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Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology
The global COVID-19 pandemic continues with continued 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,...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10301824/ https://www.ncbi.nlm.nih.gov/pubmed/37376581 http://dx.doi.org/10.3390/v15061281 |
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author | Khan, Juveriya Qamar Rohamare, Megha Rajamanickam, Karthic Bhanumathy, Kalpana K. Lew, Jocelyne Kumar, Anil Falzarano, Darryl Vizeacoumar, Franco J. Wilson, Joyce A. |
author_facet | Khan, Juveriya Qamar Rohamare, Megha Rajamanickam, Karthic Bhanumathy, Kalpana K. Lew, Jocelyne Kumar, Anil Falzarano, Darryl Vizeacoumar, Franco J. Wilson, Joyce A. |
author_sort | Khan, Juveriya Qamar |
collection | PubMed |
description | The global COVID-19 pandemic continues with continued 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 viruses rescued from molecular clones and a clinical isolate (VIDO-01 strain). 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, such as CRISPR knockout and activation screens. |
format | Online Article Text |
id | pubmed-10301824 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-103018242023-06-29 Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology Khan, Juveriya Qamar Rohamare, Megha Rajamanickam, Karthic Bhanumathy, Kalpana K. Lew, Jocelyne Kumar, Anil Falzarano, Darryl Vizeacoumar, Franco J. Wilson, Joyce A. Viruses Article The global COVID-19 pandemic continues with continued 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 viruses rescued from molecular clones and a clinical isolate (VIDO-01 strain). 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, such as CRISPR knockout and activation screens. MDPI 2023-05-30 /pmc/articles/PMC10301824/ /pubmed/37376581 http://dx.doi.org/10.3390/v15061281 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Khan, Juveriya Qamar Rohamare, Megha Rajamanickam, Karthic Bhanumathy, Kalpana K. Lew, Jocelyne Kumar, Anil Falzarano, Darryl Vizeacoumar, Franco J. Wilson, Joyce A. Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
title | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
title_full | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
title_fullStr | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
title_full_unstemmed | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
title_short | Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology |
title_sort | generation of a sars-cov-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10301824/ https://www.ncbi.nlm.nih.gov/pubmed/37376581 http://dx.doi.org/10.3390/v15061281 |
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