1. A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation.
- Author
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Kotaki T, Xie X, Shi PY, and Kameoka M
- Subjects
- Adenosine Monophosphate analogs & derivatives, Adenosine Monophosphate pharmacology, Alanine analogs & derivatives, Alanine pharmacology, Animals, CHO Cells, COVID-19, Chlorocebus aethiops, Cricetulus, Drug Evaluation, Preclinical, Electroporation, Genome, Viral, HEK293 Cells, Humans, Inhibitory Concentration 50, Kinetics, Open Reading Frames, Polymerase Chain Reaction, RNA, Viral, RNA-Dependent RNA Polymerase, SARS-CoV-2 physiology, Untranslated Regions, Vero Cells, Virion, Virus Replication drug effects, Antiviral Agents pharmacology, Drug Design, SARS-CoV-2 drug effects
- Abstract
The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC
50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.- Published
- 2021
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