Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism

  1. Hoi Tong Wong
  2. Victoria Cheung
  3. Daniel J. Salamango

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Abstract

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds nuclear pore component Nup98–Rae1 to inhibit nuclear translocation of activated STAT1 and IRF3 transcription factors. However, this direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6–Nup98 interaction domain robustly blocked nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral membrane protein, as opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.

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  1. Version published to 10.1242/jcs.259666
  2. ScreenIT

    SciScore for 10.1101/2021.12.06.471415: (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
    After blocking, samples were incubated with primary anti-STAT1 antibody (Cell Signaling Technology, 9172) in PBST with 5% BSA overnight at 4 degrees.
    anti-STAT1
    suggested: None
    The next day, samples were washed 3 times with PBS and then incubated with secondary anti-Rabbit-AlexaFluor488 (Cell Signaling Technology, 4412) and anti-mCherry-AlexaFluor594 (Invitrogen #M11240) antibodies in PBS with 5% BSA for 1 hour at room temperature.
    anti-Rabbit-AlexaFluor488 ( Cell Signaling Technology , 4412 )
    suggested: None
    anti-mCherry-AlexaFluor594
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To generate the A549 eGFP-KPNA2 stable cell line, roughly 250,000 HEK293FT cells in a 6-well plate were transfected with a pQCXIH retroviral vector containing the eGFP-KPNA2 expression cassette, an MLV-GagPol packaging vector, and a VSV-G vector.
    A549 eGFP-KPNA2
    suggested: None
    HEK293FT
    suggested: RRID:CVCL_6911)
    Media was harvested 48 hours post-transfection and frozen at −80°C for 4-6 hours, thawed and centrifuged at 1500 x g, and overlaid on A549 cells.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    In addition, localization experiments were carried out using HeLa cells as they are routinely used for microscopy experiments and localization studies due to their morphological characteristics.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Recombinant DNA
    SentencesResources
    To generate the A549 eGFP-KPNA2 stable cell line, roughly 250,000 HEK293FT cells in a 6-well plate were transfected with a pQCXIH retroviral vector containing the eGFP-KPNA2 expression cassette, an MLV-GagPol packaging vector, and a VSV-G vector.
    pQCXIH
    suggested: RRID:Addgene_37104)
    VSV-G
    suggested: RRID:Addgene_138479)
    The IFNβ reporter construct was generated by cloning an IFN responsive promoter element upstream of eGFP in the pcDNA-5TO expression vector and the constitutively active RIG-I mutant vector was generated by cloning RIG-I amino acids 1-242 into an mTagBFP2-T2A-expression cassette in a lentiviral vector.
    pcDNA-5TO
    suggested: None
    RIG-I
    suggested: None
    Software and Algorithms
    SentencesResources
    At 48 hours post-transfection, eGFP fluorescence was quantified (ImageJ software) and then graphed with Prism 6.0 (GraphPad
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    For quantification of nuclear fluorescence, individual cells expressing the indicated ORF6 proteins were scored for KPNA2 or STAT1 localization by dividing the nuclear fluorescence intensity by the cytoplasmic fluorescence intensity (n=50 for KPNA2 and n=25 for STAT1), and then graphed with Prism 6.0 (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

    About SciScore

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  3. Version published to 10.1101/2021.12.06.471415 on bioRxiv