Biochemical and structural studies of distinct conformational states of gp41

描述（由申请人提供）：HIV/SIV的包膜糖蛋白，组装成（gp120/gp41）3三聚体复合物，介导病毒附着和膜融合。生物化学和结构研究已经提供了与病毒受体和共受体结合如何触发一系列促进融合的构象变化的总体图景。在原子水平上对HIV/SIV Env构象的理解主要来自三组晶体结构：未配体的SIV gp120核心片段，受体诱导的HIV gp120核心构象，以及HIV和SIV gp41融合后的构象。除了一些低分辨率的电子显微镜图像外，病毒粒子表面的天然（gp120/gp41）3的构象，特别是其gp41组分的构象仍然未知。我们还缺乏gp41从融合前到融合后构象通路中的重要中间体的适当图片-所谓的“预发夹中间体”，它是T-20/Entfuvirtide（第一个批准的融合抑制抗病毒药物）和某些广泛中和抗体的靶标，正如我们在初步数据中所描述的那样。填补我们对包膜蛋白结构知识的这些空白将指导疫苗和治疗方法的开发。此外，即使在没有高分辨率结构的情况下，稳定、均匀的重组gp41确定构象的制备将有助于我们了解中和的结构相关性。在本提案中，我们将探讨gp41的各种构象状态对我们通过生化和结构方法理解融合机制和抗体中和至关重要的假设。我们建议研究gp41在融合前、融合中间和融合后的构象，并以结构信息为最终目标。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，我们将努力实现以下目标：开展gp41“预发夹中间体”的生化和结构研究；2. 表征预熔构象中的gp41并确定其结构；3. 研究gp41在融合后状态下与膜相互作用片段的构象。公共卫生相关性：仅在2006年，就有近4 000万人感染了人体免疫缺陷病毒（艾滋病毒），400万人新感染了艾滋病毒（艾滋病规划署/世卫组织艾滋病流行最新情况，2006年）。我们迫切需要一种有效的疫苗来阻止这一流行病。拟议的研究将使我们能够非常详细地可视化病毒包膜糖蛋白的组织，这是宿主免疫反应和一些新一代抗病毒药物（称为融合抑制剂）的主要目标。从这些实验中获得的信息有望指导艾滋病疫苗和抗病毒疗法的开发。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Nearly 40 million people are living with human immunodeficiency virus (HIV) and 4 million people are newly infected with HIV in 2006 alone (UNAIDS/WHO AIDS epidemic update, 2006). An effective vaccine is urgently needed to stop this epidemic. The proposed studies will allow us to visualize in great detail the organization of the viral envelope glycoprotein, which is the main target by host immune responses and by some new-generation antiviral drugs, called fusion inhibitors. The information obtained from these experiments is expected to guide development of both AIDS vaccine and antiviral therapeutics.