Closing coronavirus spike glycoproteins by structure-guided design
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Abstract
The recent spillover of SARS-CoV-2 in the human population resulted in the ongoing COVID-19 pandemic which has already caused 4.9 million infections and more than 326,000 fatalities. To initiate infection the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface, determining host and tissue tropism, and fusion of the viral and host membranes. Although SARS-CoV- 2 S is the main target of neutralizing antibodies and the focus of vaccine design, its stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a prefusion SARS-CoV-2 S ectodomain trimer construct covalently stabilized in the closed conformation. Structural and antigenicity analysis showed we successfully shut S in the closed state without otherwise altering its architecture. Finally, we show that this engineering strategy is applicable to other β-coronavirus S glycoproteins and might become an important tool for vaccine design, structural biology, serology and immunology studies.
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SciScore for 10.1101/2020.06.03.129817: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. 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
Experimental Models: Cell Lines Sentences Resources Recombinant S ectodomains production: All ectodomains were produced in 500mL cultures of HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37°C in a humidified 8% CO2 incubator rotating at 130 r.p.m., as previously reported18. HEK293Fsuggested: NoneHEK293T cells stably expressing human ACE270 were cultured in DMEM containing 10% FBS, 1% PenStrep and plated into 96 well plates for 16-24 hours. …SciScore for 10.1101/2020.06.03.129817: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. 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
Experimental Models: Cell Lines Sentences Resources Recombinant S ectodomains production: All ectodomains were produced in 500mL cultures of HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37°C in a humidified 8% CO2 incubator rotating at 130 r.p.m., as previously reported18. HEK293Fsuggested: NoneHEK293T cells stably expressing human ACE270 were cultured in DMEM containing 10% FBS, 1% PenStrep and plated into 96 well plates for 16-24 hours. HEK293Tsuggested: NoneSoftware and Algorithms Sentences Resources Measurements were done in duplicate and relative luciferase units (RLU) were converted to percent neutralization and plotted with a non-linear regression curve fit in PRISM. PRISMsuggested: (PRISM, RRID:SCR_005375)Data were acquired using the Leginon software71 to control a Tecnai T12 transmission electron microscope operated at 120 kV and equipped with a Gatan 4K Ultrascan CCD detector. Leginonsuggested: (Leginon, RRID:SCR_016731)CryoEM model building and analysis: UCSF Chimera77 and Coot were used to fit an atomic model (PDB 6VXX) into the cryoEM map. Cootsuggested: (Coot, RRID:SCR_014222)Results from OddPub: Thank you for sharing your data.
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 NCT03049488 Completed Dose, Safety, Tolerability and Immunogenicity of a Stabilize… 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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