The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways
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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 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.
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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 Statement IACUC: 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.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibodies were mouse anti-V5 tag [Invitrogen, R960-25, 1:2000 … 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 Statement IACUC: 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.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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-V5suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)SARS-2-Ssuggested: None229E-Ssuggested: Noneanti-β-actinsuggested: NoneA5447suggested: (ABclonal Cat# A5447, RRID:AB_2766249)As secondary antibody, we used a peroxidase-coupled goat anti-mouse antibody (Dako, P0447, 1:5000). anti-mousesuggested: (Agilent Cat# P0447, RRID:AB_2617137)P0447suggested: (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. R960-25suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)anti-MLV p30suggested: (Creative Diagnostics Cat# DCABH-2412, RRID:AB_2476319)Experimental Models: Cell Lines Sentences Resources 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-3suggested: 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 E6suggested: NoneThey were then transduced into receptor- and TMPRSS2-transfected HEK293T cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Software and Algorithms Sentences Resources Statistical analysis: Statistical analysis was performed using GraphPad Prism (version 8.4.3). GraphPad Prismsuggested: (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:
Identifier Status Title NCT04455815 Recruiting A Trial Looking at the Use of Camostat to Reduce Progression… NCT04321096 Active, not recruiting The Impact of Camostat Mesilate on COVID-19 Infection NCT04353284 Recruiting Camostat Mesylate in COVID-19 Outpatients NCT04355052 Recruiting Open Label Study to Compare Efficacy, Safety and Tolerabilit… NCT04374019 Recruiting Novel 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.
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