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An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research
Journal of Virological Methods ( IF 3.1 ) Pub Date : 2021-06-25 , DOI: 10.1016/j.jviromet.2021.114221
Peng Yang 1 , Yang Yang 2 , Yuming Wu 2 , Cong Huang 3 , Yanlei Ding 2 , Xuejun Wang 2 , Shengqi Wang 1
Affiliation  

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.



中文翻译:

用于病毒进入研究的优化且强大的 SARS-CoV-2 假病毒系统

SARS-CoV-2 是导致 2019 年冠状病毒病 (COVID-19) 的罪魁祸首。为了在 BSL-2 实验室研究 SARS-CoV-2 感染,使用带有双报告基因 eGFP 和萤火虫荧光素酶 (Luc2) 的慢病毒载体构建了 SARS-CoV-2 假病毒颗粒 (SARS2pp) 生产和感染系统) 便于观察和分析。SARS2pp不同生产条件的比较表明,通过以下方式可以大大提高假病毒滴度:1)去除刺突蛋白的最后19个氨基酸并用小鼠Igk替换信号肽信号序列;2) 使用除 CAG 之外的 CMV 启动子(由 CMV 增强子、β-肌动蛋白启动子、剪接供体和 β-珠蛋白剪接受体组成的混合启动子)表达刺突蛋白;3) 为 SARS2pp 生产筛选更好优化的刺突蛋白序列;和 4) 在 SARS2pp 生产培养基中添加 1% BSA。对于感染,该 SARS2pp 系统在 MOI 2-0.0002 之间显示出良好的线性关系,然后成功用于评估 SARS-CoV-2 感染抑制剂,包括重组人 ACE2 蛋白和 SARS-CoV-2 中和抗体。还发现肾脏、肝脏和小肠来源的细胞系对 SARSpp 和 SARS2pp 表现出不同的易感性。鉴于其稳健性和良好的性能,

更新日期:2021-06-30
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