SARS-CoV-2 Envelope protein (E) binds and activates TLR2: A novel target for COVID-19 interventions

  1. Rémi Planès
  2. Jean-Baptiste Bert
  3. Sofiane Tairi
  4. Lbachir Benmohamed
  5. Elmostafa Bahraoui

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Abstract

In this study, we present a molecular characterization of the interaction between the SARS-CoV-2 envelope protein E with TLR2. We demonstrated that E protein interacts physically with TLR2 receptor in a specific and dose-dependent manner. Furthermore, we showed that this interaction is able to engage TLR2 pathway as demonstrated by its capacity to activate NF-κB transcription factor and to stimulate the production of CXCL8 inflammatory chemokine in a TLR2-dependent manner. Furthermore, in agreement with the importance of NF-κB in TLR signaling pathway, we showed that the chemical inhibition of this transcription factor led to significant inhibition of CXCL8 production, while blockade of P38 and ERK1/2 MAP kinases resulted only in a partial CXCL8 inhibition. Overall, our findings suggest considering the envelope protein E as a novel target for COVID-19 interventions: ( i ) either by exploring the therapeutic effect of anti-E blocking/neutralizing antibodies in symptomatic COVID-19 patients, or ( ii ) as a promising non-Spike SARS-CoV-2 antigen candidate to include in the development of next generation prophylactic vaccines against COVID-19 infection and disease.

Importance

Although, the exact mechanisms of COVID-19 pathogenesis are unknown, recent data demonstrated that elevated levels of pro-inflammatory cytokines in serum is associated with enhanced disease pathogenesis and mortality. Thus, determining the molecular mechanisms responsible for inflammatory cytokine production in the course of SARS-CoV-2 infection could provide future therapeutic targets. In this context, to the best of our knowledge, our report is first to use a detailed molecular characterization to demonstrate that SARS-CoV-2 Envelope E protein binds to TLR2 receptor. Specifically, we showed that SARS-CoV-2 Envelope E protein binds to TLR2 in a direct, specific and dose-dependent manner. Investigating signalling events that control downstream activation of cytokine production show that E protein / TLR2 binding leads to the activation of NF-κB transcription factor that control the expression of multiple pro-inflammatory cytokines including CXCL8. Overall, our findings suggest considering the envelope protein E as a novel target for COVID-19 interventions.

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  1. ScreenIT

    SciScore for 10.1101/2021.11.10.468173: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: 2.1 Ethics statement: The use of human cells in this study was approved by the Research Ethical Committee of Haute-Garonne
    Consent: Written informed consent was obtained from the donors under EFS contract N° 21/PVNT/TOU/INSERM01/2011-0059, according to French Decree N° 2007–1220 (articles L1243-4, R1243-61).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    2.6 Chemical products, Proteins, and Antibodies: PAM2CSK4, PAM3CSK4, LPS-RS were purchased from InvivoGen
    Antibodies
    suggested: None
    LPS-RS
    suggested: None
    Anti-TLR2 and anti-TLR4 monoclonal antibodies were obtained from eBioscience.
    Anti-TLR2
    suggested: None
    anti-TLR4
    suggested: None
    After 5 further washes, the complexes TLR2-E-GST-anti-GST were labeled by 1 hour incubation at room temperature with 100 μl of anti-rabbit IgG antibodies coupled to horseradish peroxidase in PBS-tween 0.05% containing 5% non-fat milk (DAKOTA).
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    2.2 Cells: Human embryonic kidney cell lines stably transfected with TLR2 (HEK-TLR2), TLR4 (HEK-TLR4) and HEK-TLR2-blue and control HEK cell line (HEK-null) were purchased from InvivoGen and cultured in DMEM supplemented with 10 % FCS, 1% of P/S and selections antibiotics according to the manufacturer’s instructions (InvivoGen).
    HEK
    suggested: None
    Vero E6 and A549 cell lines were cultured in DMEM supplemented with 10% FCS and 1% of P/S. 2.3 Virus infection:
    Vero E6
    suggested: None
    A549
    suggested: None
    2.11 Cell based biological assays: Primary human monocytes or macrophages cells (106 cells) or HEK-null, HEK-TLR2 or HEK-TLR4 cell lines (2,5. 105 cells) were plated in 24 well plates and treated by E protein or PAM3CSK4 and PAM2CSK2 as positive controls at the indicated concentrations.
    HEK-TLR4
    suggested: RRID:CVCL_VI44)
    To inhibit the binding of E protein to cell membrane TLR2, E protein (at 200ng/ml) was preincubated with rTLR2 (20 ng/ml) during 1 hour at RT, before being added to HEK-TLR2 cells.
    HEK-TLR2
    suggested: RRID:CVCL_IM79)
    Software and Algorithms
    SentencesResources
    Data were acquired using FACSCalibur (BD).
    FACSCalibur
    suggested: None
    2.15 Statistical analyses: Statistical analysis was performed using GraphPad Prism software v.5.
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 26. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

  2. Version published to 10.1101/2021.11.10.468173 on bioRxiv