Screening and testing for a suitable untransfected cell line for SARS-CoV-2 studies

At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus. Author summary In the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.

It is of no debate that the overcoming of the pandemic SARS-CoV-2 pathogen spreading 2 across the continents urgently needs joint efforts in the scientific community. Within a 3 few months in the midst of spring 2020 the pandemia is accompanied with a high case 4 fatality rate of up to 20% for vulnerable risk groups [1] and high excess mortality in 5 Europe monitored by EuroMOMO (https://www.euromomo.eu/graphs-and-maps) [2]. 6 To date no specific antiviral treatment to SARS-CoV-2 has been approved not to 7 mention vaccine treatment. However, promising antiviral drugs such as remdesivir [3], 8 camostat [4], and potential vaccines such as mRNA-1273 [5] are underway. Still, as long 9 as no clinically proven drugs and vaccines are at hand, screening and testing of potential 10 drugs is a major part of the current global research effort as well as studying the 11 molecular mechanisms of viral entry to the host cell. To this, our contribution to the 12 scientific progress is to validate human cell lines of the Leibniz Institute DSMZ for their 13 permissiveness to SARS-CoV-2. Cell lines serve as valuable and valid model systems to 14 study different diseases [6,7] and have been used specifically for SARS-CoV-2 viral 15 entry [4,8] or for antiviral activities against this virus [9]. Particularly the two surface 16 proteins ACE2 and TMPRSS2 have been shown to contribute to viral binding and 17 processing [4,8] and are mainly expressed in bronchial transient secretory cells [10]. 18 Interestingly, high gene expression of ACE2 is shown for other tissues such as myocardial 19 cells, esophagus epithelial cells, and enterocytes, which may hint at COVID-19 patients, 20 infected by SARS-CoV-2, exhibiting non-respiratory symptoms [11][12][13][14]. 21 Here, in order to get hold of human cell lines helpful for SARS-CoV-2 studies, we 22 examined human cell lines with comparably high gene expression of ACE2 and 23 TMPRSS2. The rich resource Cancer Cell Line Encyclopedia (CCLE) provided publicly 24 available RNA-Seq data [15], which we screened for human cell lines expressing high 25 levels of these two surface proteins. Selected cell lines were picked for validation on 26 mRNA and protein levels and tested for the ability of SARS-CoV-2 to infect these cells. 27 With these cell lines we devise appropriate, non-overexpression cell model systems to 28 facilitate experimental SARS-CoV-2 work, further the understanding of viral entry and 29 support drug development. 30

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Selection of cell model systems 32 Inspired by the publication of Hoffmann et al. pinpointing the host factors ACE2 as 33 SARS-CoV-2 receptor and TMPRSS2 as viral S protein priming serine protease [4], we 34 sought for identifying human cell lines enriched for these two proteins. Fortunately, the 35 Cancer Cell Line Encyclopedia (CCLE) provides large-scale sequencing data including 36 mRNA-seq expression data for over 900 cell lines [15]. Screening for gene expression of 37 ACE2 and TMPRSS2 in this data set enabled us to identify potential susceptible cell 38 cultures prone to SARS-CoV-2 infection. At first, we selected for 301 human cell lines 39 readily in stock at the DSMZ cell lines repository and visualised gene expression of 40 ACE2, which was shown to be the cellular receptor for the viral S protein [16], and 41 TMPRSS2 known to cleave SARS-CoV S protein [17] (Fig 1A). Gene expression for 42 these two proteins differed between and within the various tumour entities. Of these cell 43 lines, the colon carcinoma cell lines CL-14 and CL-40 and the breast carcinoma cell line 44 HCC-1937 belonged to the top 15 ACE2 expressing cell lines along with high expression 45 for TMPRSS2 (Table 1). Since the mucosa of the upper aerodigestive tract shows a 46 high virus replication in vivo [18], we additionally selected the tongue squamous cell carcinoma cell lines CAL-27 and CAL-33, the esophageal squamous cell carcinoma cell 48 line KYSE-510, and the newly established oral squamous cell carcinoma cell lines 49 UPCI-SCC-074 and UPCI-SCC-131. Since the lung carcinoma cell lines Caco-2, Calu-3 50 and the green monkey kidney cell line Vero E6 are shown to be permissive for 51 SARS-CoV-2 [4,8], these were used as positive control cell lines.

