HIV-1 Env antagonism by ground state stabilization

 描述(申请人提供)：人类免疫缺陷病毒(HIV-1)由病毒包膜糖蛋白(Env)介导进入。Env的gp120结构域与CD4结合会导致构象变化，暴露辅助受体结合部位。与辅助受体的相互作用激活了Env的gp41结构域，促进了病毒和细胞膜之间的融合。由于HIV-1Env是暴露在HIV-1病毒粒子表面的唯一病毒蛋白，因此它仍然是疫苗和抗病毒治疗的有吸引力的靶标。基于未连接的HIV-1包膜三聚体的亚稳性质，抗病毒策略遵循三个主要概念：1)竞争抑制而不是变构激活；2)在没有靶细胞膜的情况下过早的变构激活；以及3)转移到非途径构象。这项应用提出了一种假设，即稳定Env的基态构象，防止Env的激活，是一种未得到充分利用的抗病毒策略。对这一假设的支持是基于抗体对环境构象的影响，这些抗体中和了高达98%的HIV-1分离株。为了验证这一假设，我们建立了单分子荧光共振能量转移(SmFRET)成像方法，直接显示完整病毒粒子表面天然三聚体中单个Env分子的构象动力学。将荧光团引入HIV-1株NL4-3株gp120的V1、V4和V5可变区，可以在完整病毒粒子表面的天然环境三聚体的背景下，对单个gp120结构域的结构动力学进行时间分辨监测。这些数据表明，未连接的HIV-1 env是动态的，本质上访问受体和辅助受体稳定的构象。针对HIV-1 Env的smFRET的建立使人们能够深入了解这一分子机器的内部工作原理，以及它是如何被两步受体和辅助受体机制激活以进行融合的。此外，它还为病毒粒子表面HIV-1Env的构象状态提供了一种快速、可靠的分析方法。使用这种方法，我们已经证明了广泛的中和抗体，VRC01，PG16，PGT128，PGT145和2G12，尽管以根本不同的方式与Env结合，但仍然稳定了HIV-1Env的基态构象。VRC01识别CD4结合位点的表位；PG16、PGT128和PGT145在三聚体的顶端与V1/V2结合；2G12在V3环的底部识别保守的糖链。这些结果表明，稳定基态而不是激活的中间或非途径构象代表了一种有效的抗病毒策略。值得注意的是，上述实验是用对中和敏感的NL4-3HIV-1分离株进行的。在这里，我们将测试广谱中和抗体是否对来自临床HIV-1分离株的环境病毒起类似的作用。我们还将测试进入抑制剂的构象结果，例如BMS-626529，它可能类似地稳定基态。通过这样做，所提出的研究有可能确定基态稳定是对抗病毒膜融合机的有效方法。

 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.