School of Medicine, Tsinghua University, Beijing, China.
Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Biosafety Level 3 Laboratory, Fudan University, Shanghai, China.
PLoS Pathog. 2021 Mar 12;17(3):e1009439. doi: 10.1371/journal.ppat.1009439. eCollection 2021 Mar.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）引发了 COVID-19 全球大流行。SARS-CoV-2 被归类为生物安全级别 3（BSL-3）病原体，这阻碍了对其生物学的基础研究和有效抗病毒药物的开发。在这里，我们开发了一种生物安全级别 2（BSL-2）细胞培养系统，用于生产转录和复制型 SARS-CoV-2 病毒样颗粒（trVLP）。这种 trVLP 表达报告基因（GFP）取代了病毒核衣壳基因（N），这是病毒基因组包装和病毒体组装所必需的（SARS-CoV-2 GFP/ΔN trVLP）。完整的病毒生命周期可以在细胞中实现，并且只能在异位表达 SARS-CoV 或 SARS-CoV-2 N 蛋白的细胞中进行，而不能在 MERS-CoV N 蛋白中进行。通过在表达 N 的细胞中进行一个月的连续传代后进行序列分析，没有检测到 N 提供的遗传重组进入病毒基因组。此外，我们利用内含肽介导的蛋白质反式剪接方法将病毒 N 基因分割成两个独立的载体，连接的病毒 N 蛋白可以在反式中发挥作用，重现整个病毒生命周期，进一步确保了这种细胞培养模型的生物安全性。基于这个 BSL-2 SARS-CoV-2 细胞培养模型，我们开发了一种 96 孔格式的高通量筛选方法，用于发现抗病毒药物。我们发现了盐霉素、雷公藤红素 I、莫能菌素钠、石蒜碱氯化物和硝酸钠作为抗 SARS-CoV-2 感染的有效抗病毒药物。总之，我们开发了一种方便有效的 SARS-CoV-2 反向遗传学工具，用于在 BSL-2 条件下剖析病毒生命周期。这个强大的工具应该加速我们对 SARS-CoV-2 生物学及其抗病毒药物开发的理解。
