Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline

  1. Tae Yeon Yoo
  2. Timothy Mitchison

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Abstract

SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest. Using this method, we found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting bidirectional nuclear transport, due to differences in the NUP98-binding C-terminal region that is required for the inhibition. The N-terminal region promotes membrane binding and was required for activity, but could be replaced by constructs which forced oligomerization in solution. Based on these data, we propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the FG domains of NUP98 at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

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  1. Version published to 10.1101/2021.12.10.472151v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.12.10.472151: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    AF647-conjugated anti-ALFA single-domain antibody (NanoTag, #N1502-AF647) and anti-NUP98 rabbit monoclonal antibody (C39A3, Cell Signaling, #2598) were diluted by factors of 500 and 50 in 1% BSA/PBS, respectively.
    AF647-conjugated anti-ALFA single-domain antibody
    suggested: None
    AF647-conjugated anti-ALFA single-domain
    suggested: None
    anti-ALFA
    suggested: (Antibodies-Online Cat# ABIN125862, RRID:AB_11186338)
    anti-NUP98
    suggested: (Cell Signaling Technology Cat# 2598, RRID:AB_2267700)
    Then, cells were incubated with goat anti-rabbit secondary antibody conjugated with AF568 (Thermo Fisher Scientific, #A11036
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# A-11036, RRID:AB_10563566)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: U2OS cell lines (engineered from HTB-96, ATCC) were maintained in complete DMEM (low glucose (1g/L) Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher, #10567022) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher, #A31605), 50 IU ml-1 penicillin, and 50 μg ml-1 streptomycin (Thermo Fisher, #15140122)) at 37°C in a humidified atmosphere with 5% CO2.
    U2OS
    suggested: None
    HTB-96
    suggested: None
    Recombinant DNA
    SentencesResources
    The recombinant His6-EGFP used in the GFP intensity calibration (Fig. 1b) was expressed in Rosetta 2 (DE3) competent cells (Millipore Sigma #71400) using pDual-EGFP plasmid (Addgene, #63215)
    pDual-EGFP
    suggested: RRID:Addgene_63215)
    For the calibration, the U2OS stable cell line expressing H2A-Halo was transfected with the GFP-2A-ORF6 plasmid and prepared in an 8-well chamber slide in the same way as in the dose-response characterization.
    GFP-2A-ORF6
    suggested: None
    Software and Algorithms
    SentencesResources
    Oligonucleotides for PCRs were purchased from IDT or Genewiz.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    The imaging cycle was repeated at 3–6-hour interval at different wells, which was fully automated by using Journal macro in MetaMorph Software.
    MetaMorph Software
    suggested: None
    Computational image analysis: Custom Python codes were used for quantitative image analysis of the time-lapse images.
    Python
    suggested: (IPython, RRID:SCR_001658)
    NIS-Elements software was used to control the hardware.
    NIS-Elements
    suggested: (NIS-Elements, RRID:SCR_014329)

    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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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

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  3. Version published to 10.1101/2021.12.10.472151v1 on bioRxiv