The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding

Abstract

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP(1,2)), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP(1) and GP(2) that remain after GP(1,2) is proteolytically primed by endosomal cathepsins for fusion and residues in GP(1) that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP(1,2) contains residues 33 to 190 of GP(1) and all residues of GP(2). The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP(1), by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP(1) residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP(1,2)-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP(1,2) is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP(1)) as a highly conserved region containing a two-stranded beta-sheet, the two intra-GP(1) disulfide bonds, and four critical Lys residues.

FIG. 1.

Amino acid sequence of primed 19-kDa ZEBOV GP_1,2. The 19-kDa GP_1,2 was purified from thermolysin-treated VSV^gfp-GP_1,2Δ and run on an SDS gel. A silver-stained band containing 19-kDa GP₁ and GP₂ was excised and treated with trypsin or ArgC. The sequences of the resulting proteolytic fragments, identified by mass spectrometry, are underlined (black, tryptic fragments; gray, ArgC fragments) beneath the complete amino acid sequences of GP₁ and GP₂. The sequences encompassed by the identified fragments are boxed in gray. The signal sequence cleavage site is denoted by an inverted black triangle. The potential C termini of cathepsin B- and L-cleaved 19-kDa GP₁ (K190/F193/F194) are marked by gray triangles. The raw mass spectrometry data are shown in Fig. S1 in the supplemental material.

FIG. 2.

Model of primed 19-kDa ZEBOV GP_1,2. (A) Domain architecture of EBOV GP_1,2 (based on nomenclature and color coding in reference 14). The GP₁ signal peptide (SP) (white; residues 1 to 32); base (green; residues 33 to 70, 96 to 105, 159 to 168, and 177 to 189), head (dark blue; residues 71 to 95, 106 to 158, 169 to 176, and 215 to 227), linker region (site of cathepsin B/L and thermolysin cleavage) (hatched yellow; residues 190 to 213), glycan cap (cyan; residues 228 to 313), mucin-like domain (white; residues 314 to 464), and C-terminal domain (hatched white; residues 465 to 501) are shown. The GP₂ fusion peptide (F) and transmembrane domain (T) are labeled. Domains not present in the protein used for crystallography (14) are shown in white, and regions present but unresolved in the crystal structure are shown with hatches. Pink Ys and pink Os represent sites of N- and O-glycosylation. (B) Native structure of GP_1,2Δ (left) and model of primed 19-kDa GP_1,2 (right), based on Fig. 2 of reference and colored as in panel A. Cysteine residues involved in disulfide bonds are in red, and predicted loop regions are shown with dashed lines. The asterisk (*) represents a contact point between the β1 and β13 strands (see the text for more details). Potential cathepsin B and cathepsin L cleavage sites (residues 190, 193, and 194) are labeled in the model (right). The graphic representations are based on PDB file 3CSY (14) and were produced with Pymol. Note that the depiction of primed GP_1,2 in panel B (right) and in Fig. 7A, C, and D is strictly a model that assumes no conformational changes following proteolytic priming.

FIG. 3.

Production of ZEBOV GP_1,2 RBR-Fc proteins. (A) Domain structure of full-length EBOV GP_1,2 and the regions of GP₁ included in RBR-Fc proteins. Designations are as described in the legend to Fig. 2A. Thirteen RBR proteins conjugated to rabbit Fc were generated. Set 1, RBRs initiating at residue 57, 72, or 90 and terminating at residue 149, 172, or 198. An RBR containing residues 54 to 201 (13) conjugated to rabbit Fc was also created. Set 2, RBRs initiating at residue 33 and terminating at residue 149, 193, or 198. RBR-12-Fc represents a “19-kDa GP₁-like” protein. Cys 53 (normally disulfide bonded to Cys 609) was mutated to Ser in all set 2 RBRs to avoid potential misfolding. (B) 293T cells were transfected with plasmids encoding each RBR-Fc. At 24 h posttransfection, lysates of cell pellets (P) and supernatants (S) (equal volumes for each RBR-Fc) were collected, immunoprecipitated with protein A-agarose beads, and analyzed by Western blotting for rabbit Fc. Western blots from one representative experiment of three or more are shown.

FIG. 4.

Binding activities of selected ZEBOV GP_1,2 RBR-Fc proteins. (A) RBR-Fcs 1, 4, and 7 (200 nM) were incubated with 293T or Vero E6 cells (permissive) or with Jurkat lymphocytes (nonpermissive). Cell surface binding was analyzed by flow cytometry, using protein A-Alexa Fluor 488 to detect the Fc portion of the RBR conjugate. (B) Binding of RBR-1-Fc and RBR-12-Fc to 293T, Vero E6, or Jurkat cells was determined and analyzed as in panel A. In all experiments, rabbit Fc served as a negative control. The averages of two or more experiments are shown. Bars represent the percentages of cells that bound the indicated RBR-Fc proteins. Error bars indicate standard deviations. Significance (relative to RBR-1-Fc) was determined by Student's t test. *, P < 0.05.

