A Fusion Peptide in the Spike Protein of MERS Coronavirus

Abstract

Coronaviruses represent current and emerging threats for many species, including humans. Middle East respiratory syndrome-related coronavirus (MERS-CoV) is responsible for sporadic infections in mostly Middle Eastern countries, with occasional transfer elsewhere. A key step in the MERS-CoV replication cycle is the fusion of the virus and host cell membranes mediated by the virus spike protein, S. The location of the fusion peptide within the MERS S protein has not been precisely mapped. We used isolated peptides and giant unilamellar vesicles (GUV) to demonstrate membrane binding for a peptide located near the N-terminus of the S2 domain. Key residues required for activity were mapped by amino acid replacement and their relevance in vitro tested by their introduction into recombinant MERS S protein expressed in mammalian cells. Mutations preventing membrane binding in vitro also abolished S-mediated syncytium formation consistent with the identified peptide acting as the fusion peptide for the S protein of MERS-CoV.

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

Figure 1

Multiple sequence alignment of coronavirus spike protein S2 subunit. Alignment of 17 coronavirus partial S2 sequences representing four genera of coronavirus. The isolates are: α-Alphacoronavirus (α-CoV) HCoV-229E (ABB90529.1); HCoV-NL63 (YP_003767.1); TGEV (ABG89335.1); FCoV (YP_004070194.1 AFH58021); M-Bat CoV-HKU8 (YP_001718612.1); and PEDV (NP_598310.1). Betacoronavirus (β-CoV) HCoV-HKU1 (ADN03339.1); MHV-A59 (NP_045300.1); BatCoV-HKU9 (YP_001039971.1); SARS-CoV (NP_828851.1); and MERS-CoV (AHX00731.1). Gammacoronavirus (γ-CoV) IBV (ADP06471.2); SW1 CoV (YP_001876437.1); and BdCoV-HKU22 (AHB63508.1). Deltacoronavirus (δ-CoV) NHCoV-HKU19 (AFD29226.1); PDCoV (AFD29187.1); and MCoV-HKU13 (YP_002308506.1). The identified Middle East respiratory syndrome (MERS) putative fusion peptide is boxed.

Figure 2

Effect of wild type MERS-CoV putative fusion peptide on the shape and size of giant unilamellar vesicles (GUVs). (A) Fluorescent images of electroformed GUVs treated with 10, 1 and 0.1 µM peptide 1, or 10 µM M2-Influenza and imaged at 0 and 5 min. Scale bar at bottom right indicates 5 µm. (B) GUV shape. (C) Relative GUV size. Statistical significance was shown for peptide 1 at 10 µM compared to buffer only at all time points for both size and shape of the GUV (p < 0.01; Linear Mixed Model). Error bars shown are the mean ± SEM. The scale bar indicates 20 µm. Each colored group of two columns left to right represents the data for a single peptide tested at each time point, 0 and 5 min. Some error bars are too small to be observed.

Figure 3

Comparison of wild type and mutant MERS-CoV putative fusion peptides on GUV shape and size. (A) Fluorescent images of electroformed GUVs treated with 10 µM peptides 1–6, M2-Influenza or buffer alone, and imaged at 0, 1, 2 and 5 min after peptide addition. Scale bar at lower right indicates 5 µM. (B) GUV shape. (C) GUV size. Statistical significance was shown for peptides 1, 4 and 6 at all the time points for both size and shape of the GUV (p < 0.01; Linear Mixed Model). Peptides 2, 3 and 5 did not show a statistically significant change in shape or size when compared to buffer only. All error bars shown are the mean ± SEM (Linear Mixed Model; p < 0.01). The scale bar indicates 20 µm. Each colored group of four columns, left to right, represents the data for a single peptide tested at each time point 0, 1, 2 and 5 min. Some error bars are too small to be observed.

Figure 4

MERS S expression and syncytium formation. Lenti-X 239T cells were transfected with vectors encoding WT MERS-CoV S or the alanine mutants described in the text. (A) Cells transfected with the WT S were treated with 2 µg trypsin/mL for 30 min at 37 °C prior to a 5 min acid pulse. Following recovery in complete Dulbecco’s modified Eagle medium (DMEM) for 1 h at 37 °C transfected non-treated (4A left) and transfected + acid treated (4A right) cells were processed for immunofluorescence and visualized as described. A syncytium (arrow) is shown with boundaries highlighted with a dotted line. All images were recorded using the 20× objective. (B) WT and alanine scanning mutants of S as indicated were transfected and acid pulsed before being processed for immunofluorescence as before. (C) Confirmation of DPP4 receptor expression revealed by staining Lenti-X-239T cells with a primary anti-CD26 MAb followed by an anti-mouse Fluor 488 conjugate. Nuclei were counterstained with DAPI (blue). The scale bars are 200 µm.