Acquisition of a multibasic cleavage site does not increase MERS-CoV entry into Calu-3 human lung cells

Abstract

Human-to-human transmission of the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) is currently inefficient. However, there is concern that the virus might mutate and thereby increase its transmissibility and thus pandemic potential. The pandemic SARS-CoV-2 depends on a highly cleavable furin motif at the S1/S2 site of the viral spike (S) protein for efficient lung cell entry, transmission, and pathogenicity. Here, by employing pseudotyped particles, we investigated whether augmented cleavage at the S1/S2 site also increases MERS-CoV entry into Calu-3 human lung cells. We report that polymorphism T746K at the S1/S2 cleavage site or optimization of the furin motif increases S protein cleavage but not lung cell entry. These findings suggest that, unlike what has been reported for SARS-CoV-2, a highly cleavable S1/S2 site might not augment MERS-CoV infectivity for human lung cells.IMPORTANCEThe highly cleavable furin motif in the spike protein is required for robust lung cell entry, transmission, and pathogenicity of SARS-CoV-2. In contrast, it is unknown whether optimization of the furin motif in the spike protein of the pre-pandemic MERS-CoV increases lung cell entry and allows for robust human-human transmission. The present study indicates that this might not be the case. Thus, neither a naturally occurring polymorphism that increased MERS-CoV spike protein cleavage nor artificial optimization of the cleavage site allowed for increased spike-protein-driven entry into Calu-3 human lung cells.

Keywords: MERS-CoV; cleavage; furin; protease; spike.

Fig 1

Polymorphism T746K enhances S protein cleavage. (A) Schematic illustration of the MERS-CoV S protein and mutations studied. SP, signal peptide; RBD, receptor-binding domain; FP, fusion peptide; HR, heptad repeat; TD, transmembrane domain; S1/S2 and S2′, cleavage sites. (B) T746K enhances MERS-S cleavage. Whole-cell lysates (WCL) of cells expressing the indicated S proteins (or no S protein, control; top left panel) or VSV_pp bearing the indicated S proteins (or no S protein, control; top right panel) were subjected to SDS-PAGE, and S protein cleavage was analyzed using immunoblot (via C-terminal V5-epitope tag). Detection of beta-actin (ACTB) or VSV-M protein served as loading control. The immunoblot data are representative of five (WCL) or six (VSV_pp) independent experiments. For quantification of S protein expression (bottom left panel), total S protein signals (S0 +S2) were normalized against the corresponding ACTB (WCL) or VSV-M (VSV_pp) signals and subsequently compared to EMC (WT) S (set as 1). For quantification of S protein cleavage, total S protein signals (S0 +S2) were set as 100% and the proportion of S2 signals calculated (bottom right panel). Graphs show mean ± SEM of five (WCL) or six (VSV_pp) independent experiments. Statistical significance was assessed by two-tailed, unpaired Student’s t-test with Welch correction (P > 0.05, not significant [ns]; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). (C) Peptides covering the MERS-S S1/S2 cleavage site with or without mutation T746K (Suc-TL[T/K]PRSVR-AMC), or a furin consensus sequence (Pyr-RTKR-AMC) were incubated with recombinant furin and the mean fluorescence intensity (MFI; excitation wave length: 355 nm; emission wave length: 460 nm) was recorded every 60 s for a total of 1 h, and background signals (addition of water instead of peptide) were subtracted. Graphs show average (mean) data ± SEM of four independent experiments, conducted with technical triplicates.

Fig 2

Mutation T746K does not augment cell–cell fusion. (A) A MERS-S mutant, in which the S1/S2 site was changed from RSVR to SSVR was included as control in the cell–cell fusion assay, since this mutant is known to exhibit reduced cell–cell fusion. (B) VSV particles bearing no spike, EMC WT, or mutant SSVR were concentrated, and expression of S protein and VSV-M in cell lysates was analyzed using immunoblot. Similar results were obtained in two separate experiments. (C) For analysis of S protein-driven cell-to-cell fusion, effector 293T cells transfected with plasmids encoding the indicated S proteins and β-galactosidase α-fragment were mixed with target 293T cells transfected to express DPP4 and the Ω-fragment of β-galactosidase. β-galactosidase activity in cell lysates was measured at the indicated time points post cell mixing and normalized against the assay background (effector cells expressing no S protein). The mean of three independent experiments with technical quadruplicates is shown; error bars indicate SEM. (D) Cell–cell fusion activity at 24 h post cell mixing is shown. Results obtained for MERS-S WT were set as 1. Statistical significance in panels (C) and (D) was assessed by two-way analysis of variance with Tukey’s multiple comparison test and two-tailed, unpaired Student’s t-test with Welch correction, respectively (P > 0.05, not significant [ns]; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig 3

