RT Journal Article
SR Electronic
T1 Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.03.08.531833
DO 10.1101/2023.03.08.531833
A1 Juveriya Qamar Khan
A1 Megha Rohamare
A1 Karthic Rajamanickam
A1 Kalpana K Bhanumathy
A1 Jocelyne Lew
A1 Anil Kumar
A1 Darryl Falzarano
A1 Franco J Vizeacoumar
A1 Joyce A Wilson
YR 2023
UL http://biorxiv.org/content/early/2023/03/09/2023.03.08.531833.abstract
AB The global COVID-19 pandemic continues with an increasing number of cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology and titers were comparable between rescued molecular clones and a clinical isolate (VIDO-01 strain), thus providing confidence that the rescued viruses can be used as effective replication tools. Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells, were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death and a novel lung cell line NCI-H23ACE2 A3 showed about ∼99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection and are currently being used in CRISPR knockout and activation screens in our lab.Importance We used a reverse genetics system to generate a wild type as well as a nanoluciferase-expressing reporter clone of SARS-CoV-2. The reporter virus allows for rapid transient replication assays and high throughput screens by detection of virus replication using luciferase assays. In addition, the reverse genetic system can be used to generate mutant viruses to study phenotypes of variant mutations. Additionally, unique human lung cell lines supporting SARS-CoV-2 replication will aid in studying the virus in a lung-relevant environment and based on high cytopathology induced in some cell lines, will be useful for screens that rely on virus-induced cell death for selection. Our study aims to enhance and contribute to the current replication tools available to study SARS-CoV-2 by providing rapid methods, virus clones and novel lung cell lines.