Project 3 - Genome Recognition & Packaging

总结 该项目的目标是了解HIV-1基因组识别的结构基础和机制， 细胞质运输，以及基因组在成核病毒装配中的作用。该项目汇集 几个具有广泛专业知识的高度协作小组，并与NMR紧密结合， cryoEM、X射线晶体学和计算核心。Pornillos为我们的 理解Gag的高度保守的SP1结构域如何连接衣壳和核衣壳， (NC)结构域，并形成有助于驱动颗粒形成的六聚体卷曲螺旋。萨默斯和 Telesnitsky最近确定了负责RNA包装的RNA元件的结构 （EQUCES），并显示了转录起始位点的异质性如何有助于基因组包装。这些 研究人员将联合收割机努力确定Gag如何识别和组装在RNA包装上 信号了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结构域以低于nM的亲和力结合二聚体包装信号[BICCES]2，蛋白质：RNA为6：2 这种结合是化学计量的，并且结合对于二聚体（而不是单体）RNA是选择性的。因此， 六聚体以高亲和力结合二聚体RNA，该二聚体RNA含有两个核心折叠元件， 意外的发现！该化学计量与通过流式细胞术在活的转染细胞中进行的共聚焦成像研究一致。 Simon等人的研究表明，基因组是由少量（少于10%）的 十二）Gag蛋白。与CHEETAH中心的Ganser-Pornillos一起，将使用以下方法进行cryoEM研究： 在含有SP1-NC的Gag构建体和[PICCES]2之间形成的复合物。机械假说将是 通过Telesnitsky进行的包装和组装实验进行测试。此外，西蒙还开发了 一种新的成像系统，他用它观察了细胞核中的RNA二聚体，发现大多数基因组 以二聚体的形式到达质膜。这两项初步发现都挑战了当前的教条， 指向更多的研究。现在的阶段是（1）确定基因组二聚化的主要位点 和Gag结合，（2）监测基因组从其被感染时起遵循的途径。 在细胞核中合成，直到它从质膜上发芽，（3）了解分子机制 驱动Gag：RNA组装的3D结构，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装配成核。

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.