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bioRxiv · 10.1101/2020.08.28.267567

A PCR amplicon-based SARS-CoV-2 replicon for antiviral screening

Kotaki, T.·Xie, X.·Shi, P.-Y.·Kameoka, M.
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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 screening; however, SARS-CoV-2 replicon has not been reported yet. 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 3,000 times higher luminescence than MOCK control cells at 24 hours post-electroporation, indicating robust viral translation and RNA replication. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 M, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 M). Taken together, this system could be applied to the safe and effective antiviral screening without using infectious SARS-CoV-2. Because this is a transient replicon, further improvement including the establishment of stable cell line must be achieved.

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Kotaki, T., Xie, X., Shi, P.-Y., Kameoka, M.. 2020-08-28. A PCR amplicon-based SARS-CoV-2 replicon for antiviral screening. https://doi.org/10.1101/2020.08.28.267567

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