Structural analysis of HCV E1E2 glycoproteins

摘要--项目4 尽管为了解丙型肝炎病毒(丙型肝炎病毒)的生物学做出了广泛的研究，但它仍然是人类的主要病毒 没有疫苗的病原体。此外，关于结构的信息也明显缺乏 病毒粒子及其组成蛋白的组织。我们打算用我们广泛的结构生物学 与黄病毒相关的经验，重点是确定病毒糖蛋白的结构，病毒 颗粒，以及结合到表面糖蛋白上的有效中和抗体的复合体。这个项目将依赖于 重点关注项目1并与其广泛互动，以审问结构并提供指导 糖蛋白的特征，对于诱导广泛而有效的中和抗体是重要的。迭代 将对E1E2糖蛋白进行分析和重组，以指导A型流感病毒的抗体成分 双B和T细胞丙型肝炎病毒疫苗，这是U19提案的重点。这个具体项目将使用三个目标来 确定E1E2异源二聚体的结构、病毒颗粒和与 糖蛋白。基于生物物理和免疫学分析、计算模型和结构分析， 将对E1E2糖蛋白进行改造，使其呈现能够引起强烈中和和广泛反应的表位。 抗体，并最大限度地减少呈现诱饵非生产性表位。此外，组织 病毒颗粒的糖蛋白和结构将被确定。分子病毒学的工具， 生物物理学、计算模型、x射线结晶学和冷冻电子显微镜将被用于生产 丙型肝炎病毒表位格局的原子级细节。所获得的结构信息将被用于设计 项目1中的新抗原，项目4和项目4中实验确定设计的免疫原的结构 在项目1、2和3中进行了活体测试。我们将在项目期间的不同时间指导项目1，以允许他们 利用最优的E1E2免疫原在豚鼠身上进行研究，并最终成为优化的 用于非人灵长类研究的E1E2免疫原。这些信息将对破译 抗丙型肝炎病毒的中和抗体及其作用机制，将有助于阐明 丙型肝炎病毒粒子进入。

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.