Investigating the Architecture of HIV Envelope Glycoprotein

描述（由申请人提供）：人类免疫缺陷病毒（HIV）具有一系列特性，使其能够逃避抗体中和。由于对中和抗体的主要靶点HIV包膜糖蛋白（Env）的结构了解不足，合理的疫苗设计受到限制。由于存在柔性高变环和这种蛋白质的高碳水化合物含量，结构测定的进展受到阻碍，并且完整Env复合物的排列仍然存在争议。被认为掩盖保守位点的聚糖盾的性质也是关键的兴趣，因为它介导免疫逃逸。此外，已知宿主细胞CD4受体的结合诱导Env中的大量构象变化，但这些变化的详细检查尚未得到解决。该提案旨在将联合收割机两种互补的方法，小角X射线散射（SAXS）与从头形状重建和氢氘交换（HXMS）与质谱分析相结合，以解决有关三聚体Env结构的几个问题。Hu实验室提供的构建体的广泛目录使得能够在相同分离物的背景下对Env进行从单体亚基到三聚体复合物的比较研究。三聚体界面的鉴定、受体结合后诱导的构象变化以及可变环和聚糖的表位封闭可以用这些技术来解决。还将比较几种针对CD4结合位点的充分表征的单克隆抗体的相互作用，以解决抗体结合诱导的构象变化与CD4结合导致的构象变化有何不同以及是否与中和效力存在潜在相关性的问题。了解Env的这些特性将有助于目前的努力，以确定开发有效疫苗所必需的广泛中和抗体的目标。

DESCRIPTION (provided by applicant): The human immunodeficiency virus (HIV) employs an array of properties that enable it to evade antibody neutralization. Rational vaccine design has been limited due to the insufficient structural understanding of the HIV envelope glycoprotein (Env), the primary target for neutralizing antibodies. Advances in structural determination have been hindered due to the presence of flexible hypervariable loops and the high carbohydrate content of this protein and the arrangement of the full Env complex remains controversial. The nature of the glycan shield, thought to mask conserved sites, is also of key interest as it mediates immune escape. Furthermore the binding of host cell CD4 receptor is known to induce massive conformational changes in Env, but a detailed examination of these changes has yet to be addressed. This proposal aims to combine two complementary approaches, small-angle X-ray scattering (SAXS) with ab initio shape reconstruction and hydrogen-deuterium exchange (HXMS) with mass spectrometry analysis to address several questions regarding the architecture of trimeric Env. The extensive catalog of constructs available from the Hu lab enables comparative studies of Env ranging from monomeric subunits to trimeric complexes, in the context of the same isolate. Identification of the trimeric interface, conformational changes induced upon receptor binding, and epitope occlusion by variable loops and glycans can be addressed with these techniques. The interaction of several well-characterized monoclonal antibodies directed against the CD4 binding site will also be compared to address the question of how conformational changes induced by antibody binding differ from those resulting from CD4 binding, and whether there are potentially correlates with neutralizing potency. Understanding these properties of Env will assist current efforts to identify targets for development of broadly neutralizing antibodies, necessary for an effective vaccine.