05 μg), ps2AC (0.4 μg), ps2B (0.4 μg), and the respective mentioned plasmids (0.2 μg) were cotransfected into 293T cells (cell density: 6.5 × 10⁴/cm²) seeded in a 12-well plate. The luciferase activity was detected by the luciferase reporter assay system after 48 to 54 h of transfection. (C) The gradient amounts of N-HA-expressing plasmid (consistency of plasmid amount of each group has been ensured by adding empty vector) were cotransfected into 293T cells with the combination of ps2V (0.1 μg), ps2AN (0.05 μg), ps2AC (0.4 μg), and ps2B (0.4 μg) plasmids. The luciferase activity was detected by the luciferase reporter assay system. (D) The authentic SARS-CoV-2 infection assay was performed according to Materials and Methods. At 48 h postinfection, virus RNA was isolated and RT-qPCR was performed to detect virus RNA copies. (G) The top 8 genome variations with high frequency based on the genome variation information obtained from the high-quality genome sequences of SARS-COV-2 collected from the NCBI and GISAID databases up to the date of 1 July 2020. The strain Wuhan-Hu-1 was retrieved from NCBI (NC_045512.2) as reference for variation annotation. The virus number with the variation is shown in the third column. All experimental data were analyzed using GraphPad Prism. All data are representative of at least three experiments.

FIG 4

Clinical-use compound library screening based on SARS-CoV-2 replicon system. (A) ps2V (12.5 ng), ps2AN (6.25 ng), ps2AC (50 ng), and ps2B (50 ng) were cotransfected into 293T cells (cell density: 6.5 × 10⁴/cm²) seeded in a 96-well plate. After transfection for 24 to 30 h, drugs were added into cell medium for 24-h treatment before detecting luciferase activity. After the fourth round of screening, 5 hit compounds were selected for further examination. (B to F) After transfection for 24 to 30 h, the indicated drugs were added into the cell medium. The luciferase activity was detected after drug treatment for 24 h. IC₅₀ of candidate drugs inhibiting replicon was measured using 6 to 10 different concentrations. The inhibition (%) was calculated by the formula (1 − Luc_{each concentration}/Luc_DMSO) × 100%. (G to L) The IC₅₀ of candidate drugs inhibiting authentic virus strains. The authentic SARS-CoV-2 infection assay was performed according to Materials and Methods. Drugs were added into cell medium for treatment for 24 h. Virus RNA was isolated, and RT-qPCR was performed to detect virus RNA copies. The inhibition (%) was calculated as (1 − viral RNA copies_{each concentration}/viral RNA copies_DMSO) × 100%. (M to Q) IRES-CMV-luciferase plasmid was transfected into 293T cells (cell density: 6.5 × 10⁴/cm²). After transfection for 24 to 30 h, the indicated drugs were added into the cell medium. The luciferase activity was detected after drug treatment for 24 h. IC₅₀ of the drugs was measured using different concentrations. The inhibition (%) was calculated by the formula (1 − Luc_{each concentration}/Luc_DMSO) × 100%. All experimental data were analyzed using GraphPad Prism. All data are representative of at least three experiments.