key: cord-0686477-gk9l4soq authors: Widera, Marek; Wilhelm, Alexander; Toptan, Tuna; Raffel, Johanna M.; Kowarz, Eric; Roesmann, Fabian; Siemund, Anna Lena; Luciano, Vanessa; Külp, Marius; Reis, Jennifer; Bracharz, Silvia; Pallas, Christiane; Ciesek, Sandra; Marschalek, Rolf title: Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors date: 2021-04-27 journal: bioRxiv DOI: 10.1101/2021.04.27.441606 sha: f9136f2a217d82679c84fef77ad8220cacc1731e doc_id: 686477 cord_uid: gk9l4soq 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 reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell 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 in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter 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 reporter cell line allows rapid and sensitive detection 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 reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors 3 is expanded by the usage of a process named discontinuous transcription in which subgenomic RNAs 81 (sgRNAs) are formed (Kim et al., 2020) serving as templates for the translation of downstream ORFs 82 3a-9b, including the E, M, and N genes. SgRNAs are each composed of an RNA primer that can co-83 transcriptionally jump to sequences at transcription-regulating sequences (TRSs) and create a junction 84 with downstream sequence elements that code for the other viral proteins. Quantitative reverse 85 transcriptase polymerase chain reaction (RT-qPCR) is the commonly used method for sensitive and 86 specific detection method for SARS-CoV-2. In particular, targeting the vRNA derived from cell culture 87 supernatants is suitable for the quantification of genome copy equivalents (Toptan et al., 2020) while 88 the detection of specific intracellular sgRNAs is used to quantify active viral replication (Kohmer et al., 89 2021; Shin et al., 2020; Wolfel et al., 2020) . However, high costs, excessive hands on time, and reagent 90 shortages emerging during the pandemic disqualify RT-qPCR for high-throughput testing. In vitro cell culture models that can realistically mimic the viral replication cycle to decipher the pathology 92 of COVID-19 are limited. Primary human airway epithelial cells highly express both receptors ACE2 and TMPRSS2 and are 94 permissive for SARS-CoV-2. They show cytopathic effects 96 h post infection (Hoffmann et al., 2020b; 95 Takayama, 2020) , but have limited lifespan and thus difficult to handle or expensively available from 96 commercially sources. Currently, the common cell lines for SARS-CoV-2 research are Caco2, Calu-3, Vero E6, HEK293T, and Huh7. Vero E6 cells have been shown to be susceptible to SARS-CoV-2 98 (Ogando et al., 2020) . By introducing additional gene copies of TMPRSS2 they have been rendered 99 even superior to infection when compared to the parental cell line by 2-log (Matsuyama et al., 2020) . The major prerequisites for a novel reporter cell line for high throughput SARS-CoV-2 research are easy 121 cultivation properties, reproducibility, relatively unlimited availability, but also pronounced interferon 122 responses. As a suitable starting point, we have chosen the adenocarcinomic human alveolar basal 123 epithelial cells A549 cells (Giard et al., 1973) and initially tested the susceptibility to interferons in 124 comparison to in SARS-CoV-2 research commonly used Caco2 and Vero cells. Cells were stimulated 125 with either type I or II interferons and the mRNA expression levels of known interferon stimulated genes 126 (ISGs) were examined by RT-qPCR. As surrogate markers for ISGs, we have chosen the two genes 127 Interferon-stimulated gene 15 (ISG15) and Interferon regulatory factor 1 (IRF1), which are predominantly 128 stimulated by type I / III or II interferons, respectively, and are significantly involved in host cell defense 129 against SARS- CoV-2 (Karki et al., 2021; Shin et al., 2020 Next, the time course of the ISG induction between A549 and Caco2 cells was investigated and two 139 type III interferons (IFN-λ1 and -λ2), which have a distinct receptor to type I interferons but share 140 common downstream signaling cascade, were also added. In both A549 and Caco2 cells, treatment 141 with IFN-β (type I IFN) resulted in a strong increase with higher ISG15 expression levels, while both 5 both cell lines. Using standard conditions, we found that A549 cells allowed a 70% higher transfection 160 efficiency than