The Spike Protein of SARS-CoV-2 Impairs Lipid Metabolism and Increases Susceptibility to Lipotoxicity: Implication for a Role of Nrf2

  1. Vi Nguyen
  2. Yuping Zhang
  3. Chao Gao
  4. Xiaoling Cao
  5. Yan Tian
  6. Wayne Carver
  7. Hippokratis Kiaris
  8. Taixing Cui
  9. Wenbin Tan

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Abstract

Coronavirus disease 2019 (COVID-19) patients show lipid metabolic alterations, but the mechanism remains unknown. In this study, we aimed to investigate whether the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs lipid metabolism in host cells. We generated a Spike cell line in HEK293 using the pcDNA vector carrying the Spike gene expression cassette. A control cell line was generated using the empty pcDNA vector. Gene expression profiles related to lipid metabolic, autophagic, and ferroptotic pathways were investigated. Palmitic acid (PA)-overload was used to assess lipotoxicity-induced necrosis. As compared with controls, the Spike cells showed a significant increase in lipid depositions in cell membranes as well as dysregulation of expression of a panel of molecules involving lipid metabolism, autophagy, and ferroptosis. The Spike cells showed an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a multifunctional transcriptional factor, in response to PA. Furthermore, the Spike cells exhibited increased necrosis in response to PA-induced lipotoxicity compared to control cells in a time- and dose-dependent manner via ferroptosis, which could be attenuated by the Nrf2 inhibitor trigonelline. We conclude that the Spike protein impairs lipid metabolic and autophagic pathways in host cells, leading to increased susceptibility to lipotoxicity via ferroptosis which can be suppressed by a Nrf2 inhibitor. This data also suggests a central role of Nrf2 in Spike-induced lipid metabolic impairments.

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  1. Version published to 10.3390/cells11121916
  2. ScreenIT

    SciScore for 10.1101/2022.04.19.488806: (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
    16 Anti-His tag and microtubule-associated protein 1 light chain 3 beta (LC3) Antibodies were purchased from Millipore Sigma (Burlington, MA, USA).
    16 Anti-His tag
    suggested: None
    microtubule-associated protein 1 light chain 3 beta (LC3) Antibodies
    suggested: None
    Anti-Adipose Differentiation-Related Protein (ADRP, or Perilipin-2, PLIN2), Nrf2, prostaglandin E synthase 2 (PTGS2), and PI3K-beta antibodies were obtained from ProteinTech (Rosemont, IL, USA).
    Anti-Adipose Differentiation-Related Protein ( ADRP
    suggested: None
    PLIN2
    suggested: None
    Nrf2
    suggested: None
    PTGS2
    suggested: None
    PI3K-beta
    suggested: None
    Anti-ATG7 antibody was purchased from Abcam (Waltham, MA, USA).
    Anti-ATG7
    suggested: (Abcam Cat# 2054-1, RRID:AB_991677)
    Anti-SRB1 antibody was obtained from Novus (Centennial, CO, USA).
    Anti-SRB1
    suggested: None
    Anti-Fth1, HRP-anti-rabbit or mouse secondary antibodies, and RIPA lysis buffer were obtained from Santa Cruz Biotech (Dallas, TX, USA)
    Anti-Fth1 , HRP-anti-rabbit or mouse secondary antibodies
    suggested: None
    Anti-Fth1 ,
    suggested: None
    HRP-anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Palmitic acid (PA)-induced lipotoxicity assay: The HEK293, HEK_pcDNA and HEK_Spike cells were cultured in a 96-well plate and reached 80% confluence on the next day before treatment.
    HEK293
    suggested: None
    HEK_Spike
    suggested: None
    H9C2 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (10% FBS) medium in a 96 well-plate with 80% confluence.
    H9C2
    suggested: None
    Recombinant DNA
    SentencesResources
    16 The sequence was cloned into a pcDNA3.1 vector to obtain pcDNA-Spike.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    The individual colonies with stable integration of the pcDNA-Spike (HEK_Spike) or pcDNA vector (HEK_pcDNA) were selected and expanded.
    pcDNA-Spike
    suggested: None
    pcDNA
    suggested: RRID:Addgene_66792)
    Viral production and H9C2 cell culture: The Spike gene was cleaved from pcDNA-Spike plasmid and cloned into lentiviral vector pLV-mCherry (Addgene, Watertown, MA, USA) with removal of mCherry gene to generate pLV-Spike plasmid.
    pLV-Spike
    suggested: None
    The control virus with VSV-G as the tropism and expression of mCherry was generated by co-transfection of pLV-mCherry and pMD2.G vector (Addgene, Watertown, MA, USA) into the Phoenix cells, which was referred to as VSV-G virus.
    pLV-mCherry
    suggested: RRID:Addgene_36084)
    pMD2.G
    suggested: RRID:Addgene_12259)

    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.

    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.

  3. Version published to 10.1101/2022.04.19.488806v1 on bioRxiv