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. 2021 Jan 25;17(1):e1009246.
doi: 10.1371/journal.ppat.1009246. eCollection 2021 Jan.

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

Guido Papa  1 , Donna L Mallery  1 , Anna Albecka  1 , Lawrence G Welch  1 , Jérôme Cattin-Ortolá  1 , Jakub Luptak  1 , David Paul  1 , Harvey T McMahon  1 , Ian G Goodfellow  2 , Andrew Carter  1 , Sean Munro  1 , Leo C James  1
Affiliations

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

Guido Papa et al. PLoS Pathog. .

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SARS-CoV-2 S protein mediates cell-cell fusion between different cell types.
(A) Schematic representation of S protein-mediated cell–cell fusion assay. The donor cell is identified as the cell co-expressing SARS-CoV-2 S and mCherry while the acceptor cell is green-labelled. The scheme was created with BioRender.com. (B) Merged images at 24 hours post transfection of the indicated cell lines transfected with SARS-CoV-2 S and mixed with green dye-labelled cells. Scale bars represent 200 μm. Green colour identifies the acceptor cells while red colour marks donor cells. Merged green-red colours indicate the syncytia. (C) Quantification of (B) showing percentage of green and red overlap area at 24 hours post transfection. Statistical analysis was performed using Student t-test. *** P<0.001; ** P<0.01 (D) Quantification of the cell-cell fusion kinetics shown in (B). Acceptor cells are marked in bold and italics.
Fig 2
Fig 2. Furin is not essential for the cleavage of S protein but enhances its processing.
(A) Schematic illustration of SARS-CoV-2 S including receptor binding domain (RBD) in green and proteolytic cleavage sites (S1/S2, S2’). Amino acid sequences around the S1/S2 recognition sites of SARS-CoV-2 S are indicated while the multibasic site is highlighted in purple. Amino acid mutations are highlighted in light blue while deletions are marked with orange dashes. (B) Overall structure of the SARS-CoV-2 S protein (PDB: 6VYB). RBD core is shown in green. Pro-Arg-Arg-Ala-Arg residues are shown in yellow. (C,D) Representative western blots of HIV Pseudoviruses (C) and Virus Like Particles (VLPs) (D) harbouring the indicated SARS-CoV-2 S protein mutants (detected with anti-S antibody) and produced in 293-wt and 293T-ΔFURIN cells. Expression of HIV capsid protein (p24) (C) and SARS-CoV-2 nucleoprotein (D) is shown as loading control. (E) Representative western blot analysis of spike and nucleoprotein present in SARS-CoV-2 viral particles produced in 293T-hACE2 and 293T-hACE2-ΔFURIN after 42 hours post infection. The cleaved S in (C) (D) and (E) identifies the S2 subunit.
Fig 3
Fig 3. Processing of the S protein multibasic site is essential for cell-cell fusion but furin is dispensable.
(A) and (D) Reconstructed images of the indicated cells lines transfected with the indicated S mutants and mixed with green-labelled cells at 24 hours post transfection. Scale bar 200 μm. Green colour identifies the acceptor cells while red colour marks donor cells. Merged green-red colours indicate the syncytia. (B) Quantification of green-red overlap area shown in (A) *P<0.05; **, P<0.01 analysed using Student t-test. (C) Time course of cell-cell fusion shown in (A). (E) Cell-cell fusion time course of images shown in (D). Data are expressed as mean +/- SEM (n = 2). Acceptor cells are marked in bold and italics.
Fig 4
Fig 4. Furin enhances SARS-CoV-2 replication but is not essential for S-pseudovirus entry.
(A) Infection of 293T-hACE2 cells with GFP expressing HIV pseudotyped with SARS-CoV-2 S mutants, measured as proportion of cell area expressing GFP. Viruses were produced in either 293T-wt or 293T-ΔFURIN cells. (B) and (C) Infection data of 293T-hACE2 cells with HIV pseudotyped with SARS-CoV-2 S mutants as in (A), with cells pre-treated for 2 hours with either DMSO or 25μM lysosomal inhibitor E64d as indicated. Statistical analysis was performed using Student t-test *P<0.05; **P<0.01, “ns” not significant. (D) Representative western blot of viral particles produced from SARS-CoV-2 infection of 293T-hACE2 and 293T-hACE2-ΔFURIN cells at 72 hours post infection. Spike and nucleoprotein are detected (left panel). Total protein content of virus preparation by Coomassie staining (right panel). (E) Plaque assay of Vero-hACE2-TMPRSS2 infected with viruses produced in D. (F) and (G) Immunofluorescence images displaying infection of Caco2 BVDV-Npro cells (F) and 293T-hACE2 (G) with equalised amounts of SARS-CoV-2 virus produced in 293T-hACE2 and 293T-hACE2-ΔFURIN cells. Spike (green) and nuclei (blue) are shown. Scale bar, 50 μm. (H) and (I) Quantification of SARS-CoV-2 infected cells shown in (F) and (G). *P<0.05; ***, P<0.001 analysed using Student t-test. Data are expressed as mean +/- SEM (n = 2).
Fig 5
Fig 5. TMPRSS2 protease is required on acceptor cells to trigger cell-cell fusion.
(A) Merged images of the indicated cells lines expressing the indicated S mutants and mixed with green-labelled cells at 24 hours post transfection. Scale bar 200 μm. Green colour identifies the acceptor cells while red colour marks donor cells. Merged red-green colours indicate the syncytia. (B) Quantification of percentage of green-red overlap area shown in (A) at 24 hours post transfection. Statistical analysis was performed using Student t test. **P<0.01. (C) Time course of cell-cell fusion as in (B). Data are expressed as mean +/- SEM (n = 2). Acceptor cells are marked in bold and italics.
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