Caco2 cells ( Figure 2C) . These data confirmed that A549 have cell culture properties 161 that are highly suitable for high-throughput analyses. Since parental A549 cells are less susceptible to SARS-CoV and SARS-CoV-2 infection and per se do 163 not allow CPE formation, we aimed to overexpress both receptors ACE2 and TMPRSS2 (Hoffmann et 164 al., 2020b) necessary for SARS-CoV-2 entry. We used the well-established Sleeping Beauty system 165 (Kowarz et al., 2015) , which enables transposase-mediated stable transfection of the target cells. For 166 this purpose, we first generated a construct that allows constitutive expression of ACE2 together with 167 an expression cassette for dTomato (Fehler! Verweisquelle konnte nicht gefunden werden. Figure 168 3a) using blasticidin S deaminase (BSD) as a selection marker. A549 cells were transfected together 169 with a transposase encoding plasmid to achieve a priori stable integration. Following antibiotic selection, 170 we checked for ACE2 expression via Western Blotting and investigated the extent to which A549 cells 171 can be infected in comparison to the highly susceptible Caco2. We infected parental and ACE2 To monitor SARS-CoV-2 replication non-invasively, we used an automated cell imaging system 205 (SparkCyto 400, Tecan). First, we infected the cells with SARS-CoV-2 strain FFM1 (Toptan et al., 2020) 206 and examined the cells for cytopathic effect (CPE) formation. In comparison to the Caco2 cells, we could 207 already observe CPE formation and loss of confluency after 12 hours post infection ( Figure 5A) . In AT were also suitable to display the CPE of various SARS-CoV-2 isolates including variants of concern 218 (VoCs) B.1.1.7 (incl. Spike N501Y) and P.2 (incl. Spike E484K), which have been associated with higher Quantifying the loss of dTomato signal upon viral infection constitutes a third read-out assay to monitor 221 CPE formation. This is particularly suitable for automated fluorescence intensity measurement, which is Since A549-AT was found suitable for automated readout of SARS-CoV-2 infection experiments, viral 245 outgrowth assays as well as drug screenings and neutralization tests, we next focused on the 246 investigation of cellular restrictions and dependency factors. For this purpose, we generated a third 247 vector based on the Sleeping Beauty transposase system, which has a doxycycline (Dox) inducible 248 expression cassette (Figure 7) . In addition to the gene of interest, which is expressed under the 249 inducible TCE promoter, the constructs also contain a cassette encoding the green fluorescent protein 250 (GFP) and an additional resistance cassette (PuroR) for antibiotic selection. To provide a proof of 251 principle, we cloned the known host restriction factor IFITM1, which has an antiviral effect against SASR- 8 Cellular entry of coronaviruses is a multistage process involving virus attachment to the cell surface, 282 receptor engagement, protease processing and membrane fusion (Li et al., 2021) . These stages rely on 283 distinct domains in the SARS-CoV-2 spike protein (Lan et al., 2020) . Emerging SARS-CoV-2 variants of 284 concern (VOCs) like B. In addition to the loss in fluorescence, two non-invasive factors can also be used as surrogate markers 303 for CPE formation and cell lysis, which are confluency and roughness. The former was proofed as highly 304 robust and was additionally confirmed with DAPI staining (data not shown). Exclusively in A549-AT, 305 cellular roughness was used to monitor syncytia formation which was much less pronounced in Caco2 306 cells. Furthermore, a clear CPE formation could be monitored since A549 have a less heterogenic 9 In humans, type I interferons consist of IFN-α, β, (ε, κ, and ω), while especially IFN-α consists of multiple 323 subtypes with distinct biological activities (Lavender et al., 2016) . While type I interferons are mostly 324 produced by infected cells and cells of the immune system, type II interferon (IFN-γ) is predominantly 325 produced by immune cells. In our study IRF1 expression was used as a surrogate marker of IFN-II 326 induction and was induced in Vero, Caco2 and A549 cells to a comparable extent. Importantly, IFN-γ 327 driven inflammation was shown to induce a vulnerable state in vivo allowing SARS-CoV-2 replication by 328 promoting ACE2-expression and enhanced virus production in infected cells (Heuberger et al., 2021) . Hence, A549-AT