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. 2009 Aug;83(15):7411-21.
doi: 10.1128/JVI.00079-09. Epub 2009 May 13.

Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide

Ikenna G Madu  1 , Shoshannah L Roth, Sandrine Belouzard, Gary R Whittaker
Affiliations

Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide

Ikenna G Madu et al. J Virol. 2009 Aug.

Abstract

Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of alpha-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.

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Figures

FIG. 1.
FIG. 1.
Bioinformatics analysis of the CoV S protein and the proposed S2 fusion peptide. (A) A quantitative multiple sequence alignment of the S protein from representative CoVs was performed using the program AlignX, a component of Vector NTI 10.3.1 (Invitrogen), which uses a modified Clustal W algorithm. The proposed S2 fusion peptide and other regions of interest have been identified with bars or arrows to show their location within the S gene and their various degrees of conservation across the CoV family. (B) A multiple sequence alignment of the S protein from representative CoVs was performed using AlignX as above. Conserved residues within the proposed S2 fusion peptide region are indicated in bold. Underlining indicates residues showing conserved properties. Virus abbreviations (and GenBank accession numbers) are as follows: SARS-CoV (AAP13441); HCoV-NL63, human CoV NL63 (Amsterdam accession number AAS58177); HCoV-229E, human CoV 229E (AAG48592); HCoV-OC43, human CoV OC43 ATCC VR-759 (AAR01015); HCoV-HKU I, human CoV HKU I N19 (ABD75497); IBV (Bdtte), infectious bronchitis virus Beaudette strain (AAY24433); IBV (M41), infectious bronchitis virus M41 strain (ABI26423); MHV-A59, MHV A59 (AAB86819); MHV-2, MHV 2 (AAF19386); PEDV, porcine epidemic diarrhea virus LZC (ABM64776); PRCoV, porcine CoV ISU-1 (ABG89317); PHEV, porcine hemagglutinating encephalomyelitis virus VW752 (AAY68297); bovine CoV, bovine CoV R-AH187 (ABP38295); sable antelope CoV, sable antelope CoV US/OH1/2003 (ABP38306); giraffe CoV, giraffe CoV US/OH3-TC/2006 (ABP38313); equine CoV, equine CoV NC99 (ABP87990); FIPV, feline infectious peritonitis virus WSU 79-1146 (YP239355); bat CoV, bat CoV 273/2005 (ABG47069); TGEV, transmissible gastroenteritis virus Purdue PUR46-MAD (NP058424).
FIG. 2.
FIG. 2.
Effect of alanine point mutants on spike protein surface expression. BHK cells were transfected with plasmids encoding wild-type SARS-CoV S or alanine mutants. The cells were labeled with sulfo-NHS-SS-biotin and lysed. Lysates were affinity purified with NeutraAvidin beads and resolved by SDS-PAGE and Western blotting using anti-S monoclonal antibody C9. The biotinylation assay was repeated three times, and the results from the Western blotting were quantified using IP Lab software and plotted in Sigma Plot, version 9.0. Error bars represent standard deviations of the means. WT, wild type.
FIG. 3.
FIG. 3.
Syncytium formation mediated by mutant spike proteins. BHK cells were transfected with plasmids encoding wild-type (WT) SARS-CoV S or alanine mutants and ACE2. Fusion was induced using medium containing 2 μg/ml trypsin as described in Materials and Methods, and syncytia were visualized by immunofluorescence microscopy using anti-S antibodies (green signal). Nuclei were counterstained with Hoechst 33264 (blue signal).
FIG. 4.
FIG. 4.
Quantitative assays of membrane fusion mediated by mutant spike proteins. BHK cells were cotransfected with plasmids encoding wild-type (WT) SARS-CoV S or alanine mutants and also with luciferase cDNA under the control of a T7 promoter. At 24 h posttransfection, BHK cells were then overlaid with Vero cells previously transfected with a plasmid encoding T7 polymerase. After 3 h, fusion was induced using medium containing 2 μg/ml trypsin. The cells were lysed 6 h postfusion induction, and supernatants were measured for luciferase activity. Each bar is averaged from three individual repeats, and error bars represent standard deviations from the means.
FIG. 5.
FIG. 5.
Incorporation of mutant spike proteins into MLV pseudotyped virions. Spike protein-MLV pseudotyped virions were prepared as described in Materials and Methods. Virions were concentrated using a 10% PEG precipitation, diluted in SDS sample buffer, and resolved by SDS-PAGE and Western blotting using a monoclonal antibody specific for the C9 tag. The spike protein-MLV pseudotyped virions were generated three times for wild type (WT) or mutants, and the results from the Western blotting were quantified using IP Lab software and plotted in Sigma Plot, version 9.0. Error bars represent standard deviations from the means.
