A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Abstract

The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.

Keywords: COVID-19; S1/S2; SARS-CoV-2; TMPRSS2; cleavage; entry; furin; membrane fusion; spike.

Graphical abstract

Figure 1

The Multibasic Motif at the S1/S2 Cleavage Site of SARS-2-S Is Unique among Related Group 2b Betacoronaviruses (A) Schematic illustration of a coronavirus spike glycoprotein in which functional domains and cleavage sites are highlighted (RBD, receptor-binding domain; RBM, receptor-binding motif; TD, transmembrane domain). (B) Protein models for SARS-S and SARS-2-S based on the PDB: 5X5B structure (Yuan et al., 2017) as a template. Colored in red are the S1/S2 and S2′ cleavage sites. Further, the S1 subunit (blue), including the RBD (purple), and the S2 subunit (gray) are depicted. (C and D) Amino acid sequence alignment of residues around the S1/S2 and S2′ cleavage sites of group 2b betacoronaviruses found in humans, civet cats, raccoon dog, pangolin, and bats (C) or coronaviruses that are able to infect humans (D). Basic amino acid residues are highlighted in red, while gray boxes mark the presence of multibasic motifs. Numbers refer to amino acid residues (n/a, no information available). The symbol “^∗” refers to amino acid residues that are conserved among all tested sequences, while the symbols “:” and “.” indicate positions with heterogeneous amino acid residues that share highly similar or similar biochemical properties.

Figure 2

The Multibasic S1/S2 Site of SARS-2-S Is Cleaved by Furin, and Cleavage Is Required for Syncytium Formation and Entry into Human Lung Cells (A) Overview of the SARS-S and SARS-2-S S1/S2 mutants analyzed. (B) Analysis of furin-mediated S protein priming. Rhabdoviral particles harboring the indicated S proteins containing a C-terminal V5 tag for detection were lysed and subjected to western blot analysis. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as control. (C) Rhabdoviral particles bearing MERS-S, SARS-S, or SARS-2-S equipped with a V5 or HA epitope tag at their C terminus (or no glycoprotein at all, control) were produced in the absence or presence of furin inhibitor (FI, decanoyl-RVKR-CMK; 1 μM or 10 μM) and analyzed for S protein processing by western blot analysis. Detection of VSV-M served as control. (D) Syncytium formation assay: Vero or Vero-TMPRSS2 cells were transfected to express the indicated S proteins (or no S protein, empty vector, control). At 24 h post transfection, cells were incubated in the presence or absence of trypsin (1 μg/mL) for an additional 24 h before they were fixed, stained with May-Gruenwald and Giemsa solution, and analyzed by bright field microscopy (scale bars, 200 μm). White arrowheads indicate syncytia. For (B)–(D), representative data from three (B and C) or four (D) independent experiments are shown. (E) Transduction of Vero (TMPRSS2⁻) and Calu-3 (TMPRSS2⁺) cells with rhabdoviral particles bearing the indicated S proteins or vesicular stomatitis virus glycoprotein (VSV-G). At 16 h post transduction, virus-encoded firefly luciferase was quantified in cell lysates. Presented are the mean data from three independent experiments. Transduction efficiency is shown relative to that measured for particles not bearing a viral glycoprotein. Error bars indicate the standard error of the mean. Statistical significance was tested by one-way analysis of variance with Dunnett’s post test (p > 0.05, ns; ^∗∗∗p ≤ 0.001).