Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages

  1. Nisha Kriplani
  2. Sara Clohisey
  3. Sonia Fonseca
  4. Sarah Fletcher
  5. Hui-Min Lee
  6. Jordan Ashworth
  7. Dominic Kurian
  8. Samantha J. Lycett
  9. Christine Tait-Burkard
  10. J. Kenneth Baillie
  11. Mark E. J. Woolhouse
  12. Simon R. Carding
  13. James P. Stewart
  14. Paul Digard

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-κB signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.13.456266: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Primary human CD14+ monocytes were isolated from fresh blood from volunteer donors under ethical approval from the Lothian Research Ethics Committee (11/AL/0168) as described before [34].
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were blocked using 1% BSA in PBS-T at room temperature for 1 hour and then incubated with primary antibodies, anti-FLAG (Cell Signalling Technologies, 2908S), anti-SARS-CoV-2-ORF8 (MRCPPU, University of Dundee, DA088, [37]) and anti-Tubulin (BioRad, MCA77G).
    anti-SARS-CoV-2-ORF8
    suggested: None
    anti-Tubulin
    suggested: (Bio-Rad Cat# MCA77G, RRID:AB_325003)
    Donkey anti-rabbit IRDye 800 and goat anti-rat IRDye 680 (Li-cor Biosciences) secondary antibodies were used to detect bound IgG, followed by imaging on a Licor Odyssey instrument.
    anti-rabbit
    suggested: (LI-COR Biosciences Cat# 926-32223, RRID:AB_621845)
    anti-rat IRDye 680
    suggested: (LI-COR Biosciences Cat# 926-32229, RRID:AB_1850020)
    Primary antibodies, anti-FLAG (Cell Signalling, 2368S) and anti-Calreticulin (Abcam, ab2907), were used at 1:800 dilution, Alexa Fluorconjugated secondary antibodies (Donkey α-Mouse Alexa-Fluor 488 for Flag and Donkey αMouse Alexa-Fluor 594 for Calreticulin, Life Technologies) were used at 1:2000 and nuclei were stained using Hoechst 33342 at 1:5000.
    anti-FLAG
    suggested: (Cell Signaling Technology Cat# 5407, RRID:AB_1950473)
    anti-Calreticulin
    suggested: (Abcam Cat# ab2907, RRID:AB_303402)
    Experimental Models: Cell Lines
    SentencesResources
    For treatment of X293T or A549 cells, supernatants were diluted 1:2 with fresh medium before adding to cells, for MDMs, supernatants were diluted 1:3.
    X293T
    suggested: None
    For treatment with poly I:C (Invivogen), cells were transfected with 2.5 µg (A549 cells) or 15 µg (MDMs) using Lipofectamine (Life Technologies) according to standard protocols.
    A549
    suggested: None
    Vero E6 cells were cultured in DMEM (Lonza), 10% HI FBS (Gibco), 1X ultraglutamine (Lonza), and 1X non-essential amino acids solution (Invitrogen) and infected (at confluence) with passage 2 SARS-CoV-2 EDB-2 at an MOI of 1.
    Vero E6
    suggested: RRID:CVCL_XD71)
    24 hours later, 20 µl of supernatants from the HEK-Blue™ or THP1-Blue™ cells were mixed with 180 µl of QuantiBlue reagent (InvivoGen) and optical density (O.D.) was measured at 650nm using a plate reader (CLARIOstar, BMG Labtech).
    THP1-Blue™
    suggested: RRID:CVCL_X588)
    Recombinant DNA
    SentencesResources
    Both untagged and tagged versions of ORF8 cDNAs were subcloned into a strong expression vector pVR1255 [35] by replacing a Firefly luciferase gene between NotI and BamHI restriction sites.
    pVR1255
    suggested: None
    Software and Algorithms
    SentencesResources
    For antagonist experiments IFNβ or lipopolysaccharide (LPS; Sigma Aldrich, L2630) were added at indicated concentrations.
    LPS; Sigma Aldrich
    suggested: None
    Statistical tests (one-way ANOVA using repeated measures between samples from the same experiment or donor as appropriate, or mixed-effects analysis in cases where sample loss had occurred, followed by Dunnett’s multiple comparison tests) were performed using Graphpad Prism 8.4.3.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Colour channels were adjusted individually using Adobe Photoshop during figure preparation to ensure visibility and the images were cropped to increase visibility of the chosen cells.
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)

    Results from OddPub: Thank you for sharing your data.

    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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  2. Version published to 10.1101/2021.08.13.456266v2 on bioRxiv
  3. Version published to 10.1101/2021.08.13.456266v1 on bioRxiv