Fig. 1: The suboptimal furin CS of SARS-CoV-2 spike enhances entry into mucosal epithelial and primary human airway cells.
From: The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets
a, Amino acid sequence alignment of coronavirus furin CS mutants used in this study. Mutants with potential S1/S2 furin CSs are shown in shades of orange while mutants without furin CSs are shown in shades of blue. b, Syncytia formation due to overexpression of different coronavirus spike proteins in Vero E6 cells. Percentage indicates the proportion of nuclei in each field that have formed clear syncytia. Data are plotted as mean + s.d. of three independent repeats. Statistical significance was determined by one-way ANOVA with multiple comparisons against SARS-CoV-2 WT. ****P < 0.0001. An extended figure of representative fields shown is in Extended Data Fig. 1. c, Western blot analysis of concentrated lentiviral PVs with different coronavirus spike proteins. Levels of lentiviral p24 antigen shown as loading control. Representative blot shown from n = 3 independent repeats. d, Western blot analysis of concentrated WT and ΔCS SARS-CoV-2 viruses. Levels of nucleocapsid (N) protein shown as loading control. Representative blot shown from n = 2 independent repeats. e, SARS-CoV-2 competition assay growth curve between WT and ΔCS virus in Vero E6 and Caco-2 cells. Cells were infected at an MOI of 0.1. Starting inoculum ratio is shown on the left-hand bar, while proportions of virus as determined by deep sequencing at 72 h post-inoculation are shown on the right. Virus titres determined by plaque assay at 72 h post-inoculation are shown in superimposed white data points. All results indicate triplicate repeats plotted as mean + s.d. f, SARS-CoV-2 competition assay growth curve between WT and ΔCS virus in HAEs. Cells were infected at an MOI of 0.1. Starting inoculum ratio shown at time 0; proportions of virus determined by deep sequencing. All time points were taken from triplicate repeats. Virus replication was determined by plaque assay and is shown as imposed white data points. Data are plotted as mean + s.d. g, Head-to-head replication kinetics of clonal WT and ΔCS viruses in Calu-3 human lung cells. Cells were infected at an MOI of 0.1. All time points taken from triplicate repeats are plotted as mean + s.d. Data shown are a representative replicate from (total n = 2) repeats. Virus replication was determined by plaque assay. Dotted line indicates limit of detection. h–j, Entry of lentiviral PVs containing different viral glycoproteins into 293T-ACE2 (h), Caco-2 (i) and Calu-3 (j) cells. Cells were transduced with different PVs and lysed 48 h later, and analysed by firefly luciferase luminescence. Data are shown as raw luminescence units (RLU). All assays were performed in sextuplicate (coronavirus pseudovirus) or triplicate (non-coronavirus controls) and are plotted as mean + s.d. Data shown are a representative replicate from (n = 4) independent repeats. Statistics were determined by one-way ANOVA on log-transformed data (after determining log normality by the Shapiro–Wilk test and QQ plot.) *0.05 ≥ P > 0.01; **0.01 ≥ P > 0.001; ***0.001 ≥ P > 0.0001; ****P ≤ 0.0001.
