The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells
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Abstract
SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2’ cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.
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SciScore for 10.1101/2020.09.30.318311: (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 Outbred female ferrets (16-20 weeks old), weighing 750-1000 g were used. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Near infra-red (NIR) secondary antibodies, IRDye® 680RD Goat anti-mouse (abcam; ab216776), IRDye® anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Human epithelial colorectal adenocarcarcinoma cells (Caco-2) and human lung cancer cells (Calu-3) were maintained in DMEM, 20% FCS, 1% NEAA, 1% P/S. VeroE6/TMPRSS2 cells were obtained from the Centre for AIDS Reagents (National … SciScore for 10.1101/2020.09.30.318311: (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 Outbred female ferrets (16-20 weeks old), weighing 750-1000 g were used. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Near infra-red (NIR) secondary antibodies, IRDye® 680RD Goat anti-mouse (abcam; ab216776), IRDye® anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Human epithelial colorectal adenocarcarcinoma cells (Caco-2) and human lung cancer cells (Calu-3) were maintained in DMEM, 20% FCS, 1% NEAA, 1% P/S. VeroE6/TMPRSS2 cells were obtained from the Centre for AIDS Reagents (National Institute for Biological Standards and Control) (Matsuyama et al., 2020; Nao et al., 2019), and maintained in DMEM, 10% FCS, 1% NEAA, 1% P/S, 1 mg/ml Geneticin (G418). VeroE6/TMPRSS2suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)The WT SARS-CoV-2 strain SARS-CoV-2 strain England/2/2020 (VE6-T) and the ΔCS mutant present in the original mixed stock were purified by serially diluting the stock (10-fold dilutions) in MEM supplemented with 2% FCS and adding the dilutions to either Vero E6 or Caco-2 cells in a 96 well plate. Caco-2suggested: NoneBriefly, 10cm2 dishes of 293T cells were co-transfected with a mixture of 1 μg of the HIV packaging plasmid pCAGGs-GAGPOL, 1.5 μg of the luciferase reporter construct, pCSLW and 1 μg of each envelope protein in pcDNA3.1. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Serum dilutions were incubated with 100 TCID50/well of virus in assay diluent for 1 h at RT and transferred to 96-well plates pre-seeded with Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Recombinant DNA Sentences Resources TMPRSS2 expression plasmid was a kind gift from Roger Reeves (Addgene plasmid #53887; http://n2t.net/addgene:53887; RRID:Addgene_53887,(Edie et al., 2018)). detected: RRID:Addgene_53887)Software and Algorithms Sentences Resources Sequences were analysed in Geneious (v11) and a pipeline in R. Geneioussuggested: (Geneious, RRID:SCR_010519)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Our study also confirms the limitations of relying on Vero E6 cells as a system for developing classes of drugs such as entry inhibitors as they do not accurately reflect the preferred entry mechanism of SARS-CoV-2 into human airway cells (Hoffmann, Mosbauer, et al., 2020; T. Ou et al., 2020). Indeed the data here explains why chloroquine is ineffective in clinic against SARS-CoV-2 (Hoffmann, Mosbauer, et al., 2020), since during replication in the human airway WT SARS-CoV-2 has evolved to enter cells without the need for endosomal acidification. Monitoring wild coronaviruses will likely be important in predicting and preventing future pandemics. We suggest that a furin CS in the SARS lineage is a cause for concern. The polybasic insertion to the S1/S2 CS provides a significant fitness advantage in TMPRSS2 expressing cells and is likely essential for efficient human transmission.
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… 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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