Monitoring single conformational events during HIV assembly

描述(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感染是通过病毒将其基因组包装成约120 nm的蛋白质衣壳，用脂双层包裹在其周围，并将感染颗粒传播给邻近的细胞、组织和其他人的能力来传播的。HIV的组装是由Gag多蛋白前体驱动的，这种前体单独可以组装并释放携带二聚体RNA基因组的病毒样颗粒。这一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的HIV病毒粒子的图像。使用荧光标记的GAG进行的活细胞成像可以直接显示活细胞中的艾滋病毒组装。然而，尽管取得了这一进展，我们仍面临着个体构象的现有结构快照与组装过程的动态性质之间的智力差距。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)成像来监测HIV组装过程中衣壳和基因组的构象变化。为此，我们已经建立了用Cy3和Cy5荧光团定点标记重组GAG和基因组RNA分子所需的技术，这将使smFRET成像成为可能。荧光标记的Gag和基因组RNA分子在多余的未标记物质存在的情况下，在依赖于额外的磷酸肌醇存在的体外组装反应中，组装成大小正确的HIV颗粒。有了这些工具，我们将监测单个Gag分子在组装成病毒颗粒的过程中的构象。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。对HIV组装的能量格局和动力学的详细了解将有助于识别新的结构中间体。这一新信息将与抗病毒疗法的合理设计相关。 公共卫生相关性：该应用建议建立荧光相关光谱(FCS)和单分子荧光共振能量转移(SmFRET)成像来监测HIV组装过程中衣壳和基因组的构象变化。对HIV组装的能量格局和动力学的详细了解将有助于识别新的结构中间体。这一新信息将与抗病毒疗法的合理设计相关。

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. PUBLIC HEALTH RELEVANCE: This application proposes 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. 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.