In Silico, In Vitro and In Cellulo Models for Monitoring SARS-CoV-2 Spike/Human ACE2 Complex, Viral Entry and Cell Fusion

  1. Delphine Lapaillerie
  2. Cathy Charlier
  3. Henrique S. Fernandes
  4. Sergio F. Sousa
  5. Paul Lesbats
  6. Pierre Weigel
  7. Alexandre Favereaux
  8. Véronique Guyonnet-Duperat
  9. Vincent Parissi

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.02.03.429555: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Proteins and antibodies: SARS-CoV-2 (438-516) S-RBD((HiS)6 and biotynilated human ACE2 have been purchased from Fisher Scientific (respective references 16534204 and 16545164).
    ACE2
    suggested: None
    Monoclonal anti-6X His tag antibody has been purchased from ABCAM (reference ab18184, dilution 1/200).
    anti-6X
    suggested: (Abcam Cat# ab18184, RRID:AB_444306)
    Anti α-tubulin antibody has been purchased from SIGMA (reference T6199, dilution 1/500).
    Anti α-tubulin
    suggested: None
    Secondary goat anti-mouse antibody coupled to AlexaFluor 488 has been purchase from Fisher Scientific (Reference Allo2g, dilution 1/400).
    anti-mouse
    suggested: None
    Bound proteins were detected by western blot using anti-His antibody and streptavidin-HRP conjugate (SIGMA, reference GERPN1231).
    anti-His
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lentiviral particles were produced by transient transfection of HEK293T(human embryonic kidney cells according to standard protocols.
    HEK293T(human
    suggested: None
    DNA from two different cell clones (human 293T and K562 cells), containing a single integrated copy of the provirus, was used as a normalized cell line.
    293T
    suggested: None
    DNA from a HEK cell line with one proviral insert (HEK-2C9) was used as a standard for quantification by the ΔCt method.
    HEK
    suggested: None
    Cell imaging: For cell imaging 20 000 HEK293T and HEK293T-ACE2 cells were plated on glass coverslips pretreated with poly-L-Lysine solution 0.01% (SIGMA ref P4832) 5 minutes at room temperature.
    HEK293T
    suggested: None
    For quantification of the S-RBD/ACE2 interaction in cellular context 45 000 HEK293T and HEK293T-ACE2 cells were incubated 45 minutes at 37°C in 100µl DMEM and increasing concentrations of RBD recombinant protein.
    HEK293T-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analyzed with GraphPad Prism 5.01 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Sensorgrams curves were plotted using Prism 5.0 software (Graphpad Software, La Jolla, CA) Cells and lentiviral vectors production: Lentivirus vector production was done by the service platform Vect’UB, (INSERM US 005
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    Epifluorescence microscopy was carried out on a Zeiss Axioimager Z1 driven by Metamorph.
    Metamorph
    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: 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.3390/v13030365
  3. Version published to 10.1101/2021.02.03.429555v1 on bioRxiv