The Primed Ebolavirus Glycoprotein (19-Kilodalton GP1,2): Sequence and Residues Critical for Host Cell Binding
The Primed Ebolavirus Glycoprotein (19-Kilodalton GP1,2): Sequence and Residues Critical for Host Cell Binding
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DOI：
10.1128/jvi.01956-08
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发表时间：
2009-04-01
影响因子：
5.4
通讯作者：
White, Judith M.
中科院分区：
文献类型：
--
作者：
Dube, Derek;Brecher, Matthew B.;White, Judith M.
埃博拉病毒（EBOV）进入细胞是由其糖蛋白（GP（1，2））介导的，GP（1，2）是一种I类融合蛋白，其结构最近被确定（J.E. Lee等人，Nature 454：177-182，2008）。在这里，我们证实了结构分析的两个主要预测，即GP（1）和GP（2）中的残基，其在GP（1，2）被内体组织蛋白酶蛋白水解引发以用于融合后保留，以及GP（1）中的残基，其对于与宿主细胞结合至关重要。质谱分析表明，引发的GP（1，2）含有GP（1）的33 - 190位残基和GP（2）的所有残基。通过双管齐下的方法确定受体结合位点的位置。我们通过比较四种高产RBR蛋白的细胞结合能力，鉴定了一个小的受体结合区（RBR），即GP（1）的90 - 149位残基。我们表征了最佳RBR（含有GP（1）残基57至149）的结合特性，然后进行突变分析以鉴定关键结合残基。四个赖氨酸（K95、K114、K115和K140）的取代降低了RBR蛋白的结合和抑制GP（1，2）介导的感染的能力。K114、K115和K140位于一个小区域中，该区域被建模为位于蛋白水解引发后的圣杯的顶面上; K95位于圣杯碗中更深处。结合Lee等人的研究，我们的发现提供了GP（1，2）是如何引发融合的结构见解，并将EBOV RBR的核心（GP（1）的残基90 - 149）定义为包含双链β-折叠、两个GP（1）内二硫键和四个关键的Lys残基的高度保守区域。
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.