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

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.
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用于COVID-19研究的质粒DNA启动的SARS-CoV-2反向遗传学系统和冠状病毒工具包。

DOI：
10.1371/journal.pbio.3001091
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发表时间：
2021-03
期刊：
PLoS biology
影响因子：
9.8
通讯作者：
Mahalingam S
Mahalingam S
中科院分区：
生物学1区
文献类型：
--
作者：
Rihn SJ;Merits A;Bakshi S;Turnbull ML;Wickenhagen A;Alexander AJT;Baillie C;Brennan B;Brown F;Brunker K;Bryden SR;Burness KA;Carmichael S;Cole SJ;Cowton VM;Davies P;Davis C;De Lorenzo G;Donald CL;Dorward M;Dunlop JI;Elliott M;Fares M;da Silva Filipe A;Freitas JR;Furnon W;Gestuveo RJ;Geyer A;Giesel D;Goldfarb DM;Goodman N;Gunson R;Hastie CJ;Herder V;Hughes J;Johnson C;Johnson N;Kohl A;Kerr K;Leech H;Lello LS;Li K;Lieber G;Liu X;Lingala R;Loney C;Mair D;McElwee MJ;McFarlane S;Nichols J;Nomikou K;Orr A;Orton RJ;Palmarini M;Parr YA;Pinto RM;Raggett S;Reid E;Robertson DL;Royle J;Cameron-Ruiz N;Shepherd JG;Smollett K;Stewart DG;Stewart M;Sugrue E;Szemiel AM;Taggart A;Thomson EC;Tong L;Torrie LS;Toth R;Varjak M;Wang S;Wilkinson SG;Wyatt PG;Zusinaite E;Alessi DR;Patel AH;Zaid A;Wilson SJ;Mahalingam S
来源链接：
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906417/

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最近出现的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是冠状病毒疾病2019年(新冠肺炎)的根本原因，已导致一场全球大流行，造成大量发病率、死亡率和经济破坏。作为回应，许多实验室将注意力转向SARS-CoV-2，这意味着迫切需要能够在不习惯与冠状病毒合作的实验室使用的工具。在这里，我们报告了一系列用于SARS-CoV-2研究的工具。首先，我们描述了一种简单的单质粒SARS-CoV-2反向遗传学系统，该系统易于遗传操作，可以通过瞬时转染(不需要体外转录或额外的表达载体)来拯救传染性病毒。救援系统伴随着我们的小组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许可细胞系，所有这些都对科学界开放。使用这些工具，我们在这里证明了有争议的ORF10蛋白在受感染的细胞中表达。此外，我们还发现阿必利莫特的抗病毒活性依赖于TMPRSS2的表达。总之，我们的SARS-CoV-2工具包可以通过我们的网站https://mrcppu-covid.bio/，直接访问，它构成了一个具有相当大潜力的资源，可以推动新冠肺炎疫苗设计、药物测试和发现科学的发展。为了满足随后对新冠肺炎试剂的需求，本文提供了一个开放可用的冠状病毒工具包(https://mrcppu-covid.bio))，并描述了这些工具的生成和验证，其中包括一个简单的SARS-CoV-2反向遗传学系统和一组近乎全面的SARS-CoV-2抗体。
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. To help meet the ensuing demand for COVID-19 reagents, this article presents an openly available ‘coronavirus toolkit’ (https://mrcppu-covid.bio) and describes the generation and validation of these tools, including a simple SARS-CoV-2 reverse genetics system and a near-comprehensive panel of SARS-CoV-2 antibodies.
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