Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

  1. Marek Widera
  2. Alexander Wilhelm
  3. Tuna Toptan
  4. Johanna M. Raffel
  5. Eric Kowarz
  6. Fabian Roesmann
  7. Anna Lena Siemund
  8. Vanessa Luciano
  9. Marius Külp
  10. Jennifer Reis
  11. Silvia Bracharz
  12. Christiane Pallas
  13. Sandra Ciesek
  14. Rolf Marschalek

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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 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

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    1. ScreenIT

      SciScore for 10.1101/2021.04.27.441606: (What is this?)

      Please note, not all rigor criteria are appropriate for all manuscripts.

      Table 1: Rigor

      Ethicsnot detected.
      Sex as a biological variablenot detected.
      Randomizationnot detected.
      Blindingnot detected.
      Power Analysisnot detected.
      Cell Line Authenticationnot detected.

      Table 2: Resources

      Antibodies
      SentencesResources
      Western blot analysis: Protein extracts, separated by SDS-PAGE and transferred onto PVDF membranes, were probed with antibodies against FLAG (F1804, Sigma), ACE2 (ab15248, Abcam) or GAPDH (2275-PC-100, Trevigen).
      FLAG
      suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
      ACE2
      suggested: (Abcam Cat# ab15248, RRID:AB_301789)
      GAPDH
      suggested: (R and D Systems Cat# 2275-PC-100, RRID:AB_2107456)
      Proteins of interest were detected with HRP-conjugated secondary IgG antibody and visualized with ECL Western blotting substrate (Thermo Scientific) according to the provided protocol.
      HRP-conjugated secondary IgG
      suggested: None
      Immunofluorescence experiments were performed using SARS-CoV and SARS-CoV-2 Spike antibody (40150-R007, Sino Biologicals) and Alexa-488 conjugated anti-rabbit secondary antibody (Invitrogen).
      SARS-CoV-2
      suggested: (Sino Biological Cat# 40150-R007, RRID:AB_2827979)
      anti-rabbit
      suggested: None
      Experimental Models: Cell Lines
      SentencesResources
      Cell culture and virus propagation: Vero, A549 and Caco2 cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS), 2% L-glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin at 37°C and 5% CO2.
      Vero
      suggested: None
      A549
      suggested: None
      Interferon stimulation: Vero, Caco2 and A549 cells were stimulated with the type I-III interferons (500 U / mL and 10 ng / mL, respectively.
      Caco2
      suggested: None
      Plasmids construction and cloning: Total RNA (1 μg) was isolated from HEK293T cells and reverse transcribed into cDNA using random hexamers and SuperScript II RT (Invitrogen).
      HEK293T
      suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
      Quantitative proliferation and trypsinization assay: CaCo2 and A549-based cells were seeded in 12-well plates (2×104 A549-AT and 1×105 Caco2 / well, respectively).
      A549-based
      suggested: None
      Recombinant DNA
      SentencesResources
      All amplimers were cloned into the PCR2.1-Topo-TA vector (Invitrogen) for Sanger sequencing.
      PCR2.1-Topo-TA
      suggested: None
      Correct amplimers were digested with Sfi1 and cloned into the pSBbi-RB (ACE2) or pSBbi-BH (TMPRSS2) Sleeping Beauty plasmids (Kowarz et al., 2015).
      pSBbi-RB
      suggested: RRID:Addgene_60522)
      pSBbi-BH
      suggested: RRID:Addgene_60515)
      Software and Algorithms
      SentencesResources
      Red object count, Confluency and roughness were analyzed using ImageAnalyzer Software v.1.1 (Tecan).
      ImageAnalyzer
      suggested: None

      Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

      Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
      Integration into genome occurs preferentially at TA-sites, however a certain limitation remains since a targeted integration at a predetermined location in the genome is not possible. Through precise dosing of the vector and the transposase-encoding plasmid scalable number of integrations can be achieved (Skipper et al., 2013). A further conceivable development of the system would be a construct expressing eGFP enabling the parallel expression of an inducible shRNA-mediated knock-down cassette. This would allow loss-of-function experiments to be carried out in addition to gain-of-function experiments. 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 the system theoretically allows infection and read-out within one working day performed via non-invasive roughness factor analysis.

      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.

      Results from scite Reference Check: We found no unreliable references.

      About SciScore

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    2. Version published to 10.1101/2021.04.27.441606v1 on bioRxiv