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Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening

Tomohisa Tanaka, Akatsuki Saito, Tatsuya Suzuki, View ORCID ProfileYoichi Miyamoto, Kazuo Takayama, Toru Okamoto, View ORCID ProfileKohji Moriishi
doi: https://doi.org/10.1101/2021.12.23.474055
Tomohisa Tanaka
1Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi 409-3898, Japan
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Akatsuki Saito
2Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
3Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-2192, Japan
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Tatsuya Suzuki
4Institute for Advanced Co-Creation Studies, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
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Yoichi Miyamoto
5Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085, Japan
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Kazuo Takayama
6Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
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Toru Okamoto
4Institute for Advanced Co-Creation Studies, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
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Kohji Moriishi
1Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi 409-3898, Japan
7Center for Life Science Research, University of Yamanashi, Yamanashi 409-3898, Japan
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  • ORCID record for Kohji Moriishi
  • For correspondence: kmoriishi@yamanashi.ac.jp
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Abstract

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-β but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-β. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

Competing Interest Statement

The authors have declared no competing interest.

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Posted December 25, 2021.
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Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening
Tomohisa Tanaka, Akatsuki Saito, Tatsuya Suzuki, Yoichi Miyamoto, Kazuo Takayama, Toru Okamoto, Kohji Moriishi
bioRxiv 2021.12.23.474055; doi: https://doi.org/10.1101/2021.12.23.474055
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Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening
Tomohisa Tanaka, Akatsuki Saito, Tatsuya Suzuki, Yoichi Miyamoto, Kazuo Takayama, Toru Okamoto, Kohji Moriishi
bioRxiv 2021.12.23.474055; doi: https://doi.org/10.1101/2021.12.23.474055

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