Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion
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Abstract
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.
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SciScore for 10.1101/2020.08.13.243303: (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 Contamination: All cells are regularly tested and are mycoplasma free. Table 2: Resources
Antibodies Sentences Resources Alternatively, blots probed with an anti-Alexa Fluor 488 secondary antibody were imaged using a Typhoon biomolecular imager (Cytivia). anti-Alexasuggested: NoneThe primary antibodies used were: anti-furin (1:1000, Abcam #ab3467), anti-furinsuggested: (Abcam Cat# ab3467, RRID:AB_303828)The secondary antibodies were anti-rabbit HRP conjugate (1:10000, Invitrogen 31462), anti-rabbitsuggested: None… SciScore for 10.1101/2020.08.13.243303: (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 Contamination: All cells are regularly tested and are mycoplasma free. Table 2: Resources
Antibodies Sentences Resources Alternatively, blots probed with an anti-Alexa Fluor 488 secondary antibody were imaged using a Typhoon biomolecular imager (Cytivia). anti-Alexasuggested: NoneThe primary antibodies used were: anti-furin (1:1000, Abcam #ab3467), anti-furinsuggested: (Abcam Cat# ab3467, RRID:AB_303828)The secondary antibodies were anti-rabbit HRP conjugate (1:10000, Invitrogen 31462), anti-rabbitsuggested: NoneExperimental Models: Cell Lines Sentences Resources Caco2 BVDV-Npro cell line was generated as described previously (Hosmillo et al., 2015). 293T-hACE2, 293T-hACE2-ΔFURIN, Vero-hACE2 were generated transducing 293T, 293T-ΔFURIN or Vero cells with lentiviral particles expressing hACE2 ORF and cultured in DMEM 10% FCS with 5 μg/ml blasticidin. 293T-hACE2-TMPRSS2 and Vero-hACE2-TMPRSS2 were instead generated transducing 293T-hACE2 and Vero-hACE2 cells respectively with lentiviral particles expressing TMPRSS2 ORF and maintained in DMEM 10% FCS with addition of 5 μg/ml blasticidin and 1 μg/ml puromycin. Caco2 BVDV-Nprosuggested: NoneVerosuggested: NoneVero-hACE2suggested: NoneTiters were assessed by plaque assays in Vero hACE2/TMPRSS2 cells. Vero hACE2/TMPRSS2suggested: NoneVLP production and S analysis: SARS-CoV-2 VLPs were produced by transfecting 2×106 293T-wt cells in T75 flasks with 25 μg total DNA using Fugene6 (Promega). 293T-wtsuggested: NoneSARS-CoV-2 plaque assay: Vero hACE2-TMPRSS2 cells were seeded on 12-well plates day prior infection. Vero hACE2-TMPRSS2suggested: NoneVirus infection: 293T-hACE2 or 293T-hACE2-ΔFURIN cells were infected with MOI of 0.1 or 0.01 and incubated for 42 hours or 72 hours depending on the experiment. 293T-hACE2-ΔFURINsuggested: NoneGenotyping PCR: 293T-ΔTMPRSS2 cells were screened by genotyping PCR. 293T-ΔTMPRSS2suggested: NoneGenomic DNA was extracted from wild-type and ΔTMPRSS2 293T cell lines using the Puregene cell kit according to the manufacturer’s instructions (Qiagen, #158388) 293Tsuggested: NoneSoftware and Algorithms Sentences Resources Data were then analysed using Incucyte software analysis and plotted using Prism 8 software. Prismsuggested: (PRISM, RRID:SCR_005375)Alignments of sequence trace files were generated using Snapgene. Snapgenesuggested: (SnapGene, RRID:SCR_015052)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: 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.
- No funding statement was detected.
- No protocol registration statement was detected.
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