MECHANISMS OF HIV-1 MEMBRANE FUSION

HIV-1 膜融合机制

• 批准号: 8383456
• 负责人: MICHAEL JEFFREY ROOT
• 金额: $ 43.08万
• 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 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8383456
• 关键词: AIDS prevention; Address; Affinity; Binding; Binding Sites; Biological Assay; Biological Models; C-Peptide; Cell fusion; Cells; Cellular Membrane; Complex; Development; Engineering; Generations; HIV Envelope Protein gp120; HIV-1; Immunofluorescence Microscopy; Immunoprecipitation; In Vitro; Kinetics; Knowledge; Life; Liposomes; Mediating; Membrane; Membrane Fusion; Membrane Glycoproteins; Modeling; Molecular Conformation; Monoclonal Antibodies; Mutagenesis; Mutation; Natural Selections; Nature; New Agents; Peptides; Positioning Attribute; Property; Proteins; Research; Resistance; Resistance profile; Scanning; Series; Structure; Structure-Activity Relationship; Surface; Techniques; Testing; Vaccines; Variant; Viral; Work; base; conformational conversion; design; disulfide bond; inhibitor/antagonist; insight; interest; mutant; novel; peptide T20; public health relevance; research study; resistance mechanism; therapeutic development; vaccine development

DESCRIPTION (provided by applicant): 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 application 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 well-behaved 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.

描述（由申请人提供）：HIV-1表面糖蛋白ENV（GP120/GP41）通过一系列与GP120相互作用时引发的一系列协调结构变化来介导病毒进入。最终，GP41胞外域折叠成紧凑的发病蛋白，将病毒和细胞膜带入有效的膜融合所需的近距离接近。这些构象转变已通过使用c-肽和5螺旋抑制剂的部分构象转变，这些构型的靶向GP41的HR1和HR2区域和块状均方体形成。我们以前的工作描述了C肽和5螺旋抑制背后的物理基础，并定量建模了抑制效力与融合动力学之间的关系。本应用程序中概述的实验探讨了ENV在其天然和中间构象中的结构和动力学。该研究将解决有关病毒膜融合机制的三个基本问题：CD4与GP120的结合如何触发前中间状态的形成？在这种中间构象中，env的结构是什么？ hairpin态折叠的GP41折叠的哪些方面驱动病毒和细胞膜的融合？由于ENV在其天然和中间状态中的不稳定性，这些问题通常很难使用标准的实验技术（例如扫描诱变）来解决，例如扫描诱变，这些技术需要长期存在的构象中的稳定蛋白质。我们的基本方法是生成针对HR1和HR2区域的新抑制剂，并剖析对其抗性的机制。该策略利用自然选择的力量来生成具有有趣的结构或动态属性的良好表现的突变ENV变体。具体目的是：1）使用设计为克服标准C肽耐药性曲线的新型抑制剂探索HR1区域的结构和暴露； 2）使用多价抑制剂探测HR2区域的结构和暴露，并剖析对这些药物的抗性机制； 3）探索环境融合的能量要求，并检查被困在中间构象中的突变体Env变体的结构。我们的发现将为对病毒进入机制至关重要的GP120/GP41复合物的动态特性提供新的见解，并有助于开发新的病毒进入抑制剂和HIV-1疫苗。