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

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.

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses

“…The selection of certain amino acid changes was different for CPER1, 2, and 3 viruses (Supplementary Table 1 ), with such variability in selection also reported for other reverse genetics systems 16 , 21 , 27 . As noted previously 21 , 22 , the choice of cell lines can affect these selection processes, with our recovery of CPER3 virus using ACE2-HEK293T cells showed a decrease in the number of affected sites from 4 to 1 (Supplementary Table 1 ). Given the SARS-CoV-2 genome encodes >9700 amino acids, the CPER method using ACE2-HEK293T cell thus achieved nearly complete (99.99%) amino acid sequence fidelity.…”

“…Culture of SARS-CoV-2 viruses in Vero E6 cells is well known to select rapidly for mutations, often in or around the furin cleavage site 19 – 22 . This remains true for a number of reverse genetics systems 16 , 21 , 23 , although deep sequencing of recovered viruses is not always provided 15 , 17 , 24 – 26 . Such selection was also seen for CPER viruses recovered from Vero E6 cells (cocultured with transfected HEK293T) (Supplementary Table 1 ).…”

Infectious Clones Produce SARS-CoV-2 That Causes Severe Pulmonary Disease in Infected K18-Human ACE2 Mice

“…Our validation that the RG-rescued SARS-CoV-2 viruses can be reliably used to model severe SARS-CoV-2 infection in vivo makes them valuable assets in the development of vaccines, antivirals, and therapeutics against COVID-19. In Vivo Characterization of SARS-CoV-2 Infectious Clones ® using restriction endonuclease-based cloning procedures as described in detail previously (10). Briefly, 10 mg of infectious clone DNA was transfected into BHK-21 cells using Lipofectamine LTX with Plus reagent (Life Technologies) according to the manufacturer's instructions in a T25 flask.…”

“…We recently developed and characterized a user-friendly RG infectious clone (pCC1-4K-SARS-CoV-2-Wuhan-Hu1, GenBank accession no. MT926410) from which the original SARS-CoV-2-Wuhan-Hu-1 virus (Wuhan-Hu-1) can be propagated (10). In addition, three recombinant SARS-CoV-2 infectious clones expressing various reporter cassettes, including mCherry, ZsGreen, and Nanoluciferase, were generated based on the original infectious clone.…”

Two courses of deconstructed coronavirus please

“…Endeavours like those pursued by Ju and colleagues highlight the need and potential for the democratisation of science in the face of significant societal challenges, if reagents and protocols are indeed adequately shared. There are numerous examples of this in the scientific community, such as the generation and sharing of SARS-CoV-2 pseudoparticles from the lab of Benhur Lee [8] and the construction of a Coronavirus Disease 2019 (COVID-19) toolbox from the MRC-University of Glasgow Centre for Virus Research [9]. It is hoped that efforts like this will enhance the efficiency of science, while not jeopardising the safety of researchers and their communities.…”