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Journal of Virology, April 2009, p. 2883-2891, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.01956-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Primed Ebolavirus Glycoprotein (19-Kilodalton GP_1,2): Sequence and Residues Critical for Host Cell Binding ^,

Derek Dube,¹ Matthew B. Brecher,^1, Sue E. Delos,^2, Sean C. Rose,² Edward W. Park,² Kathryn L. Schornberg,¹ Jens H. Kuhn,^3,4 and Judith M. White^1,2*

Department of Microbiology,¹ Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908,² Department of Microbiology and Molecular Genetics, Harvard Medical School, Southborough, Massachusetts 01772-9102,³ Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, 14195 Berlin, Germany⁴

Received 17 September 2008/ Accepted 5 January 2009

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.

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* Corresponding author. Mailing address: Department of Cell Biology, University of Virginia, 1340 Jefferson Park Ave., Charlottesville, VA 22908-0732. Phone: (434) 924-2593. Fax: (434) 982-3912. E-mail: jw7g{at}virginia.edu

Published ahead of print on 14 January 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

M.B.B. and S.E.D. contributed equally to this study.

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Journal of Virology, April 2009, p. 2883-2891, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.01956-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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