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一种基于质粒 DNA 的 SARS-CoV-2 反向遗传学系统和冠状病毒工具包,用于 COVID-19 研究。

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

机构信息

MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom.

Institute of Technology, University of Tartu, Tartu, Estonia.

出版信息

PLoS Biol. 2021 Feb 25;19(2):e3001091. doi: 10.1371/journal.pbio.3001091. eCollection 2021 Feb.

Abstract

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

摘要

严重急性呼吸综合征冠状病毒 2(SARS-CoV-2)的最近出现是导致 2019 年冠状病毒病(COVID-19)的根本原因,导致了全球大流行,造成了大量的发病率、死亡率和经济破坏。因此,许多实验室已将注意力重新转向 SARS-CoV-2,这意味着迫切需要能够在不习惯处理冠状病毒的实验室中使用的工具。在这里,我们报告了一系列用于 SARS-CoV-2 研究的工具。首先,我们描述了一种简便的单质粒 SARS-CoV-2 反向遗传学系统,该系统易于遗传操作,可通过瞬时转染(无需体外转录或额外的表达质粒)来拯救感染性病毒。该拯救系统伴随着我们的 SARS-CoV-2 抗体(针对几乎所有病毒蛋白)、SARS-CoV-2 临床分离株和 SARS-CoV-2 许可的细胞系,这些都向科学界公开提供。使用这些工具,我们在这里证明了有争议的 ORF10 蛋白在感染细胞中表达。此外,我们表明,有希望的再利用抗病毒药物阿匹莫德的活性依赖于 TMPRSS2 的表达。总之,我们的 SARS-CoV-2 工具包可通过我们的网站 https://mrcppu-covid.bio/ 直接访问,它构成了一个具有相当大潜力的资源,可以推进 COVID-19 疫苗设计、药物测试和发现科学。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a350/7906417/dc7696ab0272/pbio.3001091.g001.jpg

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