A second functional furin site in the SARS-CoV-2 spike protein

Abstract

The ubiquitously-expressed proteolytic enzyme furin is closely related to the pathogenesis of SARS-CoV-2 and therefore represents a key target for antiviral therapy. Based on bioinformatic analysis and pseudovirus tests, we discovered a second functional furin site located in the spike protein. Furin still increased the infectivity of mutated SARS-CoV-2 pseudovirus in 293T-ACE2 cells when the canonical polybasic cleavage site (682-686) was deleted. However, K814A mutation eliminated the enhancing effect of furin on virus infection. Furin inhibitor prevented infection by 682-686-deleted SARS-CoV-2 in 293T-ACE2-furin cells, but not the K814A mutant. K814A mutation did not affect the activity of TMPRSS2 and cathepsin L but did impact the cleavage of S2 into S2' and cell-cell fusion. Additionally, we showed that this functional furin site exists in RaTG13 from bat and PCoV-GD/GX from pangolin. Therefore, we discovered a new functional furin site that is pivotal in promoting SARS-CoV-2 infection.

Keywords: S2’ cleavage; SARS-CoV-2; cell–cell fusion; furin; infectivity; pseudovirus.

Figure 1.

Schematic of furin cleavage sites. A. Schematic of SARS-CoV-2 S protein. The alignment of SARS-CoV and SARS-CoV-2 at S1/S2 and S2′ sites was shown in the lower panel. NTD, N-terminal domain; RBM, receptor-binding motif; FP, fusion peptide; TM, transmembrane domain; CT, C-terminal endodomain. B. Identification of additional furin cleavage sites within the SARS-CoV-2 S protein. ProP 1.0 server (www.cbs.dtu.dk/services/ProP/) was used to carry out the prediction using the furin-specific prediction as the default. C. Schematic of three furin cleavage site predicted by ProP1.0.

Figure 2.

Validation of furin activation sites. A–C. Infectivity analysis of SARS-CoV-2 mutants at F1, F2, and F3 sites. Normalized chemiluminescence signals (in RLUs) in 293T-ACE2-furin cells were calculated by comparing with 293T-ACE2 cells. D. Comparison of F1 and F2 single and double mutations. E. ID50 of furin inhibitor. F–G. Infectivity analysis of F1 and F2 mutations in 293T-ACE2-furin cells, with and without cathepsin inhibitor (E64D). Chemiluminescence signals (in RLUs) were normalized against WT SARS-CoV-2. H–I. Infectivity analysis of F1 and F2 mutants. Normalized chemiluminescence signals (in RLUs) in 293T-ACE2-CathepsinL/TMPRSS2 cells were calculated by comparing with 293T-ACE2 cells. A–I. Data represent the results of three replicate experiments. Values shown indicate means ± SEM. J. WT and mutated SARS-CoV-2 pseudoviruses were centrifuged in sucrose buffer, then resuspended in PBS for SDS-PAGE. Western blotting was performed with mouse anti-S2 polyclonal antibodies. VSV-M was used as an internal control. The statistical tests were comparisons of each pseudotyped mutated virus group with pseudotyped WT virus group.

Figure 3.

Analysis of furin activation site on cell–cell fusion. A. 293T-ACE2-furin cells were transfected with indicated S-expressing plasmid. The Cell morphology was investigated under bright field microscopy. B. Diagram of dual reporter cell–cell fusion system. 293 T cells were used as donor cells and 293T-ACE2 cells were used as recipient cells. F. Time course curve of cell–cell fusion. RLU signals of Renilla luciferase are shown. Data indicate means ± SEM. Representative results of three independent experiments are shown.

Figure 4.

Analysis of furin activation site in other coronaviruses. A. Schematic of SARS-CoV-2 S protein and the alignment of SARS-CoV-2, RaTG13, and PCoV-GD/GX at S1/S2 and S2′ sites. B–D. Infectivity of mutated RaTG13 (B), PCoV-GD (C), and PCoV-GX (D) pseudoviruses in 293T-ACE2, 293T-ACE2-furin, and 293T-ACE2-TMPRSS2 cells. RLU signals were normalized to 293T-ACE2 control cells. +F indicates that PRRA was inserted into the viruses at the S1/S2 site. The statistical tests were comparisons of each pseudotyped mutated virus group with pseudotyped WT virus group.

Figure 5.

Sensitivity of furin site mutated SARS-CoV-2 to neutralization. ID₅₀ ratios normalized against WT are shown as means ± SEM. Dashed lines indicate the threshold of fourfold difference. All experiments were repeated two to four times, depending on sample availability. CS, convalescent sera. The statistical tests were comparisons of ID₅₀ of each pseudotyped mutated virus group with ID₅₀ of pseudotyped WT virus group.

Figure 6.

Effect of Δ682–686 and K814A mutations on SARS-CoV-2 infectivity of lung cell line and human ACE2 transgenic mice. A. Infectivity of pseudotyped SARS-CoV-2 mutants in Calu-3 cells. The statistical tests were comparisons of each pseudotyped mutated virus group with the pseudotyped WT virus group. The experiment was repeated twice with three duplicates each time. B. Infectivity of pseudotyped SARS-CoV-2 mutants in human ACE2 transgenic mice. The relative intensities of emitted light are presented as the photon flux values in photon/(sec/cm2/sr) and displayed as pseudocolor images, with colours ranging from blue (lowest intensity) to red (highest intensity). One of two repeated experiments is shown. C. Pseudotyped virus infection in each group as indicated by the total flux values. Statistical significance was assessed by one-way ANOVA with Tukey's post hoc test for multiple comparisons with WT.