Dataset Information

A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation.

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 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 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.

SUBMITTER: Kotaki T

PROVIDER: S-EPMC7838314 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation.

Kotaki Tomohiro T Xie Xuping X Shi Pei-Yong PY Kameoka Masanori M

Scientific reports 20210126 1

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 w ...[more]

PMID: 33500537

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Project description:The emerging coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide, resulting in global public health emergencies and economic crises. In the present study, a noninfectious and biosafety level 2 (BSL2)-compatible SARS-CoV-2 replicon expressing a nano luciferase (nLuc) reporter was constructed in a bacterial artificial chromosomal (BAC) vector by reverse genetics. The nLuc reporter is highly sensitive, easily quantifiable, and high throughput adaptable. Upon transfecting the SARS-CoV-2 replicon BAC plasmid DNA into Vero E6 cells, we could detect high levels of nLuc reporter activity and viral RNA transcript, suggesting the replication of the replicon. The replicon replication was further demonstrated by the findings that deleting nonstructural protein 15 or mutating its catalytic sites significantly reduced replicon replication, whereas providing the nucleocapsid protein in trans enhanced replicon replication in a dose-dependent manner. Finally, we showed that remdesivir, a U.S. Food and Drug Administration-approved antiviral drug, significantly inhibited the replication of the replicon, providing proof of principle for the application of our replicon as a useful tool for developing antivirals. Taken together, this study established a sensitive and BSL2-compatible reporter system in a single BAC plasmid for investigating the functions of SARS-CoV-2 proteins in viral replication and evaluating antiviral compounds. This should contribute to the global effort to combat this deadly viral pathogen. IMPORTANCE The COVID-19 pandemic caused by SARS-CoV-2 is having a catastrophic impact on human lives. Combatting the pandemic requires effective vaccines and antiviral drugs. In the present study, we developed a SARS-CoV-2 replicon system with a sensitive and easily quantifiable reporter. Unlike studies involving infectious SARS-CoV-2 virus that must be performed in a biosafety level 3 (BSL3) facility, the replicon is noninfectious and thus can be safely used in BSL2 laboratories. The replicon will provide a valuable tool for testing antiviral drugs and studying SARS-CoV-2 biology.

Dataset Information

A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation.

Publications

A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation.

Similar Datasets

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