A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis

• DOI: 10.1016/j.csbj.2022.08.044
• Publication Date: 2022-08-30T06:36:20Z
• Authors: Xiaolong Feng; Xiaofan Zhang; Shuangying Jiang; Yuanwei Tang; Chao Cheng; Parthasarathy Abi...; Xiaoting Wang; Junbiao Dai; Jianyang Zeng; Tian Xia; Dan Zhao
• Keywords: Replicon; Subgenomic mRNA; Coronavirus; Vero cell; Transcription; Coronaviridae

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome 2 (SARS-CoV-2) has seriously affected public health around world. In-depth studies on pathogenic mechanisms of SARS-CoV-2 is urgently necessary for prevention. However, most laboratory have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting progress relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method assemble replication and transcription system Vero E6 cells without virion envelope formation, thus avoiding risk exposure. Furthermore, an improved real-time quantitative reverse PCR (RT-qPCR) approach was distinguish full-length replicon RNAs subgenomic (sgRNAs). Using replicon, demonstrated that nucleocapsid (N) protein facilitates sgRNAs discontinuous synthesis process. Moreover, two high-frequency mutants N protein, R203K S194L, can obviously enhance hinting these mutations likely allow spread reproduce more quickly. addition, remdesivir chloroquine, well-known drugs effective against previous studies, also inhibited our indicating potential applications antiviral drug discovery. Overall, developed bio-safe valuable useful study viral RNA novel screening.