Abstract:

From its first report in late 2019 to September 2023, the Corona Virus Disease 2019 (COVID-19) pandemic had accumulatively infected at least 770 million people worldwide, and caused a death toll surpassing 6.96 million. The super-spreading of COVID-19 posed a huge challenge to healthcare systems all over the world, and led to a global economic recession. Later research confirmed that the causative agent is a novel coronavirus, nomenclature Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This virus possesses a similar genomic structure, and displays many common harmful characteristics of SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Due to its acute contagiousness, relevant genetic research and antiviral drug development must be carried out in laboratories with the biosafety level 3 or above, which is insufficient worldwide. Moreover, cost for such experiments is very high, and the processes are tedious, time-consuming and laborious. To overcome these limitations and enable relevant research work conducted in laboratories with the biosafety level 2, global researchers have constructed a series of SARS-CoV-2 replicons using cutting-edge reverse genetic techniques. By in vitro transcription and electroporation or liposome-related transfection, mRNAs or plasmids carrying replicons can be delivered into diverse susceptible host cells. These replicons can replicate, transcribe, and translate inside permissive cells through their transcription and translation machines. With one or more structural genes deleted, no functional virion can form to lose virus infectivity. At the same time, reporter genes, such as green fluorescent protein (GFP) and/or luciferase gene, can be inserted into the replicons to report the levels of virus replication and transcription, indicating the functions of genes or the effectiveness and efficacy of antiviral compounds. By co-transfection with trans-complemental proteins, such as stomatitis virus glycoprotein, virions can be formed with a feature of the single infection of cells without SARS-CoV-2 receptors, such as angiotensin-converting enzyme 2 (ACE2), or transmembrane serine protease 2 (TMPRSS2). Resistance-selecting genes can be introduced into the proper position of replicons to select stable cell lines expressing replicons. Thus, cell lines stably expressing replicons can be obtained through multiple passages in the presence of selectable drugs, such as G418. In this article, we summarize synthesis methods for constructing SARS-CoV-2 replicons, such as in vitro ligation by type Ⅱ or ⅡS restriction enzymes, Bacterial Artificial Chromosome (BAC), yeast transformation-associated recombination (TAR), and cyclic polymerase extension reactions (CPER). Moreover, we review single-cycle and stably expressed SARS-CoV-2 replicon systems, which provide a solid foundation for studying the SARS-CoV-2 gene function, pathogenesis, virus-host interactions, vaccine assessment, and large-scale high-throughput screening of antiviral drugs to contain transmission of this pandemic.

Key words: SARS-CoV-2, replicon, reverse genetic system, BAC, TAR

摘要：

从2019年12月初首次报道到2023年9月，新型冠状病毒在全世界已造成约7.7亿人感染和696万人死亡。由于该病毒具有极强的传染性，理论上相关的研究工作都必须在生物安全三级或以上的实验室中进行。为了克服这一局限性，研究人员应用反向遗传学技术构建了众多复制子，使得在生物安全二级实验室中就可以进行相关的研究工作。科学家们通常会在复制子中插入绿色荧光蛋白或荧光素酶等报告基因以及遗传霉素等抗性基因，方便检测病毒和建立稳定的细胞株。另外，新冠病毒复制子还可通过与共转染的病毒糖蛋白的反式互补作用形成只能单次侵染的病毒粒子，使之能侵染无新冠病毒受体的细胞。因此，本文总结了合成新冠病毒复制子的主要方法，如通过Ⅱ型或ⅡS型限制性内切酶进行体外连接、基于细菌人工染色体的构建、利用酵母转化相关重组进行克隆和应用环形聚合酶延伸反应构建，同时综述了新冠病毒的单周期和稳定表达复制子系统。总之，多种复制子系统的构建和应用为研究新冠病毒基因功能、病毒与宿主互作机制和抗病毒药物的高通量筛选奠定了坚实的基础，为阻止病毒的蔓延和保卫人类的健康做出了重大贡献。

关键词: 新冠病毒, 复制子, 反向遗传系统, BAC, TAR