Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis
Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis
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DOI：
10.1371/journal.ppat.1006735
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发表时间：
2017-12-01
期刊：
影响因子：
6.7
通讯作者：
Law, Mansun
中科院分区：
文献类型：
--
作者：
Gopal, Radhika;Jackson, Kelli;Law, Mansun
丙型肝炎病毒（HCV）包膜糖蛋白E1和E2在病毒表面形成非共价连接的异源二聚体，介导病毒进入。E1、E2和异源二聚体复合物E1E2是候选疫苗抗原，但由于通过标准蛋白表达和纯化方法难以产生天然折叠蛋白，因此研究在技术上具有挑战性。为了更好地理解这些蛋白质的抗原性，创建了包含整个E1E2（555个残基）的丙氨酸扫描突变体文库，用于评估每个残基在糖蛋白中的作用。通过基于高通量流式细胞术的测定，探测突变体库与共受体CD81的结合，以及靶向E1、E2和E1E2复合物的连续和不连续表位的一组13种人和小鼠单克隆抗体（mAb）。结合最近确定的E2核心结构域（E2c）的晶体结构，我们发现E2背层区域中的几个残基间接影响靶向E2保守中和面的CD81和mAb的结合。这些发现突出了E2背层在与E2前层相互作用以实现其生物学功能方面的意想不到的作用。我们还确定了E1和E2可能位于E1E2复合物界面处或附近的区域，并确定E2背层在E1E2复合物形成中也起着重要作用。CD81和抗体组对E1E2突变体文库的构象依赖性反应性提供了每个氨基酸（aa）对E1E2表达和折叠影响的全局视图。这一信息是有价值的指导蛋白质工程的努力，以提高疫苗抗原的开发和结构研究的抗原特性和稳定性的E1E2。
The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a highthroughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformationdependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.