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Construction of a non-infectious SARS coronavirus replicon for application in drug screening and analysis of viral protein function.

ABSTRACT: Severe acute respiratory syndrome virus (SARS-CoV) was the causative agent of the SARS outbreaks in 2002-2003. A safer in vitro system is desirable for conducting research on SARS-CoV and to screen for antiviral drugs against the virus. Based on the infectious cDNA clone of rSARS-CoV-DeltaE, in which the E gene has been deleted, a safe non-infectious replicon was constructed by replacing the S gene with the enhanced green fluorescent protein (eGFP) gene. Successful replication was achieved as evident from continuous expression of eGFP detected by both fluorescence and Western blot. Treatment with antiviral drugs demonstrated that the replication could be significantly inhibited by 0.4 mg/ml of cysteine proteinase inhibitor E-64D, but not by ribavirin. The same replicons containing further deletion of the coding regions for non-structural proteins (nsp) 1, 2 or 16 confirmed previous observation that nsp16, but not nsp1 or nsp2, was essential for efficient viral replication or transcription.

SUBMITTER: Wang JM

PROVIDER: S-EPMC7092913 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Construction of a non-infectious SARS coronavirus replicon for application in drug screening and analysis of viral protein function.

Wang Jian-Min JM Wang Lin-Fa LF Shi Zheng-Li ZL

Biochemical and biophysical research communications 20080711 1

Severe acute respiratory syndrome virus (SARS-CoV) was the causative agent of the SARS outbreaks in 2002-2003. A safer in vitro system is desirable for conducting research on SARS-CoV and to screen for antiviral drugs against the virus. Based on the infectious cDNA clone of rSARS-CoV-DeltaE, in which the E gene has been deleted, a safe non-infectious replicon was constructed by replacing the S gene with the enhanced green fluorescent protein (eGFP) gene. Successful replication was achieved as ev ...[more]

PMID: 18619943

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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

Construction of a non-infectious SARS coronavirus replicon for application in drug screening and analysis of viral protein function.

Publications

Construction of a non-infectious SARS coronavirus replicon for application in drug screening and analysis of viral protein function.

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