HIV-1 Env antagonism by ground state stabilization

通过基态稳定对抗 HIV-1 Env

• 批准号: 9029296
• 负责人: James B Munro
• 金额: $ 10.64万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-05 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9029296
• 关键词: Antibodies; Antiviral Agents; Antiviral Therapy; Binding; Binding Sites; Biological Assay; Cell membrane; Cells; Cellular Membrane; Clinical; Color; Data; Development; Dyes; Epitopes; Fluorescence Microscopy; HIV; HIV Envelope Protein gp120; HIV-1; Health; Image; Immunoglobulin Variable Region; Individual; Kinetics; Laboratories; Mediating; Membrane Fusion; Molecular Conformation; Molecular Machines; Monitor; Nature; Pathway interactions; Polysaccharides; Shapes; Structure; Surface; Testing; Time; V3 Loop; Vaccines; Viral; Viral Proteins; Virion; Work; base; design; env Glycoproteins; fluorophore; imaging modality; inhibitor/antagonist; insight; neutralizing antibody; novel; premature; prevent; receptor; receptor binding; research study; single molecule; single-molecule FRET; small molecule therapeutics; therapeutic development

 DESCRIPTION (provided by applicant): Human Immunodeficiency Virus (HIV-1) entry is mediated by the viral envelope glycoprotein (Env). Binding of the gp120 domain of Env to CD4 leads to conformational changes that expose the coreceptor binding site. Interaction with the coreceptor activates the gp41 domain of Env to promote fusion between the viral and cellular membranes. HIV-1 Env remains an attractive target for vaccines and antiviral therapies as it is the only viral protein exposed on the surface of HIV-1 virions. Based on the metastable nature of the unliganded HIV-1 Env trimer, antiviral strategies have followed three main concepts: 1) competitive inhibition without allosteric activation; 2) premature allosteric activation in the absence of target cell membranes; and 3) diversion into off- pathway conformations. This application proposes to test the hypothesis that the stabilization of the ground state conformation of Env, which prevents the activation of Env, is an underutilized antiviral strategy. Support for this hypothesis is based on the impact on Env conformation of antibodies that neutralize up to 98% of all HIV-1 isolates. To test this hypothesis, we have established single-molecule fluorescence resonance energy transfer (smFRET) imaging methods to directly visualize the conformational dynamics of single Env molecules within the native trimer on the surface of intact virions. Fluorophores introduced into variable regions V1, V4, and V5 of gp120 of the NL4-3 strain of HIV-1 allowed the time-resolved monitoring of structural dynamics of individual gp120 domains within the context of the native Env trimer on the surface of intact virions. These data reveal that the unliganded HIV-1 Env is dynamic and intrinsically accesses the receptor- and coreceptor- stabilized conformations. The establishment of smFRET for HIV-1 Env allows insights into the inner workings of this molecular machine and how it is activated for fusion by the two-step receptor and coreceptor mechanism. Moreover, it offers a fast and reliable assay for the conformational state of HIV-1 Env on the surface of virions. Using this approach we have shown that the broadly neutralizing antibodies, VRC01, PG16, PGT128, PGT145, and 2G12, stabilize HIV-1 Env in its ground-state conformation despite engaging Env in fundamentally different ways. VRC01 recognizes an epitope in the CD4-binding site; PG16, PGT128, and PGT145 bind V1/V2 at the apex of the trimer; and 2G12 recognizes conserved glycans at the base of the V3 loop. These results suggest that stabilization of the ground state but not of an activated intermediate or off- pathway conformation represents a powerful antiviral strategy. Notably, the above experiments were performed with the neutralization-sensitive NL4-3 HIV-1 isolate. Here, we will test whether broadly neutralizing antibodies similarly function on Env from clinical HIV-1 isolates. We will also test the conformational consequences of entry inhibitors, such as BMS-626529 that may similarly stabilize the ground state. In so doing, the proposed studies have the potential to establish that ground state stabilization is an effective way to antagonize viral membrane fusion machines.

 描述（由申请人提供）：人类免疫缺陷病毒（HIV-1）的进入是由病毒包膜糖蛋白（Env）介导的。 Env 的 gp120 结构域与 CD4 的结合导致构象变化，从而暴露出辅助受体结合位点。与辅助受体的相互作用激活 Env 的 gp41 结构域，促进病毒和细胞膜之间的融合。 HIV-1 Env 仍然是疫苗和抗病毒治疗的一个有吸引力的目标，因为它是唯一暴露在 HIV-1 病毒颗粒表面的病毒蛋白。基于未配体的HIV-1 Env三聚体的亚稳态性质，抗病毒策略遵循三个主要概念：1）无变构激活的竞争性抑制； 2）在靶细胞膜不存在的情况下过早变构激活； 3) 转向非途径构象。本申请旨在测试这样的假设：稳定 Env 基态构象可防止 Env 激活，是一种未充分利用的抗病毒策略。这一假设的支持是基于抗体对 Env 构象的影响，这些抗体可以中和高达 98% 的 HIV-1 分离株。为了检验这一假设，我们建立了单分子荧光共振能量转移（smFRET）成像方法，可以直接可视化完整病毒粒子表面天然三聚体内单个 Env 分子的构象动力学。将荧光团引入 HIV-1 NL4-3 毒株的 gp120 可变区 V1、V4 和 V5 中，可以在完整病毒粒子表面的天然 Env 三聚体背景下对单个 gp120 结构域的结构动态进行时间分辨监测。这些数据表明，未配体的 HIV-1 Env 是动态的，并且本质上接近受体和辅助受体稳定的构象。 HIV-1 Env 的 smFRET 的建立可以深入了解该分子机器的内部运作以及它如何被两步受体和共受体机制激活以进行融合。此外，它还提供了一种快速可靠的检测病毒颗粒表面 HIV-1 Env 构象状态的方法。使用这种方法，我们已经证明，广泛中和抗体 VRC01、PG16、PGT128、PGT145 和 2G12 可以将 HIV-1 Env 稳定在其基态构象，尽管以根本不同的方式参与 Env。 VRC01 识别 CD4 结合位点中的表位； PG16、PGT128 和 PGT145 在三聚体的顶端结合 V1/V2； 2G12 识别 V3 环底部的保守聚糖。这些结果表明，基态的稳定而不是激活的中间或离路构象的稳定代表了一种强大的抗病毒策略。值得注意的是，上述实验是用中和敏感的 NL4-3 HIV-1 分离株进行的。在这里，我们将测试广泛中和抗体对临床 HIV-1 分离株的 Env 是否具有类似的功能。我们还将测试进入抑制剂的构象后果，例如 BMS-626529，它可能同样稳定基态。通过这样做，拟议的研究有可能确定基态稳定是对抗病毒膜融合机器的有效方法。