A comprehensive library of fluorescent constructs of SARS‐CoV‐2 proteins and their initial characterisation in different cell types

  1. Stéphanie Miserey‐Lenkei
  2. Katarina Trajkovic
  3. Juan Martín D'Ambrosio
  4. Amanda J Patel
  5. Alenka Čopič
  6. Pallavi Mathur
  7. Kristine Schauer
  8. Bruno Goud
  9. Véronique Albanèse
  10. Romain Gautier
  11. Melody Subra
  12. David Kovacs
  13. Hélène Barelli
  14. Bruno Antonny

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Abstract

Comprehensive libraries of plasmids for SARS‐CoV‐2 proteins with various tags ( e.g ., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein–protein interactions between the SARS‐CoV‐2 virus and host proteins.

Results

We present here a large library of SARS CoV‐2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS‐2B), as well as in budding yeast. The localisation of a few SARS‐CoV‐2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria.

Conclusions and Significance

This library should facilitate further cellular investigations, notably by imaging techniques.

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  1. Version published to 10.1111/boc.202000158
  2. ScreenIT

    SciScore for 10.1101/2020.12.19.423586: (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
    For staining with antibodies against EEA1, Lamp1, M6PR and calnexin, cells were permeabilized with 0.05 % Triton X-100 in PBS for 5 min at room temperature.
    EEA1
    suggested: None
    Lamp1, M6PR
    suggested: None
    calnexin
    suggested: None
    Primary antibodies used in this study were mouse anti-GM130 (BD Biosciences, ref 610823 and Roche, ref 11814460001), rabbit anti-GalNacT2 (Sigma, HPA01122), sheep anti12 TGN46 (Biorad,
    anti-GM130
    suggested: None
    anti-GalNacT2
    suggested: None
    HPA01122
    suggested: None
    anti12 TGN46
    suggested: None
    Mouse anti-clathrin antibodies were a kind gift from Michel Franco.
    anti-clathrin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Material and methods Mammalian cell expression A549, HeLa, HEK293, Caco-2, BEAS-2B and hTert-RPE1 cell lines were used for testing the localization of SARS-Cov-2 proteins.
    A549
    suggested: None
    HeLa
    suggested: None
    HEK293
    suggested: None
    Caco-2
    suggested: None
    BEAS-2B
    suggested: BCRJ Cat# 0395, RRID:CVCL_0168)
    Software and Algorithms
    SentencesResources
    Z-series of 16 to 20 images were compressed into two dimensions using the maximum projection of Volocity software (Fig 1, 3, 5, S1, S3B and C, S4C, S5A). 2. A 3D deconvolution microscope (Leica DM-RXA2), equipped with a piezo z-drive (Physik Instrument) and a 100x1.4NA-PL-APO objective lens for optical sectioning.
    Volocity
    suggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)
    3D or 1D multicolor image stacks were acquired using the Metamorph software (MDS) through a cooled CCD-camera (Photometrics Coolsnap HQ) (Fig 2A, 2B, 6C, 7A). 3. A Zeiss Axio Observer microscope with a EC Plan NEOFLUAR
    Metamorph
    suggested: None
    Images were acquired with MetaMorph 7 software (Molecular Devices) and processed with ImageJ (NIH, MD).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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 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.

    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.

  3. Version published to 10.1101/2020.12.19.423586 on bioRxiv