Biochemical and Structural Characterization of Cathepsin L-Processed Ebola Virus Glycoprotein: Implications for Viral Entry and Immunogenicity
Biochemical and Structural Characterization of Cathepsin L-Processed Ebola Virus Glycoprotein: Implications for Viral Entry and Immunogenicity
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DOI：
10.1128/jvi.02151-09
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发表时间：
2010-03-01
影响因子：
5.4
通讯作者：
Nabel, Gary J.
中科院分区：
文献类型：
--
作者：
Hood, Chantelle L.;Abraham, Jonathan;Nabel, Gary J.
埃博拉病毒(Ebola Virus，EBOV)的细胞附着和进入是由病毒表面的包膜糖蛋白(GP)启动的。这种病毒的进入依赖于pH，并与内体或细胞膜中的组织蛋白L(Cathepsin L，CATL)和/或CatB等蛋白酶对GP的切割有关。在这里，我们表征了扎伊尔EBOV GP的CATL裂解产物(ZEBOV-GP)，并评估了它与进入的相关性。利用连接到可切割组氨酸标签的三聚化结构域产生稳定的重组形式的EBOV gp三聚体。该三聚体被纯化为均一，并与CATL切割。N-末端测序和质谱分析表明，该三聚体产物有三个裂解片段，质量分别为23、19和4 kDa。对组织蛋白L裂解的ZEBOV-GP的结构辅助模拟表明，裂解去除了糖基化的糖链和粘蛋白样结构域(MUC域)，暴露了与受体结合有关的保守的核心残基。CATL裂解的ZEBOV-GP中间体与中和抗体KZ52高亲和力结合，并诱导中和抗体，支持加工的中间体是病毒进入所必需的概念。综上所述，这些数据表明，CATL裂解EBOV gp暴露了它的受体结合域，从而在导致膜融合的步骤中促进了对假定的细胞受体的访问。
Ebola virus (EBOV) cellular attachment and entry is initiated by the envelope glycoprotein (GP) on the virion surface. Entry of this virus is pH dependent and associated with the cleavage of GP by proteases, including cathepsin L (CatL) and/or CatB, in the endosome or cell membrane. Here, we characterize the product of CatL cleavage of Zaire EBOV GP (ZEBOV-GP) and evaluate its relevance to entry. A stabilized recombinant form of the EBOV GP trimer was generated using a trimerization domain linked to a cleavable histidine tag. This trimer was purified to homogeneity and cleaved with CatL. Characterization of the trimeric product by N-terminal sequencing and mass spectrometry revealed three cleavage fragments, with masses of 23, 19, and 4 kDa. Structure-assisted modeling of the cathepsin L-cleaved ZEBOV-GP revealed that cleavage removes a glycosylated glycan cap and mucin-like domain (MUC domain) and exposes the conserved core residues implicated in receptor binding. The CatL-cleaved ZEBOV-GP intermediate bound with high affinity to a neutralizing antibody, KZ52, and also elicited neutralizing antibodies, supporting the notion that the processed intermediate is required for viral entry. Together, these data suggest that CatL cleavage of EBOV GP exposes its receptor-binding domain, thereby facilitating access to a putative cellular receptor in steps that lead to membrane fusion.