The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

  1. Manon Laporte
  2. Annelies Stevaert
  3. Valerie Raeymaekers
  4. Ria Van Berwaer
  5. Katleen Martens
  6. Stefan Pöhlmann
  7. Lieve Naesens

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Abstract

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33°C instead of 37°C. Even stronger preference for the upper airway temperature of 33°C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37°C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37°C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology.

SIGNIFICANCE STATEMENT

The rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33°C) or lower (37°C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.

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    SciScore for 10.1101/2020.11.09.374603: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Ethics statement: Lung tissue samples from eight different healthy donors and nasal tissue samples from one healthy donor and one patient with chronic rhinosinusitis with nasal polyps were obtained under the approval of the ethical committee from the University Hospital Leuven (UZ Leuven Biobanking S51577 and S59864).
    Consent: All patients were adult and provided written informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The primary antibodies were mouse anti-V5 tag [Invitrogen, R960-25, 1:2000 (SARS-1-S, MERS-S and SARS-2-S) or 1:5000 (229E-S)] and mouse anti-β-actin (Sigma-Aldrich, A5447, 1:5000).
    anti-V5
    suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)
    SARS-2-S
    suggested: None
    229E-S
    suggested: None
    anti-β-actin
    suggested: None
    A5447
    suggested: (ABclonal Cat# A5447, RRID:AB_2766249)
    As secondary antibody, we used a peroxidase-coupled goat anti-mouse antibody (Dako, P0447, 1:5000).
    anti-mouse
    suggested: (Agilent Cat# P0447, RRID:AB_2617137)
    P0447
    suggested: (Agilent Cat# P0447, RRID:AB_2617137)
    The samples were heated for 5 min at 95°C and subjected to SDS-PAGE and immunoblotting, as above, with mouse anti-V5 tag (Invitrogen, R960-25) and mouse anti-MLV p30 antibody (Abcam, ab130757) as primary antibodies.
    R960-25
    suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)
    anti-MLV p30
    suggested: (Creative Diagnostics Cat# DCABH-2412, RRID:AB_2476319)
    Experimental Models: Cell Lines
    SentencesResources
    Cells, media and compounds: Calu-3 (ATCC HTB-55) cells were grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 10 mM HEPES.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    HEK293T cells (HCL4517; Thermo Fisher Scientific) and Vero E6 (ATCC CRL-1586) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 0.075% sodium bicarbonate and (only for Vero E6) 0.1 mM non-essential amino acids.
    Vero E6
    suggested: None
    They were then transduced into receptor- and TMPRSS2-transfected HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Statistical analysis was performed using GraphPad Prism (version 8.4.3).
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04455815RecruitingA Trial Looking at the Use of Camostat to Reduce Progression…
    NCT04321096Active, not recruitingThe Impact of Camostat Mesilate on COVID-19 Infection
    NCT04353284RecruitingCamostat Mesylate in COVID-19 Outpatients
    NCT04355052RecruitingOpen Label Study to Compare Efficacy, Safety and Tolerabilit…
    NCT04374019RecruitingNovel Agents for Treatment of High-risk COVID-19 Positive Pa…

    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.

    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 10.1101/2020.11.09.374603 on bioRxiv