Project 3 - Genome Recognition & Packaging

项目3-基因组识别

• 批准号: 10245116
• 负责人: MICHAEL FINLEY SUMMERS
• 金额: $ 42.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-17 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245116
• 关键词: Affinity; Binding; Capsid; Cell Nucleus; Cell membrane; Cells; Complex; Cryoelectron Microscopy; Dimerization; Elements; Genome; Goals; HIV; HIV Genome; Heterogeneity; Interest Group; Molecular; Monitor; Nucleocapsid; Pathway interactions; Proteins; RNA; Research Personnel; Ribonucleoproteins; Role; SP1 gene; Signal Transduction; Site; Structure; Testing; Time; Transcription Initiation Site; Virus Assembly; X-Ray Crystallography; confocal imaging; dimer; experimental study; gag Gene Products; imaging study; imaging system; membrane assembly; particle; programs; spatiotemporal; stoichiometry; three dimensional structure; trafficking

Summary The goal of this Project is to understand the structural basis and mechanism of HIV-1 genome recognition and cytoplasmic trafficking, and the role of the genome in nucleating virus assembly. The Project brings together several highly collaborative groups with a broad range of expertise, and is tightly integrated with the NMR, cryoEM, X-ray crystallography, and Computational Cores. Pornillos has contributed significantly to our understanding of how the highly conserved SP1 domain of Gag, which bridges the capsid and nucleocapsid (NC) domains, and forms a hexameric coiled-coil that helps drive particle formation. Summers and Telesnitsky have recently determined the structure of the RNA element that is responsible for RNA packaging (ΨCES) and shown how transcriptional start site heterogeneity contributes to genome packaging. These investigators will combine efforts to determine how Gag recognizes and assembles on the RNA packaging signal. Exciting preliminary studies reveal that hexameric Gag constructs that minimally include the SP1 and NC domains bind with sub-nM affinity to the dimeric packaging signal [ΨCES]2 with 6:2 protein:RNA stoichiometry, and that binding is selective for the dimeric (rather than monomeric) RNA. Thus, a single Gag hexamer binds with high affinity to the dimeric RNA that contains two core encapsidation elements, an unexpected finding! This stoichiometry is consistent with confocal imaging studies in living, transfected cells by Simon, which show that the genome is anchored to the plasma membrane by a small number of (fewer than a dozen) Gag proteins. With Ganser-Pornillos of the CHEETAH Center, cryoEM studies will be conducted with complexes formed between [ΨCES]2 and Gag constructs containing SP1-NC. Mechanistic hypotheses will be tested by packaging and assembly experiments conducted by Telesnitsky. In addition, Simon has developed a new imaging system with which he has observed RNA dimers in the nucleus and found that most genomes arrive at the plasma membrane as dimers. Both of these preliminary findings challenge current dogma and point to additional studies. The stage is now set to (1) determine the primary site(s) of genome dimerization and Gag binding in infected cells, (2) monitor the pathway(s) that the genome follows from the time it is synthesized in the nucleus until it buds from the plasma membrane, (3) understand the molecular mechanisms that drive Gag:RNA assembly, and (4) determine the 3D structure of the ribonucleoprotein complex that anchors the dimeric genome to the plasma membrane and nucleates virus assembly.

概括 该项目的目标是了解HIV-1基因组识别和识别的结构基础和机制。 该项目汇集了细胞质运输和基因组在成核病毒组装中的作用。 几个高度协作的团队拥有广泛的专业知识，并与 NMR 紧密结合， 冷冻电镜、X 射线晶体学和计算核心为我们做出了重大贡献。 了解 Gag 高度保守的 SP1 结构域如何桥接衣壳和核衣壳 (NC) 域，并形成六聚体卷曲螺旋，有助于驱动萨默斯和粒子的形成。 Telesnitsky 最近确定了负责 RNA 包装的 RNA 元件的结构 (ΨCES) 并展示了转录起始位点异质性如何影响基因组包装。 研究人员将共同努力确定 Gag 如何在 RNA 包装上识别和组装 令人兴奋的初步研究表明，六聚体 Gag 构建体至少包含 SP1 和 NC 结构域以亚 nM 亲和力与 6:2 蛋白质:RNA 的二聚体包装信号 [ΨCES]2 结合 化学计量，并且该结合对二聚体（而不是单体）RNA 具有选择性，因此，单个 Gag。 六聚体以高亲和力与含有两个核心衣壳化元件的二聚体 RNA 结合， 意外的发现！该化学计量与活体转染细胞的共聚焦成像研究一致 Simon，这表明基因组通过少量（少于一个）锚定在质膜上 十几种）Gag 蛋白，将与 CHEETAH 中心的 Ganser-Pornillos 一起进行冷冻电镜研究。 [ΨCES]2 和包含 SP1-NC 的 Gag 构建体之间形成的复合物将是机械假设。 此外，Simon 还通过 Telesnitsky 进行的封装和组装实验进行了测试。 他用一种新的成像系统观察了细胞核中的 RNA 二聚体，并发现大多数基因组 这两个初步发现都挑战了当前的教条并以二聚体形式到达质膜。 现在的阶段是 (1) 确定基因组二聚化的主要位点。 和受感染细胞中的 Gag 结合，(2) 监测基因组从被感染时起所遵循的途径 在细胞核中合成直至从质膜中出芽，（3）了解分子机制 驱动 Gag:RNA 组装，以及 (4) 确定核糖核蛋白复合物的 3D 结构 将二聚体基因组锚定在质膜上并使病毒组装成核。