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A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
Tomohiro Kotaki1, Xuping Xie2, Pei-Yong Shi2
1Department of Public Health, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe, Hyogo, 654-0142, Japan. tkotaki@people.kobe-u.ac.jp.
Scientific Reports
|January 27, 2021
Summary
A new SARS-CoV-2 replicon system allows safe antiviral testing without infectious virus. This PCR-based tool demonstrated robust replication and inhibition by remdesivir, paving the way for efficient drug discovery.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- Developing antiviral compounds against SARS-CoV-2 is critical.
- Handling infectious SARS-CoV-2 requires specialized biosafety level-3 containment, hindering antiviral development.
- SARS-CoV-2 replicons offer a safer alternative for evaluating antiviral efficacy.
Purpose of the Study:
- To generate a PCR-based SARS-CoV-2 replicon system for safe and efficient antiviral evaluation.
- To validate the functionality of the replicon system through translation and RNA replication assays.
- To assess the utility of the replicon system in testing antiviral compounds like remdesivir.
Main Methods:
- Generated a PCR-based SARS-CoV-2 replicon lacking S, E, and M genes, incorporating a HiBiT-tag.
- Amplified and ligated eight genomic fragments, followed by in vitro transcription to produce replicon RNA.
- Electroporated cells with replicon RNA and measured luminescence to assess translation and replication.
- Determined the IC50 of remdesivir against the SARS-CoV-2 replicon.
Main Results:
- The SARS-CoV-2 replicon demonstrated robust translation and RNA replication, indicated by over 3000-fold higher luminescence compared to controls.
- Remdesivir, an RNA polymerase inhibitor, significantly inhibited replicon replication.
- The calculated IC50 for remdesivir was 0.29 μM, consistent with previous studies using infectious SARS-CoV-2.
Conclusions:
- The developed PCR-based SARS-CoV-2 replicon system provides a safe and effective platform for antiviral evaluation without requiring infectious virus.
- This system facilitates rapid screening of antiviral compounds.
- Further optimization, such as establishing a stable cell line, is recommended for enhanced utility.
