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

本项目所追求的针对HIV Env的有效抗病毒疗法的设计需要一种快速的方法来确定小分子抑制剂抑制HIV Env的潜在分子机制。为此，我们建立了单分子荧光共振能量转移（smFRET）方法，在HIV病毒粒子表面完整三聚体的背景下，直接可视化HIV Env的结构动力学。作为概念的证明，我们已经成功地将smFRET应用于X4-tropic HIV-1 NL4-3。我们的工作表明，非配体HIV Env具有构象动态，并且本质上能够采样受体CD4-和17b-稳定的构象。重要的是，共受体结合位点更有可能从cd4诱导状态进入。CD4的结合降低了CD4稳定构象的能量，使HIV Env为共受体结合做好了准备。这些数据解释了HIV Env是如何被CD4和共受体逐步激活的。在这里，我们将扩展smFRET的应用，包括生理相关的R5-tropic HIV-1 Env分离株。双重修饰的HIV-1 Env变异将用于确定CD4模拟物和拮抗剂诱导的HIV Env的构象轨迹。这将区分CD4模拟物（如诱导CD4样构象变化的NBD556）和拮抗剂（如DMJ-l-228）。smFRET成像还可以通过过早激活Env、动力学陷阱或变构转移Env进入通路来了解HlV-1失活的构象分子事件。因此，这些smFRET技术将为确定项目项目组开发的HIV Env拮抗和失活的潜在分子机制提供关键工具，并有助于合理设计利用HIV内在脆弱性的抗病毒疗法。smFRET技术将通过筛选阻断、捕获或灭活HIV-1 Env的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.