Abstract
The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.
Highlights
The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic
As the reporter firefly luciferase gene was under the control of the M protein transcription regulation sequence (TRS), it can be expressed only from subgenomic RNAs but not from the replicon genome RNA
It has been reported that nsp3, nsp4, and nsp6 induce the formation of doublemembrane vesicles (DMV) that are dedicated to viral RNA synthesis [39]
Summary
The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. The screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses This replicon system provides a biosafety-worryfree platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors. We developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. This replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs. During negative-strand sgRNA synthesis, RdRp (nsp12) pauses at any one of the TRS-B sequences and continues elongation to the TRS, or it switches to binding the TRS-L located in the 59 terminus of the genome and elongation to the 39 terminus [10, 11]
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