Mutation T746K does not augment S protein-driven entry into Calu-3 lung cells. The indicated cell lines were inoculated with VSV_pp bearing the indicated S proteins. S protein-driven cell entry was analyzed by quantification of virus-encoded luciferase activity in cell lysates. For normalization, cell entry of particles bearing EMC (WT) S was set as 1. Mean ± SEM of three independent experiments conducted with four technical replicates is shown. Statistical significance was assessed by two-tailed, unpaired Student’s t-test with Welch correction, respectively (P > 0.05, not significant [ns]; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig 4

Mutation T746K reduces DPP4 binding. (A) Spike scheme and location of mutation I529T. A spike protein harboring mutation I529T, which is known to reduce DPP4 binding, was included as control in the DPP4 binding assay. (B) To measure S protein binding to DPP4, 293T cells were transfected with expression plasmids for the indicated S proteins or empty vector and incubated with DPP4-Fc fusion protein and labeled antibody and subjected to flow cytometric analysis. Left panel: Mean fluorescence intensities were normalized to cells expressing no S protein. The average of three independent experiments conducted with unicates is shown; error bars indicate SEM. Right panel: Data were normalized against binding of MERS-S WT to DPP4, which was set as 1. Error bars indicate the SEM. Statistical significance was assessed by two-tailed, unpaired Student’s t-test with Welch correction (P > 0.05, not significant [ns]; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig 5

Optimization of the S1/S2 site increases S protein cleavage but not lung cell entry. (A) Spike scheme and location of mutations. The minimal furin motif at the S1/S2 site in MERS-S was either optimized (mutant S1/S2_OPT), or the complete S1/S2 loop was exchanged against the corresponding sequences found in the hemagglutinin of H5N1 influenza A virus (mutant S1/S2_H5) or the S protein of SARS-CoV-2 (mutant S1/S2_SARS2). (B) Optimization of the furin motif at the S1/S2 site of MERS-S increased S protein cleavage. Whole-cell lysates (WCL) of cells expressing the indicated S proteins (or no S protein, control; left panel), or vesicular stomatitis virus pseudovirus particles (VSV_pp) bearing the indicated S proteins (or no S protein, control; right panel) were subjected to SDS-PAGE. and S protein cleavage was analyzed using immunoblot (via C-terminal V5-epitope tag). Detection of beta-actin (ACTB) or VSV-M protein served as loading control. Representative immunoblot data of three independent experiments are shown. Bands corresponding to uncleaved (S0) and cleaved (S2) MERS-S are indicated. (C) For quantification of S protein expression (left panel), total S protein signals (S0 +S2) were normalized against the corresponding ACTB (WCL) or VSV-M (VSV_pp) signals and subsequently compared against EMC (WT) S (set as 1). For quantification of S protein cleavage, total S protein signals (S0 +S2) were set as 100% and the proportion of S2 signals calculated (right panel). Graphs show the mean of three independent experiments. Error bars indicate SEM. (D) S protein binding to DPP4 was analyzed as described for panel B of Fig. 4. Left panel: The average of four independent experiments conducted with unicates is shown, error bars indicate SEM. Right panel: Data were normalized against binding of MERS-S WT to DPP4, which was set as 1. Error bars indicate the SEM. (E) The indicated cell lines were inoculated with VSV_pp bearing the indicated S proteins. S protein-driven cell entry was analyzed by quantification of virus-encoded luciferase activity in cell lysates. For normalization, cell entry of particles bearing EMC (WT) S was set as 1. Mean ± SEM of three independent experiments conducted with four technical replicates are shown. Statistical significance in panels (C)–(E) was assessed by two-tailed, unpaired Student’s t-test with Welch correction (P > 0.05, not significant [ns]; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).