tetano
Editor, Senior Moderator
PLoS Pathog
. 2021 Mar 12;17(3):e1009439.
doi: 10.1371/journal.ppat.1009439. Online ahead of print.
A novel cell culture system modeling the SARS-CoV-2 life cycle
Xiaohui Ju[SUP] 1 [/SUP], Yunkai Zhu[SUP] 2 [/SUP], Yuyan Wang[SUP] 2 [/SUP], Jingrui Li[SUP] 3 [/SUP], Jiaxing Zhang[SUP] 4 [/SUP], Mingli Gong[SUP] 1 [/SUP], Wenlin Ren[SUP] 1 [/SUP], Sai Li[SUP] 4 5 [/SUP], Jin Zhong[SUP] 6 [/SUP], Linqi Zhang[SUP] 1 [/SUP], Qiangfeng Cliff Zhang[SUP] 4 5 [/SUP], Rong Zhang[SUP] 2 [/SUP], Qiang Ding[SUP] 1 5 [/SUP]
Affiliations
Abstract
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/?N 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.
. 2021 Mar 12;17(3):e1009439.
doi: 10.1371/journal.ppat.1009439. Online ahead of print.
A novel cell culture system modeling the SARS-CoV-2 life cycle
Xiaohui Ju[SUP] 1 [/SUP], Yunkai Zhu[SUP] 2 [/SUP], Yuyan Wang[SUP] 2 [/SUP], Jingrui Li[SUP] 3 [/SUP], Jiaxing Zhang[SUP] 4 [/SUP], Mingli Gong[SUP] 1 [/SUP], Wenlin Ren[SUP] 1 [/SUP], Sai Li[SUP] 4 5 [/SUP], Jin Zhong[SUP] 6 [/SUP], Linqi Zhang[SUP] 1 [/SUP], Qiangfeng Cliff Zhang[SUP] 4 5 [/SUP], Rong Zhang[SUP] 2 [/SUP], Qiang Ding[SUP] 1 5 [/SUP]
Affiliations
- PMID: 33711082
- DOI: 10.1371/journal.ppat.1009439
Abstract
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/?N 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.