TY  - JOUR
A1  - Widera, Marek
A1  - Wilhelm, Alexander
A1  - Toptan, Tuna
A1  - Raffel, Johanna M.
A1  - Kowarz, Eric
A1  - Roesmann, Fabian
A1  - Grözinger, Finn
A1  - Siemund, Anna Lena
A1  - Luciano, Vanessa
A1  - Külp, Marius
A1  - Reis, Jennifer
A1  - Bracharz, Silvia
A1  - Pallas, Christiane
A1  - Ciesek, Sandra
A1  - Marschalek, Rolf
T1  - Generation of a sleeping beauty transposon-based cellular system for rapid and sensitive screening for compounds and cellular factors limiting SARS-CoV-2 replication
T2  - Frontiers in microbiology
N2  - 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. HIGHLIGHTS: Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection; The versatile model cell line A549-AT is suitable for rapid and sensitive high-throughput assays; Additional gene specific expression cassettes allow the screening for compounds and cellular factors limiting SARS-CoV-2 replication.
KW  - SARS-CoV-2
KW  - COVID-19
KW  - corona virus
KW  - high throughput
KW  - drug screening
KW  - CPE
KW  - infection model
KW  - live cell imaging
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/62383
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-623839
SN  - 1664-302X
N1  - MW and RM were supported by the Deutsche Forschungsgemeinschaft (DFG, WI 5086/1–1, DFG MA 1876/13-1). MW was supported by the Federal Ministry of Education and Research (BMBF, COVIDready, 02WRS1621C).
VL  - 12
IS  - art. 701198
SP  - 1
EP  - 15
PB  - Frontiers Media
CY  - Lausanne
ER  -