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: 7115449
• 负责人: MICHAEL JEFFREY ROOT
• 金额: $ 3.45万
• 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/7115449
• 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）：随着更多人类免疫缺陷1型（HIV-1）的菌株（引起AIDS，对现有疗法具有抗药性）的病毒，重要的是要识别和表征新的病毒抑制靶标。 HIV-1糖蛋白ENV（GP120/GP41）是一个有吸引力的靶标，因为它介导了感染 - 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，最终导致形成至关重要的发病剂结构，其中GP4 1 ectodobain coopiatient的N-和C末端区域。通过与gp41 n末端区域结合并防止形成三聚蛋白酶的肽，肽衍生成C末端区域（C肽）抑制膜融合。最近，我们设计了5-螺旋蛋白来靶向GP41胞外域的C肽区域，并表现出对HIV-1膜融合的有效，宽光谱抑制。尽管这些研究确定了GP41胞外域的N和C末端区域作为HIV-1进入抑制的靶标，但C肽和5螺旋活性的基础物理碱基仍然不清楚。此处提出的研究旨在探讨GP41抑制的物理原理和机械细节。该项目的具体目的是：1）使用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得的抑制效果相关联； 3）绘制和表征对5-螺旋和C肽抑制的抗性的决定因素；和4）使用功能弥补的Env异质 - 异构体对5螺旋和C肽的抑制性化学计量法，这些异质异构体包含AIM 3中获得的逃生突变。这些研究将探索5-螺旋和C肽抑制是热力学驱动的过程还是动力学驱动的过程和动力学驱动的过程，以及每个分子对每个分子都需要每个分子。这些实验将测试两个GP41抑制性表位的敏感性以避免诱变，结果将为GP41的结构和功能提供见解，因为它折叠成融合活性构象。这些机械信息对于针对HIV-1 GP4 1胞外域的抗病毒疗法和HIV-1疫苗的开发将很有价值。