Biochemical and structural studies of distinct conformational states of gp41

gp41 不同构象状态的生化和结构研究

• 批准号: 8243563
• 负责人: Bing Chen
• 金额: $ 35.39万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8243563
• 关键词: AIDS Vaccines; Acquired Immunodeficiency Syndrome; Affinity Chromatography; Antibodies; Antiviral Agents; Binding; Biochemical; Biological Assay; Cell membrane; Cells; Characteristics; Complement; Complex; Crystallization; Data; Detergents; Development; Electron Microscopy; Electrons; Epidemic; Epitopes; Escherichia coli; Generations; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV-1; Image; Immune response; In Vitro; Insecta; Knowledge; Life; Light; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutate; NMR Spectroscopy; Pathway interactions; Peptides; Preparation; Production; Property; Proteins; Recombinants; Residual state; Resolution; SIV; SIV envelope protein gp41; Screening procedure; Series; Site; Structure; Surface; T-20; Testing; Therapeutic; Transmembrane Domain; Update; Vaccines; Viral; Virion; Virus Receptors; base; conformational conversion; design; env Gene Products; glycosylation; in vivo; inhibitor/antagonist; insight; neutralizing antibody; protein S precursor; protein structure; receptor; research study; retinal rods; simian immunodeficiency virus gp120; therapeutic vaccine; urinary gonadotropin fragment; vaccine development

The envelope glycoproteins of HIV/SIV, assembled as a (gp120/gp41)3 trimeric complex, mediate viral attachment and membrane fusion. Biochemical and structural studies have provided a general picture of how binding to viral receptor and co-receptor triggers a cascade of fusion-promoting conformational changes. Understanding of HIV/SIV Env conformations at the atomic level has come mainly from three sets of crystal structures: that of an unliganded SIV gp120 core fragment, those of receptor-induced conformations of the HIV gp120 core, and those of the postfusion form of HIV and SIV gp41. The conformation of the native (gp120/gp41)3 on the surface of the virion, especially that of its gp41 component, remains unknown, except for some low-resolution images from electron microscopy. We also lack a proper picture of an important intermediate in the pathway from the prefusion to postfusion conformation of gp41 - the so-called "prehairpin intermediate" that is the target of T-20/Entfuvirtide (the first approved fusion-inhibiting antiviral drug) and of certain broadly neutralizing antibodies, as we describe in the Preliminary Data. Filling these gaps in our knowledge of envelope protein structures will guide development of vaccines and therapeutics. Moreover, production of stable, homogeneous preparations of recombinant gp41 in defined conformations will help us understand structural correlates for neutralization, even in the absence of high-resolution structures. In this proposal, we will explore the hypothesis that the various conformational states of gp41 hold key to our understanding of fusion mechanism and antibody neutralization by biochemical and structural approaches. We propose here to study gp41 in its prefusion, fusion-intermediate, and postfusion conformations, with structural information as our ultimate goal. We will build upon preliminary results that show we can express and characterize suitable forms of each. In particular, we will pursue the following specific aims: 1. To carry out biochemical and structural studies of the "prehairpin intermediate" of gp41; 2. To characterize gp41 in the prefusion conformation and determine its structure; 3. To study the conformation of the membrane-interacting segments of gp41 in the postfusion state.

HIV/SIV 的包膜糖蛋白组装为 (gp120/gp41)3 三聚体复合物，介导病毒传播 附着和膜融合。生物化学和结构研究提供了一个关于如何 与病毒受体和辅助受体的结合引发一系列促进融合的构象变化。 对 HIV/SIV Env 构象在原子水平上的理解主要来自于三组晶体 结构：未配体的 SIV gp120 核心片段的结构、HIV 受体诱导构象的结构 gp120 核心，以及 HIV 和 SIV 融合后形式的 gp41。原生的构象 病毒颗粒表面的 (gp120/gp41)3，尤其是其 gp41 成分，仍然未知，除了 一些来自电子显微镜的低分辨率图像。我们还缺乏对重要事件的正确认识 gp41 从融合前构象到融合后构象途径的中间体 - 所谓的“前发夹” 中间体”，是 T-20/Entfuvirtide（第一个批准的融合抑制抗病毒药物）和 正如我们在初步数据中所描述的，某些广泛中和抗体。填补我们的这些空白 对包膜蛋白结构的了解将指导疫苗和治疗方法的开发。而且， 生产具有确定构象的稳定、均质的重组 gp41 制剂将有助于我们 即使没有高分辨率结构，也能了解中和的结构相关性。在这个 提案中，我们将探索 gp41 的各种构象状态对我们的研究至关重要的假设 通过生化和结构方法了解融合机制和抗体中和。我们 在此建议研究 gp41 的融合前、融合中间和融合后构象，以及结构 信息作为我们的最终目标。我们将建立在初步结果的基础上，这些结果表明我们可以表达和 描述每种形式的合适形式。具体而言，我们将追求以下具体目标： 1. 开展 gp41“前发夹中间体”的生化和结构研究； 2. 表征 gp41 预融合构象并确定其结构； 3. 研究膜相互作用的构象 处于融合后状态的 gp41 片段。