FIG. 6.
FIG. 6.
Infectivity of spike protein-MLV pseudotyped virions. (A) Endosomal infectivity of spike protein-MLV pseudotyped virions. Virions were bound to Vero cells at 4°C in RPMI medium for 2 h. After binding, cells were rinsed in RPMI medium; the medium was then replaced with complete DMEM, and cells were incubated at 37°C. At 48 h postbinding cells were lysed and assayed for luciferase activity. (B) Trypsin-mediated infectivity of spike protein-MLV pseudotyped virions. Virions were bound to Vero cells at 4°C in RPMI medium and treated with 25 mM ammonium chloride. Fusion was induced using serum-free medium containing 2 μg/ml trypsin. The cells were lysed at 48 h postfusion induction, and supernatants were assayed for luciferase activity. Each bar is averaged from three individual infectivity assays, and error bars represent standard deviations of the means. WT, wild type.
FIG. 7.
FIG. 7.
Summary of effects of S2 mutations on SARS-CoV S fusion activity. The fusion activity of SARS-CoV S wild-type (WT) and mutants in cell-cell fusion assays, as well as in pseudovirus assays of endosomal (− trypsin) and nonendosomal (+ trypsin) infection are summarized. +++, 80 to 100% of wild-type level; ++, 79 to 50% of wild-type level; +, 49 to 20% of wild-type level; and −, <20% of wild-type level. ND, not determined.
FIG. 8.
FIG. 8.
The proposed SARS-CoV S2 fusion peptide induces lipid mixing in liposomes. (A) Extent of lipid mixing was determined by varying the ratios of SARS-CoV S2 fusion peptide or control peptide concentrations with the total concentration of labeled and unlabeled POPC-POPS-cholesterol (1:3:1) liposomes at pH 5 and pH 7. The kinetics of mixing was followed by monitoring fluorescence intensity at 530 nm upon addition of peptide. Each data point is averaged from three individual assays, and error bars represent standard deviations of the means. (B) Effect of SARS-CoV S2 fusion peptide on lipid mixing at various pH values. The kinetics of lipid mixing was followed by monitoring NBD-PE fluorescence intensity at 530 nm in decreasing-pH environments (from pH 7.0 to pH 4.5). Each data point is averaged from three individual assays, and error bars represent standard deviations of the means. (C) Extent of lipid mixing at pH 7 was determined by varying the ratios of wild-type or modified LLF-AAA SARS-CoV S2 fusion peptide with POPC-POPS-cholesterol (1:3:1) liposomes at various ratios of peptide to lipid. Each data point is averaged from three individual assays, and error bars represent standard deviations of the means. (D) Extent of lipid mixing at pH 5 was determined by varying the ratios of wild type or modified LLF-AAA SARS-CoV S2 fusion peptide with POPC-POPS-cholesterol (1:3:1) liposomes at various ratios of peptide to lipid. Each data point is averaged from three individual assays, and error bars represent standard deviations of the means.
FIG. 9.
FIG. 9.
The proposed SARS-CoV S2 fusion peptide has the propensity to form helical secondary structure. (A) CD spectrum (mean residue ellipticity) of the SARS-CoV S2 fusion peptide (solid line) in a TFE/fusion buffer ratio of 50% (vol/vol) at 37°C or in fusion buffer alone (dotted line). (B) CD spectrum (mean residue ellipticity) of the SARS-CoV S2 fusion peptide (FP; solid line) alongside a control peptide (dotted line) in a TFE/fusion buffer ratio of 50% (vol/vol) at 37°C. (C) CD spectrum (mean residue ellipticity) of the short SARS-CoV S2 fusion peptide (solid line) in a TFE/fusion buffer ratio of 50% (vol/vol) at 37°C or in fusion buffer alone at either pH 5.0 (dotted line) or pH 7.0 (dashed line). deg, degree.
FIG. 10.
FIG. 10.
Structural model of the SARS-CoV S2. (A) A three-dimensional model of the SARS-CoV S protein ectodomain trimer is shown in cartoon form, based on Protein Data Bank file 1T7G. The figure was generated in MacPyMOL (DeLano Scientific), with the proposed fusion peptide shown in white and the S2 cleavage site (R797) shown in magenta. (B) The side chains of the proposed fusion-active core (L803, L804, and F805) are shown in enlarged form.
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