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: 6944848
• 负责人: MICHAEL JEFFREY ROOT
• 金额: $ 26.09万
• 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 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6944848
• 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

DESCRIPTION (provided by applicant): As more strains of human immunodeficiency type 1 (HIV-1), the virus that causes AIDS, become resistant to existing therapies, it is important to identify and characterize new targets for viral inhibition. The HIV-1 glycoprotein Env (gp120/gp41) is an attractive target because it mediates the initial stages of infection-viral attachment and membrane fusion. The interaction of Env with cellular receptors triggers conformational changes that ultimately lead to formation of a crucial trimer-of-hairpins structure in which the N- and C- terminal regions of the gp4 1 ectodomain associate. Peptides derived fiom the C-terminal region (C-peptides) inhibit membrane fusion by binding to the gp41 N-terminal region and preventing formation of the trimer-of- hairpins. Recently, we designed the 5-Helix protein to target the C-peptide region on the gp41 ectodomain and demonstrated potent, broad-spectrum inhibition of HIV-1 membrane fusion. While these studies have established the N- and C-terminal regions of the gp41 ectodomain as targets for HIV-1 entry inhibition, the physical bases underlying the activities of C-peptides and 5-Helix remain unclear. The studies proposed here are designed to explore the physical principles and mechanistic details of gp41 inhibition. The project's specific aims are: 1) To determine a high-resolution structure of 5-Helix protein using X-ray crystallography to validate the design strategy; 2) To characterize the interaction of 5-Helix and C-peptides by scanning mutagenesis and to correlate observed binding parameters with measured inhibitory potencies; 3) To map and characterize the determinants of resistance to 5-Helix and C-peptide inhibition; and 4) To determine the inhibitory stoichiometry of 5-Helix and C-peptides using functionally-complemented Env hetero-oligomers containing escape mutations obtained in Aim 3. These studies will explore whether 5-Helix and C-peptide inhibition is a thermodynamically or a kinetically driven process and how many molecules are needed to inhibit each gp41 trimer. The experiments will test susceptibilities of the two gp41 inhibitory epitopes to escape mutagenesis, and the results will provide insights into the structure and function of gp41 as it folds into a fusion-active conformation. This mechanistic information will be valuable for development of antiviral therapeutics and HIV- 1 vaccines that target the HIV- 1 gp4 1 ectodomain.

描述（由申请人提供）：随着越来越多的 1 型人类免疫缺陷病毒 (HIV-1)（导致艾滋病的病毒）对现有疗法产生抗药性，识别和表征病毒抑制的新靶标非常重要。 HIV-1 糖蛋白 Env (gp120/gp41) 是一个有吸引力的靶点，因为它介导感染病毒附着和膜融合的初始阶段。 Env 与细胞受体的相互作用触发构象变化，最终导致关键的发夹三聚体结构的形成，其中 gp4 1 胞外域的 N 端和 C 端区域结合在一起。源自 C 末端区域的肽（C 肽）通过与 gp41 N 末端区域结合并防止发夹三聚体的形成来抑制膜融合。最近，我们设计了 5-螺旋蛋白来靶向 gp41 胞外域上的 C 肽区域，并证明对 HIV-1 膜融合具有有效、广谱的抑制作用。虽然这些研究已将 gp41 胞外域的 N 端和 C 端区域确定为 HIV-1 进入抑制的靶点，但 C 肽和 5 螺旋活性的物理基础仍不清楚。这里提出的研究旨在探索 gp41 抑制的物理原理和机制细节。该项目的具体目标是：1）利用X射线晶体学确定5-螺旋蛋白的高分辨率结构，以验证设计策略； 2) 通过扫描诱变来表征 5-螺旋和 C 肽的相互作用，并将观察到的结合参数与测量的抑制效力相关联； 3) 绘制和表征抗 5-螺旋和 C 肽抑制的决定因素； 4) 使用目标 3 中获得的含有逃逸突变的功能互补的 Env 异源寡聚物来确定 5-螺旋和 C 肽的抑制化学计量。这些研究将探讨 5-螺旋和 C 肽抑制是热力学抑制还是热力学抑制动力学驱动的过程以及抑制每个 gp41 三聚体需要多少分子。这些实验将测试两个 gp41 抑制性表位逃避诱变的敏感性，结果将深入了解 gp41 折叠成融合活性构象时的结构和功能。该机制信息对于开发针对 HIV-1 gp4 1 胞外域的抗病毒疗法和 HIV-1 疫苗非常有价值。