A novel cell culture system modeling the SARS-CoV-2 life cycle.
A novel cell culture system modeling the SARS-CoV-2 life cycle.
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一种模拟SARS-CoV-2生命周期的新型细胞培养系统
DOI：
10.1371/journal.ppat.1009439
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发表时间：
2021-03
期刊：
影响因子：
6.7
通讯作者：
Ding Q
中科院分区：
文献类型：
--
作者：
Ju X;Zhu Y;Wang Y;Li J;Zhang J;Gong M;Ren W;Li S;Zhong J;Zhang L;Zhang QC;Zhang R;Ding Q
严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)导致新冠肺炎的全球大流行。SARS-CoV-2被列为生物安全3级(BSL-3)毒剂，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二级(BSL-2)细胞培养系统，用于生产具有转录和复制能力的SARS-CoV-2病毒样颗粒(TrVLP)。它表达一个报告基因(Gfp)，取代病毒核衣壳基因(N)，这是病毒基因组包装和病毒粒子组装所必需的(SARS-CoV-2gfp/ΔN trVLP)。病毒的整个生命周期可以实现，并且仅限于异位表达SARS-CoV或SARS-CoV-2N蛋白的细胞中，而不是MERS-CoV N蛋白。N-表达细胞经过一个月的连续传代后，序列分析表明，没有检测到反式供应的N进入病毒基因组的基因重组。此外，利用内含素介导的蛋白质反式剪接方法将病毒N基因切割成两个独立的载体，连接的病毒N蛋白可以反式作用于整个病毒生命周期，进一步确保了该细胞培养模型的生物安全性。基于建立的BSL-2 SARS-CoV-2细胞培养模型，我们建立了用于抗病毒药物筛选的96孔法高通量筛选。我们确定盐霉素、土贝母苷I、莫能菌素钠、氯化石蒜碱和黑素钠是有效的抗SARS-CoV-2感染的药物。我们共同开发了一种方便高效的SARS-CoV-2反向遗传学工具来剖析BSL-2条件下的病毒生命周期。这一强大的工具应该会加速我们对SARS-CoV-2生物学及其抗病毒开发的理解。严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)的生物安全3级(BSL-3)分类阻碍了抗病毒药物的研究和开发。我们报道了一种新的细胞培养系统，用于生产转录和复制能力强的SARS-CoV-2病毒样颗粒(TrVLP)，可用于BSL-2实验室的高通量中和和抗病毒筛选。该系统由两部分组成：含有核衣壳(N)基因缺失的基因组病毒RNA和表达N蛋白的生产细胞系。完整的病毒生命周期是可以实现的，并且完全局限于产生细胞。此外，内含素介导的蛋白质反式剪接将N裂解成两个载体，进一步确保了该系统的生物安全性。基于这一系统，我们发现N蛋白的残基特异性磷酸化是病毒感染的关键。此外，还开发了高通量抗病毒筛选，并发现了新药。因此，该实验系统将为SARS-CoV-2生物学的研究及其抗病毒药物的开发提供便利。
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. The biosafety level-3 (BSL-3) classification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impedes the research and antivirals development. We report a novel cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) that can be used at BSL-2 laboratory for high-throughput neutralization and antiviral screening. This system consists of two components: a genomic viral RNA containing a deletion of nucleocapsid (N) gene, and a producer cell line expressing the N protein. The complete viral life cycle can be achieved and exclusively confined in the producer cells. Moreover, intein-mediated protein trans-splicing that splits N into two vectors further secures the biosafety of this system. Based on this system, we found residue-specific phosphorylation of N protein is critical for viral infection. Besides, high-throughput antiviral screening was developed and new drugs were discovered. Thus, this experimental system will facilitate the studies of SARS-CoV-2 biology and its antiviral development.
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影响因子：
64.5
作者：
Hou, Yixuan J.;Okuda, Kenichi;Baric, Ralph S.
通讯作者：
Baric, Ralph S.
影响因子：
5
作者：
Almazán F;Sola I;Zuñiga S;Marquez-Jurado S;Morales L;Becares M;Enjuanes L
通讯作者：
Enjuanes L
影响因子：
4.7
作者：
Ianevski, Aleksandr;Yao, Rouan;Kainov, Denis E.
通讯作者：
Kainov, Denis E.
DOI：
10.1126/science.abc0870
发表时间：
2020-09-04
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Chan KK;Dorosky D;Sharma P;Abbasi SA;Dye JM;Kranz DM;Herbert AS;Procko E
通讯作者：
Procko E
影响因子：
4.8
作者：
Cascarina, Sean M.;Ross, Eric D.
通讯作者：
Ross, Eric D.