The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding.
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.
Dube
Derek
D
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908-0732, USA.
Brecher
Matthew B
MB
Delos
Sue E
SE
Rose
Sean C
SC
Park
Edward W
EW
Schornberg
Kathryn L
KL
Kuhn
Jens H
JH
White
Judith M
JM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D004252:DNA Mutational Analysis; D029043:Ebolavirus / Q000235:genetics / Q000502:physiology*; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053586:Virus Internalization
D006023:Glycoproteins; D014760:Viral Fusion Proteins