cells are suitable to study cell culture based responses to IFN-γ. Type III interferons consist of four subtypes of IFN-λ. Although some immune cells produce type III 331 interferons, these are also produced by epithelial cells or cells that are from the same developmental 332 stage, which are the respiratory tract, the gastrointestinal tract, and the urogenital tract derived cells. Type III IFNs were effective in preventing SARS-CoV-2 infection but are in contrast to IFN-I locally 334 limited in the respiratory tract (Andreakos and Tsiodras, 2020), which makes them relevant for treatment 335 in early infected patients. Hence, it will be also interesting to analyse the impact of type III interferons 336 on different SARS-CoV-2 variants in future. Although lentiviral transduction is relatively quick, the compatibility of a gene of interest, which has to be In conclusion, we developed a reporter cell system with optimal ACE2/TMPRSS2 ratio for high SARS- CoV-2 susceptibility, syncytia and CPE formation. The confluency and particularly roughness of A549- AT cells represents a highly sensitive marker for early syncytia formation and cytopathic effects. Thus, A549-AT cells allow the infection and readout of an experiment on the same working day. Using 1 MOI A549 cells were shown in 12-well plates with low density (2x10 4 A549-AT / well; 1x10 5 Caco2 / well). The relative confluence was monitored automatically using live cell imaging SparkCyto 400 (Tecan) over Relative expression of SARS-CoV-2 sgRNA was used as surrogate markers to quantify active viral 404 replication. C and D) Relative expression of ISG15 and IFITM1 (fold change) were determined using 405 RT-qPCR to monitor interferon signaling in SARS-CoV and SARS-CoV-2 infected cells. CaCo2 and A549-based cells were seeded in 12-well plates (2x10 4 A549-AT and 1x10 5 Caco2 / well, 484 respectively). Cells were incubated for 16 days at 37°C and 5% CO2 and cell count and confluency was 485 optically determined using SparkCyto 400 (Tecan) at regular intervals starting after 48 hours after 486 seeding. To allow attachment, cells were incubated for 48 h before monitoring. Prior to imaging the wells 487 were washed using PBS and refilled with 2 ml culture medium. Trypsinization assay was performed in 488 96 well plates. After rinsing twice with PBS, confluent cells were treated with 100 µl trypsin (2 mg/ml) 489 and incubated for different intervals. Cells were incubated at room temperature and 37°C for incubation 490 times longer than 10 min. Subsequently cells were rinsed with cell culture medium, remaining cells were 491 fixed with 3% PFA, and nuclei were stained with DAPI and analyzed with a plate reader (SparkCyto Statistical significance compared to untreated control was determined using unpaired student's t-test. Asterisks indicated p-values as * (p < 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.005). Optimized Sleeping Beauty transposons rapidly 605 generate stable transgenic cell lines Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo Caco-2 Cell Line The Impact of Food Bioactives on Health: 616 in vitro and ex vivo models Angiotensin-converting enzyme 2 is a 619 functional receptor for the SARS coronavirus SARS-CoV-2 induces 623 double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and 624 cardiomyocytes Efficient activation 626 of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease 627 TMPRSS2 Natural Products against Type II Transmembrane Serine Protease (TMPRSS2), the Priming Agent of 646 Coronavirus 2 (SARS-CoV-2) Cell entry mechanisms of 648 Proceedings of the National Academy of Sciences of the United States of America Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity DNA transposon-based gene 655 vehicles -scenes from an evolutionary drive Critical Role of Type III Interferon in Controlling 658 SARS-CoV-2 Infection in Human Intestinal Epithelial Cells Deep Mutational Scanning 661 of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding In Vitro and Animal Models for SARS-CoV-2 research Optimized qRT-PCR Approach for the Detection of Intra-and Extra-667 Cellular SARS-CoV-2 RNAs. International journal of molecular sciences 21 The role of IFN-gamma in regulation of IFN-gamma-inducible 669 protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures Virological assessment of hospitalized patients with 688 COVID-2019 A Novel Coronavirus from Patients with Pneumonia in China