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Of note, gene expression of different splice variants of the ACE2 protein became 53 evident comparing the read numbers of the exons as seen in the CCLE RNA-seq data 54 for CAL-27, CAL-33 and KYSE-510 ( Fig 1B). Furthermore, missense mutations were 55 detected in TMPRSS2 for CL-40, HCC-1937, CAL-33 and Calu-3 ( Fig 1C). We assume 56 that these variations may have a considerable effect on the virus-host interaction.

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Recently published data suggest, that cells need interferon stimulation to produce 58 ACE2, thereby changing the cell's permissiveness to SARS-CoV-2 and hinting on the 59 dynamic levels of this host factor [19] [20].

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All selected cell lines exhibited marked ACE2 and TMPRSS2 mRNA expression 61 according to CCLE data or/and RQ-PCR (Table 1 and Fig 2A). Beside qualitative 62 verification for mRNA expression via RNA-Seq data and RQ-PCR (Fig 2A) protein 63 occurrence of ACE2 and TMPRSS2 was confirmed via western blot ( Fig 2B).  No co-expression of ACE2 and TMPRSS2 was found in the LL-100 data set, another 65 data set which comprises RNA-seq and WES data of 100 human leukemia and 66 lymphoma cell lines [6] (Table S1). This is reflected in the CCLE results, in which cell 67 lines of the hematopoetic lineages exhibited lower gene expression of both genes 68 compared to other cell lines of different origin (see Fig 1A,   Finally, we assessed the permissiveness of these cell lines by assessing the release of 122 mature, infectious virions in a plaque assay (Fig 3D). Supernatants of infected cells were 123 taken 5 days post infection (dpi) and plaques were counted on Vero E6 cells. We found 124 that all tested cell lines, except CL-40, support the production of infectious  So far, clinically proven therapeutics specifically addressing SARS-CoV-2 are missing for 135 the treatment of COVID-19 patients. It is getting clear, that the virus attacks different 136 organs and cell types [14,21,22], which explains diverse symptoms of COVID-19 [11][12][13]. 137 Many virus-host-interaction studies rely on ACE2/TMPRSS2-transfected cell lines such 138 as 293T and HeLa cells or non-human material such as green monkey cells Vero 139 E6 [4,28,29]. By screening RNA-seq data of human cell lines for higher levels of ACE2 140 and TMPRSS2 gene expression, we selected candidate cell lines, of which the human were split, trimmed and reassigned (GenomeAnalysisTK, 3.7-0, SplitNCigarReads [32]); 159 mutations were called by the HaplotypeCaller (GenomeAnalysisTK, 3.7-0,) [32]; and 160 mutation effects were predicted via the Ensembl VEP (GRCh38, v90) [33]. Mutations 161 were filtered ≥10 depth and predicted missense mutations.

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Cultivation of human cell lines 163 The continuous cell lines were provided for accessioning to the biological resource center 164 DSMZ by the original or secondary investigators [34]. Cell lines were grown at 37°C in a 165 humidified atmosphere of air containing 5% CO2. The basic growth media (Life 166 Technologies, Darmstadt, Germany) were supplemented with 10-20% fetal bovine serum 167 (Sigma Aldrich, Taufkirchen, Germany). No antibiotics were added to the cultures. All 168 cell lines were free of mycoplasmas and other bacterial, yeast and fungi contaminations 169 as tested by PCR and microbiological growth assays [35]. The authenticity of the cell  Supernatants of infected cells were taken 5 dpi, serially diluted, and used to infect 213 confluent Vero E6 cells on 96-well cell culture plates for one hour. Then, the inoculum 214 was removed and cells were overlaid with cell culture medium (MEM, 10% FCS, 2 mM 215 glutamine) containing 1.5% methyl-cellulose. After 3 days, virus plaques were counted 216 from phase contrast microscopic images.

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Immunofluorescence staining 218 SARS-CoV-2 infected cells were fixed with 6% paraformaldehyde in PBS for one hour at 219 room temperature, followed by washing three times with PBS. Cell permeabilisation 220 was done with PBS containing 0.15% Triton X-100 for 10 min at RT. Primary antibody 221 used to stain dsRNA was mouse anti-dsRNA, clone rJ2 (Merck Millipore, Cat. no. 222 MABE1134); secondary antibody was goat anti-rabbit-Cy5 (Life Technologies, Cat. no. 223 A10523).