Biochemical and structural studies of distinct conformational states of gp41

HIV/SIV包膜糖蛋白组装成(gp120/gp41)3三聚体复合体，介导病毒 附着和膜融合。生化和结构研究提供了一个大致的图景 与病毒受体和辅受体的结合触发了一系列促进融合的构象变化。 在原子水平上对HIV/SIV环境构象的理解主要来自三组晶体 结构：未连接的SIV gp120核心片段、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。原住民的构象 (gp120/gp41)3病毒粒子表面，特别是其gp41组分的表面仍然未知，除了 一些来自电子显微镜的低分辨率图像。我们也缺乏一张重要的 Gp41从融合前到融合后构象的中间产物--所谓的“前发夹” 中间体“，这是T-20/恩福韦肽(第一个被批准的融合抑制抗病毒药物)和 某些广谱中和抗体，正如我们在初步数据中所描述的那样。填补我们在以下方面的空白 对包膜蛋白结构的了解将指导疫苗和疗法的发展。此外， 生产稳定、均一的具有特定构象的重组gp41制剂将有助于我们 了解中和的结构相关性，即使在没有高分辨率结构的情况下也是如此。在这 提案中，我们将探讨gp41的各种构象状态对我们的 通过生化和结构方法了解融合机制和抗体中和。我们 建议在此研究gp41的前融合构象、融合中间构象和融合后构象。 信息是我们的终极目标。我们将以初步结果为基础，表明我们可以表达和 描述每一种的适当形式。特别是，我们将追求以下具体目标：1.开展 Gp41“发夹前中间体”的生化和结构研究 预融合构象及其结构测定；3.膜相互作用构象的研究 融合后状态的gp41片段。

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.