Assembly, maturation, and structures of HIV Gag, Gag-Pol and Pol, and PFV polyproteins

摘要 Gag能够在体外自组装，尽管人们普遍认为宿主因素起着决定性作用 用于不成熟的笑话贩运。尽管许多细胞蛋白和机制选择了病毒出芽， 和释放是很好的理解，完整的身份和细胞蛋白质的作用，参与未成熟的Gag 组装仍不完整。本项目的重点是确定细胞因子的组成 在整个Gag组装途径中与Gag/Gag-Pol相互作用，从而促进动态的 组装和细胞运输。为了提供关于Gag组装的基本信息，早期步骤将 在体外以单分子水平和随后在细胞中进行询问。计算方法也将 将结构和生物物理发现整合到高阶组装的连贯结构模型中。 重要的是，第一种逆转录病毒多蛋白，即蛋白酶-逆转录酶融合蛋白的结构 泡沫病毒原型的问题已经解决了这为其他重要的结构性项目奠定了基础。 表征更复杂的逆转录病毒多聚蛋白，这是了解细胞宿主如何 因子相互作用有助于Gag和Gag-Pol组装。 拟议的调查将有力地利用内部的互补科学专门知识， HIVE中心的协作性质，并利用尖端技术来解决 具体目标：目标1。脉冲标记和定量质谱法表征Gag 和Gag-Pol，组装中间体，包括相关的蛋白质和RNA，以及随后的功能性 分析（合作者是：威廉姆森，Lyumkis，Marcotrigiano，Arnold，Griffin，Torbett）;目标2。单- 体外和体内Gag组装的分子分析，以深入了解特异性RNA贡献和Gag Gag组装所需的寡聚化（合作者为：Millar、Musier-Forsyth、Torbett、威廉姆森）; Aim 3. HIV-1和PFV的Gag、Gag-Pol和Pol的结构研究（合作者是：Arnold，Marcotrigiano， Lyumkis，Griffin，Torbett，威廉姆森）;和Aim 4.装配过程中Gag的中尺度建模（合作者 Olson，Levy，Goodsell）。 理解导致不成熟Gag组装的细胞因子的时间相互作用， 进展，以及Gag-Pol的战略作用，并阐明这些相互作用的结构基础 将提供一个更全面的了解，在逆转录病毒生物学领域，目前还不好 明白阐明与未成熟Gag相互作用的已鉴定细胞因子的结构基础， Gag-Pol将允许开发新的小化学探针，用于进一步询问这些相互作用。

ABSTRACT Gag is capable of self-assembly in vitro, although it is generally accepted that host factors play a decisive role for immature Gag trafficking. Although many of the cellular proteins and mechanisms co-opted for viral budding and release are well understood, the full identity and the role of cellular proteins participating in immature Gag assembly remains incomplete. This Project focuses on identifying the composition of cellular factors interacting with Gag/Gag-Pol throughout the Gag assembly pathway contributing to both the dynamic assembly and cellular trafficking of Gag. To provide fundamental information on Gag assembly, early steps will be interrogated in vitro at the single molecule level and later in cells. Computational methodologies will also integrate the structural and biophysical findings into coherent structural models of higher-order assemblies. Importantly, the structure of the first retroviral polyprotein, the protease-reverse transcriptase fusion protein of prototype foamy virus has been solved. This sets the stage for additional and important structural characterization of more complex retroviral polyproteins, which is required for understanding how cellular host factor interactions contribute to Gag and Gag-Pol assembly. The proposed investigations will strongly leverage the complementary scientific expertise within and collaborative nature of the HIVE Center and take advantage of cutting-edge technologies available to address the following Specific Aims: Aim 1. Pulse-labeling and quantitative mass spectrometry to characterize Gag and Gag-Pol, assembly intermediates including associated proteins, and RNA, with subsequent functional analysis (collaborators are: Williamson, Lyumkis, Marcotrigiano, Arnold, Griffin, Torbett); Aim 2. Single- molecule analysis of Gag assembly in vitro and in vivo to gain insight into specific RNA contributions and Gag oligomerization required for Gag assembly (collaborators are: Millar, Musier-Forsyth, Torbett, Williamson); Aim 3. Structural studies of Gag, Gag-Pol and Pol of HIV-1 and PFV (collaborators are: Arnold, Marcotrigiano, Lyumkis, Griffin, Torbett, Williamson); and Aim 4. Mesoscale modeling of Gag during assembly (collaborators are: Olson, Levy, Goodsell). Understanding the temporal interplay of cellular factors contributing to immature Gag assembly and progression, as well as the strategic role of Gag-Pol, and elucidating the structural basis for these interactions will provide a more comprehensive understanding of an area in retroviral biology that is currently not well understood. Elucidating the structural basis of identified cellular factors that interact with immature Gag and Gag-Pol will allow development of novel small chemical probes for further interrogation of these interactions.