Single molecule imaging of HIV Env

描述(申请人提供)：尽管作出了巨大努力，但目前还没有有效的艾滋病毒/艾滋病疫苗可用。这在很大程度上是由于病毒能够逃避中和抗体。三聚体HIV包膜糖蛋白gp160(HIV Env)暴露于病毒粒子表面的宿主免疫反应中，有效地隐藏了抗体的表位，同时保持了促进病毒进入的能力。少数暴露的抗体结合位点在结构上是不稳定的，并保持着相当大的序列可变性。因此，很少有广谱中和抗体被鉴定出来。在这里，我们将开发单分子荧光共振能量转移(SmFRET)成像技术来监测单个天然环境病毒分子在HIV病毒表面的构象。我们将应用新的酶促方法，在不干扰传染性的位置将有机荧光团引入HIV Env。将两种染料放置在一个环境分子中将允许 应用smFRET从多角度报道未连接的HIV env的构象变化。在鉴定了未连接的HIV env之后，我们将确定它的构象动力学如何随着受体和抗体的结合而变化。我们的方法将确定HIV Env的关键构象状态，这些状态应该作为疫苗开发的目标，揭示免疫逃避的分子机制，并了解现有中和抗体的效力。

DESCRIPTION (provided by applicant): Despite enormous efforts, no effective vaccine is currently available against HIV/AIDS. This is largely due to the virus's ability to evade neutralizing antibodies. The trimeric HIV envelope glycoprotein gp160 (HIV Env), which is exposed to the host immune response on the surface of the virion, effectively conceals epitopes from antibodies while maintaining its ability to promote viral entry. The few exposed antibody-binding sites are structurally unstable and sustain substantial sequence variability. Consequently, few broadly neutralizing antibodies have been identified. Here we will develop single molecule Fluorescence Resonance Energy Transfer (smFRET) imaging to monitor the conformational landscape of individual native Env molecules on the surface of an HIV virus. We will apply new enzymatic methods to introduce organic fluorophores into HIV Env at positions that don't interfere with infectivity. The placement of two dyes within one Env molecule will allow the application of smFRET to report conformational changes of the unliganded HIV Env from various angles. Following the characterization of the unliganded HIV Env, we will determine how its conformation dynamics change in response to receptor and antibody binding. Our approach will identify critical conformational states of HIV Env that should be targeted for vaccine development, reveal the molecular mechanism underlying immune evasion, and understand the potency of existing neutralizing antibodies.