A novel cell culture system modeling the SARS-CoV-2 life cycle.
Xiaohui Ju, Yunkai Zhu, Yuyan Wang, Jingrui Li, Jiaxing Zhang, Mingli Gong, Wenlin Ren, Sai Li, Jin Zhong, Linqi Zhang, Qiangfeng Cliff Zhang, Rong Zhang, Qiang Ding
Author Information
Xiaohui Ju: School of Medicine, Tsinghua University, Beijing, China. ORCID
Yunkai Zhu: 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.
Yuyan Wang: 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.
Jingrui Li: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China.
Jiaxing Zhang: School of Life Sciences, Tsinghua University, Beijing, China.
Mingli Gong: School of Medicine, Tsinghua University, Beijing, China.
Wenlin Ren: School of Medicine, Tsinghua University, Beijing, China. ORCID
Sai Li: School of Life Sciences, Tsinghua University, Beijing, China. ORCID
Jin Zhong: Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China. ORCID
Linqi Zhang: School of Medicine, Tsinghua University, Beijing, China. ORCID
Qiangfeng Cliff Zhang: School of Life Sciences, Tsinghua University, Beijing, China. ORCID
Rong Zhang: 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. ORCID
Qiang Ding: School of Medicine, Tsinghua University, Beijing, China.
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.
References
J Biol Chem. 2009 Feb 20;284(8):5229-39
[PMID: 19106108]
Lancet. 2020 Feb 15;395(10223):470-473
[PMID: 31986257]
Cell Host Microbe. 2014 Oct 8;16(4):462-72
[PMID: 25299332]
J Med Chem. 2017 Mar 9;60(5):1648-1661
[PMID: 28124907]