FIG. 5.

Binding properties of RBR-1-Fc. (A) RBR-1-Fc was incubated with 293T cells at the indicated concentration, and binding was determined as described in the legend to Fig. 4. (i) Percentage of cells that bound RBR-1-Fc (black) or Fc (gray). (ii) Mean fluorescence intensity (MFI) of cells incubated with RBR-1-Fc, with background values for control Fc subtracted. Data for one representative experiment of three are shown. (B) 293T cells were lifted as indicated with a solution containing EDTA (PEEG, as for all other binding experiments) or with 0.5% trypsin-EDTA for 15 min and then processed for RBR-1-Fc (200 nM) binding. The averages of three experiments are shown. Error bars indicate standard deviations. Significance (between RBR-1-Fc binding to trypsin- and EDTA-treated cells) was determined by Student's t test. *, P < 0.05. (C) 293T cells were incubated with RBR-1-Fc (200 nM), exposed to medium at the indicated pH for 10 min (at 4°C), returned to normal medium, and then processed for cell surface binding. Values were normalized to those for RBR-1-Fc at pH 7.0. The averages of two or more experiments are shown. Error bars indicate standard deviations. Significance (relative to RBR-1-Fc binding at pH 7) was determined by Student's t test. *, P < 0.05.

FIG. 6.

Binding of WT and mutant RBR-1-Fc proteins. Ala substitutions were made individually and in combination within RBR-1-Fc at K95, K114 and K115, K140, and G143 (3, 15, 17). 3mer, K114A/K115A/K140A mutant; 4mer, K95A/K114A/K115A/K140A mutant; 5mer, K95A/K114A/K115A/K140A/G143A mutant. 293T cells were incubated with the indicated RBR-Fc or control Fc (200 nM) and analyzed for cell surface binding as described in the legend to Fig. 4. Binding values were normalized to those for WT RBR-1-Fc. The number of experiments performed is shown above each bar. Significance (relative to WT RBR-1-Fc) was determined by Student's t test. *, P < 0.04; **, P < 0.0004.

FIG. 7.

Model of 19-kDa GP₁ and locations of key receptor binding residues. (A) Ribbon diagram of 19-kDa GP₁ showing key recombinant RBRs used in this study, including RBR-7 (residues 90 to 149; dark blue), RBR-1 (residues 57 to 149; dark blue and green), and RBR-12 (residues 33 to 193; dark blue, green, and pink). The side chains of K95, K114, K115, and K140 are shown. Cysteines involved in disulfide bonds are shown in red. An asterisk (*) denotes a contact point between the β1 and β13 strands. (B) Surface rendering (top view) of the GP_1,2Δ trimer structure. One monomer is colored dark gray for clarity. (C and D) Model of GP_1,2Δ trimer after cleavage by cathepsins B and L, viewed from the top (C) and side (80° rotation) (D). Residues that decreased RBR-1-Fc binding when replaced with Ala are shown in all figures (K95, orange; K114, K115, and K140, cyan). The graphic representations were based on PDB file 3CSY (14) and rendered with Pymol. Note that the depictions in panels A, C, and D are only models that assume no conformational changes in GP₁ following proteolytic priming.

FIG. 8.

Inhibition of EBOV GP_1,2Δ-mediated infection by RBR-1-Fc and RBR-12-Fc. (A) Vero E6 cells were incubated for 3 h with VSV^gfp-GP_1,2Δ in the presence or absence of RBR-1-Fc, RBR-12-Fc, or control rabbit Fc at the indicated concentration. Unbound virus was removed by washing, the cells were incubated overnight, and GFP expression was quantified by flow cytometry. Samples were analyzed in triplicate. The average values from one representative experiment are shown. Error bars represent standard deviations. Significance (relative to the rabbit Fc control tested at the same concentration) was determined by Student's t test. *, P < 0.05. The exact same experiment was conducted two times with similar results. RBR-1-Fc and RBR-12-Fc were tested five additional times (with HIV gp120-Fc as a negative control), with very similar results. (B) Vero E6 cells were infected with HIV^blam-GP_1,2Δ or HIV^blam-G in the presence or absence of WT or 4mer mutant RBR-12-Fc at 800 nM. Cells were loaded with the beta-lactamase substrate CCF2/AM. Cells loaded only with CCF2/AM served as a negative control (no virus). The extent of CCF2/AM cleavage by beta-lactamase introduced into the cytoplasm was evaluated by flow cytometry (detected by the change in dye emission from green to blue). Samples were analyzed in duplicate. The averages for duplicate samples from one representative experiment are shown. Error bars represent standard deviations.