Bornholdt Zachary A, Ndungo Esther, Fusco Marnie L, Bale Shridhar, Flyak Andrew I, Crowe James E, Chandran Kartik, Saphire Erica Ollmann
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
mBio. 2016 Feb 23;7(1):e02154-15. doi: 10.1128/mBio.02154-15.
The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.
Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs.
丝状病毒表面糖蛋白（GP）介导病毒进入宿主细胞。病毒内化进入内体后，GP被宿主半胱氨酸蛋白酶切割，暴露出一个受体结合位点（RBS），否则该位点会免受免疫监视。在此，我们展示了经蛋白水解切割的埃博拉病毒GP的晶体结构，分辨率为3.3埃。我们将此结构与大量携带突变GP蛋白的假型病毒的功能分析相结合，以分子分辨率绘制埃博拉病毒GP内体RBS图谱。我们的研究表明，GP与其内体受体尼曼-匹克C1（Niemann-Pick C1）的结合分两个不同阶段进行：最初的静电相互作用之后是与内体切割的GP1亚基上暴露的疏水凹槽的特异性相互作用。最后，我们证明靶向丝状病毒RBS的单克隆抗体可中和所有已知的丝状病毒GP，使这个保守的口袋成为开发泛丝状病毒疗法的有希望的靶点。
埃博拉病毒利用其糖蛋白（GP）进入新的宿主细胞。在进入过程中，GP必须被人类酶切割才能发生受体结合。在此，我们提供了埃博拉病毒GP切割形式的晶体结构。我们证明切割暴露了GP顶部的一个位点，该位点结合受体的关键结构域C，即尼曼-匹克C1（NPC1）。我们进行诱变以找到结合NPC1所必需的位点部分，并绘制切割后的GP中上部带电荷的嵴和下部疏水凹槽的不同作用。我们发现这个三维位点在丝状病毒家族中是保守的，针对该位点的抗体能够结合所测试的每种丝状病毒切割后的GP，并中和携带这些GP的病毒。
