Investigating the Architecture of HIV Envelope Glycoprotein

描述(由申请人提供)：人类免疫缺陷病毒(HIV)使用一系列使其能够逃避抗体中和的特性。由于对HIV包膜糖蛋白(Env)的结构了解不足，合理的疫苗设计一直受到限制。由于存在灵活的高变环和该蛋白的高碳水化合物含量，以及完整的Env复合体的排列仍然存在争议，结构测定的进展受到了阻碍。被认为可以掩盖保守部位的葡聚糖屏障的性质也是人们关注的关键，因为它介导了免疫逃避。此外，宿主细胞CD4受体的结合已知会导致Env的大量构象变化，但对这些变化的详细研究尚未得到解决。这一建议旨在将两种互补的方法结合起来，即小角X射线散射(SAXS)与从头计算形状重建和氢-氚交换(HXMS)与质谱分析相结合，以解决与三聚体环境的结构有关的几个问题。HU实验室提供的广泛的构建物目录使在同一分离株的背景下对从单体亚单位到三聚体复合体的环境蛋白进行了比较研究。三聚体界面的鉴定、受体结合引起的构象变化以及可变环和多糖对表位的封闭都可以用这些技术来解决。还将比较针对CD4结合部位的几种特征良好的单抗之间的相互作用，以解决抗体结合引起的构象变化与CD4结合引起的构象变化有何不同，以及是否存在潜在的与中和效力相关的问题。了解Env的这些特性将有助于目前的努力，以确定开发广泛中和抗体的目标，这是有效疫苗所必需的。

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.