Bulk and single-cell gene expression profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for therapeutic intervention
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Abstract
The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health threat with more than two million infected people since its emergence in late 2019. Detailed knowledge of the molecular biology of the infection is indispensable for understanding of the viral replication, host responses, and disease progression. We provide gene expression profiles of SARS-CoV and SARS-CoV-2 infections in three human cell lines (H1299, Caco-2 and Calu-3 cells), using bulk and single-cell transcriptomics. Small RNA profiling showed strong expression of the immunity and inflammation-associated microRNA miRNA-155 upon infection with both viruses. SARS-CoV-2 elicited approximately two-fold higher stimulation of the interferon response compared to SARS-CoV in the permissive human epithelial cell line Calu-3, and induction of cytokines such as CXCL10 or IL6. Single cell RNA sequencing data showed that canonical interferon stimulated genes such as IFIT2 or OAS2 were broadly induced, whereas interferon beta (IFNB1) and lambda (IFNL1-4) were expressed only in a subset of infected cells. In addition, temporal resolution of transcriptional responses suggested interferon regulatory factors (IRFs) activities precede that of nuclear factor-κB (NF-κB). Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 charperone activity by Tanespimycin/17-N-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in a reduction of viral replication, and of TNF and IL1B mRNA levels. In summary, our study established in vitro cell culture models to study SARS-CoV-2 infection and identified HSP90 protein as potential drug target for therapeutic intervention of SARS-CoV-2 infection.
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SciScore for 10.1101/2020.05.05.079194: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Primary detection of hACE-2 was done using a goat anti-hACE-2 antibody (1:1,250; #AF933, R&D Systems), a horseradish peroxidase (HRP)-labeled donkey anti-goat antibody (1:5,000, Dianova) and Super Signal West Femto Chemiluminescence Substrate (Thermo Fisher Scientific). anti-hACE-2suggested: NoneHRP)-labeled donkey anti-goat antibody (1:5,000, Dianova)suggested: NoneAs loading control, samples were analyzed for β-actin expression using a mouse anti-β-actin … SciScore for 10.1101/2020.05.05.079194: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Primary detection of hACE-2 was done using a goat anti-hACE-2 antibody (1:1,250; #AF933, R&D Systems), a horseradish peroxidase (HRP)-labeled donkey anti-goat antibody (1:5,000, Dianova) and Super Signal West Femto Chemiluminescence Substrate (Thermo Fisher Scientific). anti-hACE-2suggested: NoneHRP)-labeled donkey anti-goat antibody (1:5,000, Dianova)suggested: NoneAs loading control, samples were analyzed for β-actin expression using a mouse anti-β-actin antibody (1:5,000, Sigma Aldrich) and a HRP-labeled goat anti-mouse antibody (1:10,000, Sigma-Aldrich). anti-β-actinsuggested: Noneanti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources (ATCC HTB-37) and H1299 (ATCC CRL-5803) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 1% non-essential amino acids, 1% L-glutamine and 1% sodium pyruvate (all Thermo Fisher Scientific) in a 5% CO2 atmosphere at 37 °C. H1299suggested: ATCC Cat# CRL-5803, RRID:CVCL_0060)Cells inoculated with cell culture supernatants from uninfected Vero cells mixed with OptiPro serum-free medium supplemented with 0.5% gelatine and PBS, in accordance to virus stock preparation, serves as mock infected controls. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)For the assay Vero E6 cells were seeded to confluence and infected with serial dilution of virus-containing cell culture supernatant diluted in OptiPro serum-free medium. Vero E6suggested: RRID:CVCL_XD71)Infections for RNA sequencing experiments: Calu-3 cells and H1299 cells were seeded at a concentration of 6 x 10^5 cells/mL and 5 x 10^4 cells/mL, respectively. Calu-3suggested: NoneResults from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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