Single molecule imaging of HIV Env

描述（由申请人提供）：尽管付出了巨大的努力，目前还没有有效的疫苗可用于预防艾滋病毒/艾滋病。这在很大程度上是由于病毒逃避中和抗体的能力。三聚体HIV包膜糖蛋白gp 160（HIV Env）暴露于病毒体表面的宿主免疫应答，有效地隐藏抗体的表位，同时保持其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，并维持实质性的序列变异性。因此，很少有广泛的中和抗体已被确定。在这里，我们将开发单分子荧光共振能量转移（smFRET）成像，以监测HIV病毒表面单个天然Env分子的构象景观。我们将应用新的酶促方法将有机荧光团引入HIV Env中不干扰感染性的位置。在一个Env分子内放置两种染料将允许 应用smFRET从多个角度报道了未配体的HIV Env的构象变化。在对未配体的HIV Env进行表征之后，我们将确定其构象动力学如何响应于受体和抗体结合而变化。我们的方法将确定HIV Env的关键构象状态，这些构象状态应该成为疫苗开发的目标，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.