SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation

  1. Taiwei Li
  2. Adam D. Kenney
  3. Helu Liu
  4. Guillaume N. Fiches
  5. Dawei Zhou
  6. Ayan Biswas
  7. Jianwen Que
  8. Netty Santoso
  9. Jacob S. Yount
  10. Jian Zhu

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.26.445787: (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
    Compounds and antibodies: Recombinant human TNF-α (Cat. # 554618) was purchased from BD.
    TNF-α
    suggested: None
    Anti-V5 (Cat. # R960-25)
    Anti-V5
    suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)
    Anti-GAPDH antibody (Cat. # sc-32233) was purchased from Santa Cruz Biotechnology. Anti-FLAG (Cat. # 2368) antibody, anti-H3 antibody (Cat. # 9715S), and goat HRP-conjugated anti-rabbit IgG antibody (Cat. # 7074) were purchased from Cell Signaling Technology.
    Anti-FLAG
    suggested: None
    anti-H3
    suggested: None
    anti-rabbit IgG
    suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)
    Anti-IL8 antibody (Cat. # 554717) was purchased from BD. Biosciences. Plasmids: pLEX-IMPDH2-V5 vector was picked from the MISSION TRC3 human LentiORF library from Sigma-Aldrich.
    Anti-IL8
    suggested: None
    Anti-GAPDH and anti-histone H3 immunoblotting were used as internal controls to determine the cytoplasmic and nuclear fractions.
    Anti-GAPDH
    suggested: None
    A normal mouse IgG antibody (Cat. # sc-2025, Santa Cruz) was used as the control in parallel.
    mouse IgG
    suggested: (Santa Cruz Biotechnology Cat# sc-2025, RRID:AB_737182)
    Slides were incubated with an anti-IL-8 antibody (Cat. # 550419, BD Pharmingen™) in 5% NGS with PBS at 4°C for overnight.
    anti-IL-8
    suggested: (BD Biosciences Cat# 550419, RRID:AB_393672)
    Slides were washed with PBST and incubated with Alexa 488 coated goat anti-mouse antibody in 5% NGS/PBS for 2 h at room temperature.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549 cells (Cat. # CCL-185, ATCC) were cultured in F12K medium (Cat. # 21127030, Gibco™).
    A549
    suggested: None
    HEK293T cells stably expressing ACE2-GFP were previously described (28).
    HEK293T
    suggested: None
    A549-ACE2 cells were obtained through BEI Resources, NIH, NIAID (Cat # NR53821).
    A549-ACE2
    suggested: None
    A WT virus plaque was then propagated on Vero E6 cells stably expressing TMPRSS2 (kindly provided by Dr. Shan-Lu Liu, Ohio State University) for 72 h.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    The pcDNA-FLAG-V5-Nsp10/14/16 vectors were constructed from pDONR223 SARS-CoV-2 Nsp10 (Cat. # 141264, Addgene), Nsp14 (Cat. # 141267, Addgene), and Nsp16 (Cat. # 141269, Addgene) vectors to the pcDNA3.1-3xFLAG-V5-ccdB (Cat. # 87064, Addgene) destination vector using Gateway™ LR Clonase™ II Enzyme Mix (Cat. # 11791020, Invitrogen).
    pcDNA-FLAG-V5-Nsp10/14/16
    suggested: None
    pDONR223
    suggested: RRID:Addgene_60532)
    pcDNA3.1-3xFLAG-V5-ccdB
    suggested: RRID:Addgene_87064)
    pEZY-FLAG-Nsp14 vector was constructed from pDONR223 SARS-CoV-2 Nsp14 vector to the pEZY-FLAG (Cat # 18700, Addgene) destined vector.
    pEZY-FLAG-Nsp14
    suggested: None
    pDONR223 SARS-CoV-2 Nsp14
    suggested: RRID:Addgene_141267)
    SARS-CoV-2
    suggested: RRID:Addgene_164583)
    pEZY-FLAG
    suggested: None
    The pLEX-FLAG-V5 vector was constructed by cloning the FLAG sequence to the pLEX-307 (Cat # 41392, Addgene) vector.
    pLEX-FLAG-V5
    suggested: None
    pLEX-307
    suggested: None
    The pNF-κB-luciferase vector (PRDII4–luc in the pGL3 vector) was the gift from Dr. Jacob Yount’s lab (61).
    pNF-κB-luciferase
    suggested: None
    pGL3
    suggested: RRID:Addgene_48743)
    The pIRES-luciferase vector (Cat. # 219092) was acquired from Agilent Technologies.
    pIRES-luciferase
    suggested: None
    The pRL-TK Renilla Luciferase vector (Cat. # AF025846) was purchased from Promega.
    pRL-TK
    suggested: RRID:Addgene_11313)
    Luciferase reporter assays: HEK293T cells were transfected with ISRE or NF-κB luciferase vector along with pRL-TK renilla luciferase vector with or without the indicated vector expressing Nsp14.
    pRL-TK renilla luciferase
    suggested: None
    Nuclear and cytoplasmic extraction: HEK293T cells were transfected by pcDNA-FLAG-V5-Nsp14 or control vector pLex307-FLAG-V5 for 24h and changed fresh completed DMEM medium for further 24 h culture.
    pcDNA-FLAG-V5-Nsp14
    suggested: None
    pLex307-FLAG-V5
    suggested: None
    Software and Algorithms
    SentencesResources
    Luciferase/renilla signal intensity was detected using Biotek Cytation5 and analyzed by GEN5 software (Biotek).
    GEN5
    suggested: (Gen5, RRID:SCR_017317)
    Data were analyzed using FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistics: Statistical analysis was performed using the GraphPad PRISM.
    GraphPad PRISM
    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

    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 10.1101/2021.05.26.445787v1 on bioRxiv