HIV-1 Entry Inhibition by C-Peptide and 5-Helix Protein

C 肽和 5 螺旋蛋白抑制 HIV-1 进入

• 批准号: 7115225
• 负责人: MICHAEL JEFFREY ROOT
• 金额: $ 26.83万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7115225
• 关键词: HIV envelope protein gp41; X ray crystallography; antiAIDS agent; chemical kinetics; human immunodeficiency virus 1; membrane fusion; peptide structure; peptides; protein protein interaction; protein structure function; site directed mutagenesis; stoichiometry; thermodynamics

The HIV-1 surface glycoprotein Env (gp120/gp41) mediates viral entry through a series of coordinated structural changes initiated when CD4 interacts with gp120. Ultimately, the gp41 ectodomain folds into a compact trimer-of-hairpins that brings the viral and cellular membranes into the close proximity required for efficient membrane fusion. These conformational transitions have been delineated, in part, through the use of C-peptide and 5-Helix inhibitors that target the HR1 and HR2 regions of gp41 and block trimer-of-hairpins formation. Our previous work has described the physical basis behind C-peptide and 5-Helix inhibition and quantitatively modeled the relationship between inhibitory potency and fusion kinetics. The experiments outlined in this revised proposal explore the structure and dynamics of Env in its native and intermediate conformations. The research will address three fundamental questions regarding the mechanism of viral membrane fusion: How does CD4 binding to gp120 trigger formation of the prehairpin intermediate state? What is the structure of Env in this intermediate conformation? What aspects of gp41 folding from the prehairpin state drive the fusion of viral and cellular membranes? Owing to the unstable nature of Env in its native and intermediate states, these questions are often difficult to address using standard experimental techniques, such as scanning mutagenesis, that require stable proteins in long lived conformations. Our basic approach will be to generate new inhibitors that target the HR1 and HR2 regions, and dissect the mechanism of resistance to them. This strategy takes advantage of the power of natural selection to generate wellbehaved mutant Env variants with interesting structural or dynamic properties. The specific aims are: 1) to explore the structure and exposure of the HR1 region using novel inhibitors engineered to overcome standard C-peptide resistance profiles; 2) to probe the structure and exposure of the HR2 region using multivalent inhibitors, and to dissect the mechanisms of resistance to these agents; and 3) to explore the energetic requirements of Env-mediated membrane fusion, and to examine the structure of mutant Env variants trapped in intermediate conformations. Our findings will provide new insights into the dynamic properties of the gp120/gp41 complex critical to the viral entry mechanism and will aide the development of new viral entry inhibitors and HIV-1 vaccines.

HIV-1 表面糖蛋白 Env (gp120/gp41) 通过一系列介导病毒进入 CD4 与 gp120 相互作用时启动协调的结构变化。最终，gp41 胞外域折叠成紧凑的发夹三聚体，将病毒和细胞膜带入 达到有效膜融合所需的紧密距离。这些构象转变 部分是通过使用靶向 C 肽和 5-螺旋抑制剂 gp41 的 HR1 和 HR2 区域并阻断发夹三聚体的形成。我们之前的工作有 描述了 C 肽和 5-螺旋抑制背后的物理基础并进行了定量建模 抑制效力和融合动力学之间的关系。本文概述的实验 修订后的提案探索了 Env 在其原生和中间体中的结构和动态 构象。该研究将解决有关该机制的三个基本问题 病毒膜融合的过程：CD4 与 gp120 结合如何触发前发夹的形成 中间状态？这个中间构象中Env的结构是什么？哪些方面 gp41 从发夹前状态折叠驱动病毒和细胞膜的融合？欠 由于 Env 在其自然状态和中间状态下的不稳定性质，这些问题常常是 使用标准实验技术（例如扫描诱变）很难解决这一问题 需要具有长寿命构象的稳定蛋白质。我们的基本方法是产生新的 针对 HR1 和 HR2 区域的抑制剂，并剖析它们的耐药机制。 该策略利用自然选择的力量来产生行为良好的突变体 具有有趣的结构或动态特性的环境变体。具体目标是：1）探索 使用新型抑制剂来克服 HR1 区域的结构和暴露 标准 C 肽耐药性概况； 2) 探测HR2区域的结构和暴露 使用多价抑制剂，并剖析对这些药物的耐药机制； 3) 至 探索 Env 介导的膜融合的能量需求，并检查 陷入中间构象的突变型 Env 变体的结构。我们的研究结果将提供 对 gp120/gp41 复合物动态特性的新见解对病毒进入至关重要 机制并将有助于新病毒进入抑制剂和 HIV-1 疫苗的开发。