Structural analysis of HCV E1E2 glycoproteins

摘要-项目4 尽管广泛的研究努力，以了解丙型肝炎病毒（HCV）的生物学，它仍然是一个主要的人类 没有疫苗的病原体此外，还明显缺乏关于其结构的资料。 病毒体及其组成蛋白的结构。我们打算利用我们丰富的结构生物学 与相关的黄病毒的经验，专注于确定病毒糖蛋白的结构，病毒 颗粒，以及与表面糖蛋白结合的有效中和抗体的复合物。该项目将依靠 与项目1进行了广泛的互动，以询问结构并为项目1提供指导。 这些糖蛋白的特征对于引发广泛和有效的中和抗体是重要的。迭代 将进行E1E2糖蛋白的分析和再工程，以指导抗体组分的合成。 双B和T细胞HCV疫苗，这是U19提案的重点。这个具体项目将使用三个目标， 确定E1E2异二聚体、病毒颗粒和结合至E1E2异二聚体的中和抗体Fab的结构。 糖蛋白基于生物物理学和免疫学测定、计算建模和结构分析， E1E2糖蛋白将被工程化以呈递引起强中和和广泛反应性的表位 抗体并使诱饵非生产性表位的呈递最小化。此外，组织 将确定病毒颗粒的糖蛋白和结构。分子病毒学的工具， 生物物理学、计算建模、x射线晶体学和低温电子显微镜将用于产生 HCV表位景观的原子级细节。所获得的结构信息将用于设计 项目1中的新抗原，项目4中实验性地确定设计的免疫原的结构， 在项目1、2和3中进行了体内测试。我们将在项目期间的不同时间指导项目1，以使他们能够 利用最佳的E1E2免疫原在豚鼠中进行研究，并最终成为优化的 用于非人灵长类动物研究的E1E2免疫原。这些信息将是非常宝贵的解密的性质， 中和抗体及其对HCV的作用机制，并将揭示组装和 丙型肝炎病毒粒子的进入。

ABSTRACT – PROJECT 4 Despite extensive research efforts to understand the biology of hepatitis C virus (HCV), it remains a major human pathogen without a vaccine. In addition, there is a remarkable absence of information regarding the structure of the virion and organization of its component proteins. We intend to use our extensive structural biology experience with the related flaviviruses, to focus on determining the structure of the viral glycoproteins, the virus particle, and complexes of potent neutralizing antibodies bound to the surface glycoproteins. This project will rely heavily on and interact extensively with Project 1 to interrogate the structure and provide guidance into the features of the glycoproteins that are important for eliciting broad and potent neutralizing antibodies. Iterative analysis and re-engineering of the E1E2 glycoproteins will be conducted to guide the antibody component of a dual B and T cell HCV vaccine, the focus of this U19 proposal. This specific project will use three aims to determine the structure of the E1E2 heterodimer, the virus particle, and neutralizing antibody Fabs bound to the glycoproteins. Based on biophysical and immunological assays, computational modeling and structure analyses, the E1E2 glycoproteins will be engineered to present epitopes that elicit strong neutralizing and broadly reactive antibodies and minimize the presentation of decoy non-productive epitopes. Furthermore, the organization of the glycoproteins and the structure of the virus particle will be determined. The tools of molecular virology, biophysics, computational modeling, x-ray crystallography and cryo-electron microscopy will be used to produce atomic-level details of the epitope landscape of HCV. The gained structural information will be utilized to design novel antigens in Project 1, determine the structures of designed immunogens experimentally in Project 4 and tested in vivo in Projects 1, 2 and 3. We will guide Project 1 at various times during the project to allow them to utilize the optimal E1E2 immunogen for studies in guinea pigs and, ultimately, a lead candidate for an optimized E1E2 immunogen for nonhuman primate studies. This information will be invaluable in deciphering the nature of neutralizing antibodies and their mechanisms of action against HCV and will shed light on the assembly and entry of hepatitis C virions.