Monitoring single conformational events during HIV assembly

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV）是艾滋病的病原体。HIV感染通过病毒将其基因组包装成~120 nm蛋白质衣壳，用脂质双层包围它，并将感染性颗粒传播到邻近细胞，组织和其他人的能力进行传播。HIV组装由Gag多聚蛋白前体驱动，Gag多聚蛋白前体可以单独组装和释放携带二聚体RNA基因组的病毒样颗粒。该领域的关键进展为我们提供了HIV Gag各个结构域的结构快照，RNA基因组及其包装信号的结构模型，六聚体衣壳晶格的结构，以及完整的未成熟和成熟HIV病毒粒子的图像。使用荧光标记的Gag的活细胞成像允许活细胞中HIV组装的直接可视化。然而，尽管取得了这一进展，我们面临着一个知识差距之间的可用结构快照的个别构象，和动态性质的组装过程。在这里，我们建议建立荧光相关光谱（FCS）和单分子荧光共振能量转移（smFRET）成像监测HIV组装过程中的衣壳和基因组的构象变化。为此，我们已经建立了用Cy 3和Cy 5荧光团位点特异性标记重组Gag和基因组RNA分子所需的技术，这将允许smFRET成像。在过量未标记物质的存在下，标记的Gag和基因组RNA分子在体外组装反应中组装成正确大小的HIV颗粒，该反应取决于磷酸肌醇的额外存在。有了这些工具，我们将在组装成病毒颗粒的过程中监测单个Gag分子的构象。我们同样标记了HIV包装信号，以监测HIV RNA基因组作为二聚体包装成不断增长的HIV颗粒时的构象动力学。详细的知识的能量景观和动力学的艾滋病毒组装将有助于识别新的结构中间体。这一新的信息将与抗病毒治疗的合理设计有关。

DESCRIPTION (provided by applicant): The human immunodeficiency virus type 1 (HIV) is the etiological agent of AIDS. HIV infection is spread through the virus's ability to package its genome into a ~120 nm protein capsid, surround it with a lipid bilayer, and transmit infectious particles to neighboring cells, tissues and other persons. HIV assembly is driven by Gag polyprotein precursor that alone can assemble and release virus-like particles carrying a dimeric RNA genome. Critical progress in the field has provided us with structural snapshots of individual domains of HIV Gag, structural models of the RNA genome and its packaging signal, the structure of the hexameric capsid lattice, as well as images of intact immature and mature HIV virions. Live cell imaging using fluorescently tagged Gag has allowed direct visualization of HIV assembly in living cells. Yet despite this progress, we face an intellectual gap between available structural snapshots of individual conformations, and the dynamic nature of the assembly process. Here we propose to establish fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET) imaging to monitor the conformational changes of the capsid and the genome during HIV assembly. Towards this end, we have already established the technology needed to site-specifically label recombinant Gag and genomic RNA molecules with Cy3 and Cy5 fluorophores, which will permit smFRET imaging. Fluorescently labeled Gag and genomic RNA molecules, in the presence of excess unlabeled material, assemble into HIV particles of the correct size in an in vitro assembly reaction that depends on the additional presence of phosphoinositols. With these tools in hand, we will monitor the conformation of a single Gag molecule during the assembly into a viral particle. We have similarly labeled the HIV packaging signal to monitor the conformational dynamics of HIV RNA genomes as they are packaged as dimers into growing HIV particles. A detailed knowledge of the energy landscape and the kinetics of HIV assembly will aid in the identification of novel structural intermediates. This new information will be relevant for the rational design of antiviral therapies.