Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

Abstract

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca²⁺) play an important role in fusogenic activity via a Ca²⁺ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca²⁺ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca²⁺ environments. Our results demonstrate that intracellular Ca²⁺ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca²⁺ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca²⁺ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca²⁺ ratio, as opposed to an 1:2 SARS-CoV FP to Ca²⁺ ratio, suggesting significant differences in FP Ca²⁺ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

Keywords: MERS; coronavirus; fusion; fusion peptide; membrane fusion; spike protein; viral fusion.

FIG 1

Sequence and model of MERS-CoV S protein fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FPs). FP1 and FP2 designate the two different domains in the FP. Sequences illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E; in green are the A substitutions. (B) Modeling of the MERS-CoV S protein monomer with an emphasis on the FP. Negatively charges D and E are depicted as atomic bonds in red. The S2’ site is orange, and the FP1 and FP2 domains are labeled blue and pink, respectively.

FIG 2

Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.

FIG 3

Immunofluorescence assay of MERS-CoV S protein WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding the respective MERS-CoV S protein variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleave MERS-CoV S proteins for fusion, no further protease treatment was needed to induce syncytium formation. WT + furin inhibitor (FI) indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection to block fusion. Syncytia were visualized using immunofluorescence microscopy by staining the MERS-CoV S protein with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI; in blue). Images were taken at a magnification of ×25. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted, and the average number of nuclei per syncytium was calculated. Error bars represent standard deviations (n = 9). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against each of the respective mutant *, P > 0.5; **, P > 0.05; ***, P > 0.005.

FIG 4

Western blot analysis of S proteins incorporated into PPs. A total of 1 ml of DMEM containing PPs per each tested S protein was ultracentrifuged, washed in PBS, and resuspended in SDS Laemmli buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S protein antibodies.

FIG 5

Pseudoparticle assays of MERS-CoV S protein WT and mutants. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S protein WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays. (C) Impact of intracellular Ca²⁺ on MERS-CoV fusion. Cells were pretreated with growth medium containing either 50 μM calcium chelator BAPTA-AM or dimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PPs in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessment for luciferase activity. (D) Impact of extracellular Ca²⁺ on MERS-CoV fusion. Cells were pretreated with growth medium either with or without 1.8 mM Ca²⁺ for 1 h. The infection protocol is as described above except PPs were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviations (n = 3). Statistical analysis was performed using an unpaired Student’s t test, as indicated. *, P > 0.5; **, P > 0.05; ***, P > 0.005.

FIG 6

Pseudoparticle assays of MERS-CoV S protein WT and E891A/D896A. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to the WT sample. Error bars represent standard deviations (n = 3). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against the respective mutant (for B and C, the untreated WT was compared to each sample). *, P > 0.5; **, P > 0.05; ***, P > 0.005. (A) Infectivity of PPs without pretreatment of cells. (B) Impact of intracellular Ca²⁺ on MERS-CoV fusion. Cells and PPs were treated as described for Fig. 5C. (C) Impact of extracellular Ca²⁺ on MERS-CoV fusion. Cells and PPs were treated as described for Fig. 5D.

FIG 7

ESR and ITC analysis of the MERS-CoV FP. (A and B) Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol of 3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca²⁺ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca²⁺ at pH 5; and purple, scrambled peptide, 1 mM Ca²⁺ and at pH 5. (C) Plot of the difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca²⁺ concentration in POPC/POPS/Chol of 3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S₀ ranges from 1 × 10⁻³ to 5 × 10⁻³, while the uncertainties from repeated experiments were 5 × 10⁻³ to 8 × 10⁻³ or less than ±0.01. We show the standard deviation bars in A and B. (D) ITC analysis of Ca²⁺ binding to MERS-CoV FP. The peptides were titrated with CaCl₂. The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.