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，以A（GP120/GP41）3三聚体复合物组装，介导病毒 附着和膜融合。生化和结构研究提供了有关如何 与病毒受体和共受体的结合触发了一系列融合构象变化的级联反应。 对原子水平上的艾滋病毒/siv env构象的理解主要来自三组晶体 结构：无配合的SIV GP120核心片段的结构，艾滋病毒构象的碎片 GP120核心，以及HIV和SIV GP41的后发现形式的核心。当地人的构象 （gp120/gp41）3在病毒体的表面，尤其是其gp41成分的表面，除了 电子显微镜的一些低分辨率图像。我们也缺乏重要的图片 从预融合到gp41的后灌注构象的途径中的中间体 - 所谓的“前” 中级“这是T-20/entfuvirtide（首次批准抑制抗病毒药物）和的目标 正如我们在初步数据中所描述的那样，某些广泛中和抗体。在我们的 包膜蛋白结构的知识将指导疫苗和治疗剂的开发。而且， 在定义的构象中生产重组GP41的稳定，均质制剂将有助于我们 即使在没有高分辨率结构的情况下，也可以理解中和的结构相关。在这个 提案，我们将探讨以下假设：GP41的各种构象状态符合我们的关键 通过生化和结构方法了解融合机制和抗体中和。我们 在这里提议以结构性研究GP41在其预融合，融合中间和融合后构象中进行研究 信息作为我们的最终目标。我们将以初步结果为基础，表明我们可以表达并 表征每种的合适形式。特别是，我们将追求以下特定目标：1。执行 GP41的“前中间体”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 GP41在灌注后状态中的细分市场。