The Primed Ebolavirus Glycoprotein (19-Kilodalton GP 1,2 ): 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 GP₁ and GP₂ that remain after GP_1,2 is proteolytically primed by endosomal cathepsins for fusion and residues in GP₁ that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP_1,2 contains residues 33 to 190 of GP₁ and all residues of GP₂. 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 GP₁, by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP₁ 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 GP₁) as a highly conserved region containing a two-stranded β-sheet, the two intra-GP₁ disulfide bonds, and four critical Lys residues.