Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication.

Abstract

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.

• Authors: Marek Widera, Alexander Wilhelm, Tuna Toptan, Johanna M Raffel, Eric Kowarz, Fabian Roesmann, Finn Grözinger, Anna Lena Siemund, Vanessa Luciano, Marius Külp, Jennifer Reis, Silvia Bracharz, Christiane Pallas, Sandra Ciesek, Rolf Marschalek
• Journal: Frontiers in microbiology (Front Microbiol) Vol. 12 Pg. 701198 ( 2021) ISSN: 1664-302X [Print] Switzerland
• PMID: 34394046 (Publication Type: Journal Article)
• Copyright: Copyright © 2021 Widera, Wilhelm, Toptan, Raffel, Kowarz, Roesmann, Grözinger, Siemund, Luciano, Külp, Reis, Bracharz, Pallas, Ciesek and Marschalek.