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（由申请方提供）：人类免疫缺陷病毒（HIV-1）进入由病毒包膜糖蛋白（Env）介导。Env的gp 120结构域与CD 4的结合导致暴露辅助受体结合位点的构象变化。与辅助受体的相互作用激活Env的gp 41结构域，以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有吸引力的靶标，因为它是暴露在HIV-1病毒体表面上的唯一病毒蛋白。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）竞争性抑制而无变构活化; 2）在不存在靶细胞膜的情况下过早变构活化;和3）转向为途径外构象。本申请提出测试Env的基态构象的稳定化（其防止Env的活化）是未充分利用的抗病毒策略的假设。对这一假设的支持是基于中和高达98%的所有HIV-1分离株的抗体对Env构象的影响。为了验证这一假设，我们已经建立了单分子荧光共振能量转移（smFRET）成像方法，直接可视化的天然三聚体内的完整病毒体表面上的单个Env分子的构象动力学。引入HIV-1的NL 4 -3株的gp 120的可变区V1、V4和V5中的荧光团允许在完整病毒体表面上的天然Env三聚体的背景下对单个gp 120结构域的结构动力学进行时间分辨监测。这些数据表明，未配体的HIV-1 Env是动态的，并且内在地接近受体和辅助受体稳定的构象。HIV-1 Env的smFRET的建立允许深入了解这种分子机器的内部工作原理以及它如何通过两步受体和辅助受体机制激活融合。此外，它提供了一种快速和可靠的测定HIV-1 Env在病毒体表面的构象状态。使用这种方法，我们已经表明，广泛中和抗体，VRC 01，PG 16，PGT 128，PGT 145和2G 12，稳定HIV-1 Env在其基态构象，尽管以根本不同的方式接合Env。VRC 01识别CD 4结合位点中的表位; PG 16、PGT 128和PGT 145在三聚体的顶点处结合V1/V2; 2G 12识别V3环底部的保守聚糖。这些结果表明，稳定的基态，而不是一个激活的中间或关闭途径构象代表了一个强大的抗病毒策略。值得注意的是，上述实验使用中和敏感性NL 4 -3 HIV-1分离株进行。在这里，我们将测试广泛中和抗体是否对来自临床HIV-1分离株的Env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，其可能类似地稳定基态。在这样做时，所提出的研究有可能建立基态稳定化是拮抗病毒膜融合机器的有效方法。