Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication.
COVID-19
CPE
SARS-CoV-2
corona virus
drug screening
high throughput
infection model
live cell imaging
Journal
Frontiers in microbiology
ISSN: 1664-302X
Titre abrégé: Front Microbiol
Pays: Switzerland
ID NLM: 101548977
Informations de publication
Date de publication:
2021
2021
Historique:
received:
27
04
2021
accepted:
28
06
2021
entrez:
16
8
2021
pubmed:
17
8
2021
medline:
17
8
2021
Statut:
epublish
Résumé
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication.
Identifiants
pubmed: 34394046
doi: 10.3389/fmicb.2021.701198
pmc: PMC8362758
doi:
Types de publication
Journal Article
Langues
eng
Pagination
701198Informations de copyright
Copyright © 2021 Widera, Wilhelm, Toptan, Raffel, Kowarz, Roesmann, Grözinger, Siemund, Luciano, Külp, Reis, Bracharz, Pallas, Ciesek and Marschalek.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Références
Med Microbiol Immunol. 2021 Aug;210(4):235-244
pubmed: 34196781
Nature. 2003 Nov 27;426(6965):450-4
pubmed: 14647384
Cell Host Microbe. 2021 Mar 10;29(3):463-476.e6
pubmed: 33592168
Nature. 2020 Nov;587(7835):657-662
pubmed: 32726803
J Natl Cancer Inst. 1973 Nov;51(5):1417-23
pubmed: 4357758
J Virol. 2010 Dec;84(24):12658-64
pubmed: 20926566
Cardiovasc Res. 2007 Feb 1;73(3):463-9
pubmed: 17049503
Cell. 2020 Sep 3;182(5):1295-1310.e20
pubmed: 32841599
Respir Res. 2007 Nov 08;8:80
pubmed: 17996064
Trends Pharmacol Sci. 2020 Aug;41(8):513-517
pubmed: 32553545
J Infect Dis. 2013 Jun 1;207(11):1743-52
pubmed: 23532101
Cell Rep. 2020 Jul 7;32(1):107863
pubmed: 32610043
EBioMedicine. 2020 Nov;61:103104
pubmed: 33158808
J Virol. 2014 Jan;88(2):1293-307
pubmed: 24227843
Cell. 2021 Apr 29;184(9):2372-2383.e9
pubmed: 33743213
Clin Sci (Lond). 2014 Jun;126(12):815-27
pubmed: 24593683
Mol Cell. 2020 May 21;78(4):779-784.e5
pubmed: 32362314
N Engl J Med. 2020 Feb 20;382(8):727-733
pubmed: 31978945
Cell Res. 2020 Mar;30(3):269-271
pubmed: 32020029
Cell. 2021 Apr 29;184(9):2384-2393.e12
pubmed: 33794143
Cell. 2020 May 14;181(4):914-921.e10
pubmed: 32330414
J Infect Dis. 2021 Jul 05;:
pubmed: 34223909
Microorganisms. 2021 Apr 02;9(4):
pubmed: 33918332
N Engl J Med. 2003 May 15;348(20):1967-76
pubmed: 12690091
Molecules. 2020 May 12;25(10):
pubmed: 32408547
J Clin Med. 2021 Jan 17;10(2):
pubmed: 33477365
Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7001-7003
pubmed: 32165541
Nature. 2020 May;581(7809):465-469
pubmed: 32235945
J Virol. 2016 Jun 10;90(13):6001-6013
pubmed: 27099312
Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5871-6
pubmed: 19321428
Cells. 2020 Oct 30;9(11):
pubmed: 33143316
J Gen Virol. 2020 Sep;101(9):925-940
pubmed: 32568027
Proc Natl Acad Sci U S A. 2020 May 26;117(21):11727-11734
pubmed: 32376634
Int J Mol Sci. 2020 Jun 20;21(12):
pubmed: 32575728
Biotechnol J. 2015 Apr;10(4):647-53
pubmed: 25650551
J Biomed Sci. 2013 Dec 09;20:92
pubmed: 24320156
EBioMedicine. 2021 Mar;65:103255
pubmed: 33676899
DNA Res. 2014 Dec;21(6):673-83
pubmed: 25267831
EMBO Mol Med. 2021 Apr 9;13(4):e13191
pubmed: 33544398
N Engl J Med. 2020 Mar 26;382(13):1278-1280
pubmed: 32069388
Cell. 2020 Apr 16;181(2):271-280.e8
pubmed: 32142651
PLoS One. 2013 Oct 03;8(10):e76469
pubmed: 24098509
EMBO Mol Med. 2020 Jun 8;12(6):e12465
pubmed: 32333818
Cell. 2021 Jan 7;184(1):149-168.e17
pubmed: 33278357
Nature. 2020 Jul;583(7816):469-472
pubmed: 32408336
Proc Natl Acad Sci U S A. 2021 Apr 20;118(16):
pubmed: 33811184
Nature. 2020 May;581(7807):215-220
pubmed: 32225176
EMBO J. 2020 Dec 1;39(23):e106267
pubmed: 33051876
Life Sci Alliance. 2020 Jul 23;3(9):
pubmed: 32703818
Cell. 2020 May 14;181(4):894-904.e9
pubmed: 32275855