A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery.
A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery.
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带有 SARS-CoV-2 辅助基因的方便且生物安全的复制子及其在抗病毒药物发现中的潜在应用
DOI：
10.1007/s12250-021-00385-9
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发表时间：
2021-10
影响因子：
5.5
通讯作者：
Guo D
中科院分区：
文献类型：
--
作者：
Jin YY;Lin H;Cao L;Wu WC;Ji Y;Du L;Jiang Y;Xie Y;Tong K;Xing F;Zheng F;Shi M;Pan JA;Peng X;Guo D
SARS-CoV-2导致COVID-19的大流行，迄今为止还没有针对这种疾病的有效药物。由于该病毒的高感染性和致病性，所有关于活病毒的研究都严格限制在生物安全三级（BSL 3）实验室，但这将阻碍SARS-CoV-2的基础研究和抗病毒药物开发，因为BSL 3设施并不常见，并且在遏制中的工作既昂贵又费力。本研究通过将SARS-CoV-2的S基因缺失后，将病毒cDNA组装到细菌人工染色体（BAC）载体中，构建了SARS-CoV-2的反向遗传学系统。将cDNA转染到细胞中导致RNA复制子的产生，该RNA复制子保持基因组或亚基因组复制的能力，但由于S蛋白的缺失而在病毒体组装和感染中有缺陷。因此，这种复制子系统没有感染性，可以在普通的生物实验室中使用。我们通过证明具有与野生型病毒相似特征的亚基因组RNA的表达来证实复制子的有效复制。通过nsp 12和nsp 14的突变分析，我们表明RNA聚合酶、核酸外切酶和cap N7甲基转移酶在基因组复制和sgRNA产生中起重要作用。我们还创建了携带荧光素酶报告基因的SARS-CoV-2复制子，并通过已知抗SARS-CoV-2抑制剂的抑制试验验证了该系统。因此，这种单质粒系统是生物安全的，并且使用方便，这将有利于基础研究和抗病毒药物的开发。
SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.
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