Structural dynamics of HIV Env in unliganded, receptor-bound, inhibited

项目六：Mothes/Blanchard结合的HIV-1包膜的构象轨迹总结该项目所追求的针对HIV Env的有效抗病毒疗法的设计需要一种快速方法来确定小分子抑制剂抑制HIV Env的潜在分子机制。为此，我们已经建立了单分子荧光共振能量转移（smFRET）方法，直接可视化的HIV Env的结构动力学的背景下，完整的三聚体的表面上的HIV病毒粒子。作为概念的证明，我们已经成功地将smFRET应用于X4-tropic HIV-1 NL 4 - 3。我们的工作表明，未配体的HIV Env是构象动态的，并且本质上能够对受体CD 4和17 b稳定的构象进行采样。重要的是，辅助受体结合位点更可能从CD4诱导状态进入。CD4的结合降低了CD4稳定构象的能量，并使HIV Env准备好与共受体结合。这些数据解释了HIV Env是如何被CD4和辅助受体以逐步的方式激活的。在这里，我们将扩展应用smFRET，包括生理相关的R5嗜性HIV-1 Env分离株。双重修饰的HIV-1 Env变体将用于确定由CD4模拟物和拮抗剂诱导的HIV Env的构象轨迹。这将允许在诱导CD4样构象变化的CD4模拟物如NBD 556和拮抗剂如DMJ-1 - 228之间进行区分。smFRET成像还将允许理解通过过早激活Env、动力学陷阱或变构地将Env转移到关闭途径中而导致HIV-1失活的构象分子事件。因此，这些smFRET技术将提供一个关键的工具，以确定HIV Env拮抗和灭活的潜在分子机制，并帮助合理设计利用HIV内在脆弱性的抗病毒疗法。smFRET技术将通过筛选阻断、捕获或抑制HIV-1 Env的所需构象效应来加速药物发现。

Project 6: Mothes/Blanchard Conformational trajectories of inhibitor-bound HIV-1 envelope Summary The design of effective antiviral therapies directed against HIV Env pursued by this program requires a fast method to determine the underlying molecular mechanism of HIV Env inhibition by small molecule inhibitors. Towards this end, we have established single-molecule Fluorescence Resonance Energy Transfer (smFRET) methods to visualize directly the structural dynamics of HIV Env in the context of the complete trimer on the surface of HIV virions. As a proof of concept we have successfully applied smFRET to the X4-tropic HIV-1 NL4-3- Our work reveals that the unliganded HIV Env is conformationally dynamic and intrinsically capable of sampling receptor CD4- and 17b-stabilized conformations. Importantly, the co-receptor binding site is more likely to be accessed from the CD4-induced state. Binding of CD4 lowers the energy of the CD4-stabilized conformation and prepares the HIV Env for co-receptor binding. These data explain how HIV Env is activated in a stepwise manner by CD4 and the co-receptor. Here, we will extend the application of smFRET to include physiologically relevant R5-tropic HIV-1 Env isolates. Dually modified HIV-1 Env variants will be used to determine the conformational trajectories of HIV Env induced by CD4 mimetics and antagonists. This will allow distinction between CD4 mimics such as NBD556 that induce CD4-like conformational changes and antagonists such as DMJ-l-228. smFRET imaging will also permit an understanding of conformational molecular events underlying HlV-1 inactivation either by prematurely activating Env, kinetic traps or allosterically diverting Env into off-pathways. As such, these smFRET technologies will provide a critical tool to determine the underlying molecular mechanism of HIV Env antagonism and inactivation developed by the Program Project Team and aid in a rational design of antiviral therapies that exploit the intrinsic vulnerabilities of HIV. smFRET technologies will accelerate pharmaceutical discovery by enabling screening for desired conformational effects that block, trap or inactivate HIV-1 Env.