Proteomic Tools to Uncover the Role of Chromatin Remodeling in HIV-1 Infection

描述(申请人提供)：HIV的基因容量只有104个碱基对，已经引发了毁灭性的获得性免疫缺陷综合征(AIDS)疫情。在很大程度上，控制被病原体入侵的细胞命运的精细机制仍不清楚。为了在分子水平上了解这种疾病并开发治疗方法，我们正在开发新的方法来揭示这些时间和空间蛋白质相互作用的性质。这些方法需要一种基于快速冷冻的策略，使用单一的荧光标记(GFP)来显示活细胞中的病毒蛋白，并在感染过程中捕获它们相互作用的伙伴。我们对HIV-1宿主蛋白相互作用的初步工作表明，染色质重塑复合体是受病毒调节的其他细胞功能之一。这一发现表明，病毒已经发展出通过篡改这些复合体来控制宿主基因表达的机制。事实上，最近的报告表明，组蛋白脱乙酰酶可能介导了HIV的潜伏期。我们建议对HIV-1感染过程中已知的11个组蛋白脱乙酰酶进行全面的分析。为了深入了解这些多蛋白质复合体的位置、组成、结构和功能，我们将把新的蛋白质组学方法与分子生物学、病毒学和生物化学方法结合起来。使用融合了绿色荧光蛋白的表达HDAC的人类细胞系，我们将首先在未感染的细胞中观察和分离这些嵌合体，然后在感染HIV-1时进行分离。我实验室中最先进的质谱学配置是此类配置中的第一台，它将分析速度与MALDI(基质辅助激光解吸电离)的健壮性、线性离子陷阱中的浓缩以及Orbitrap中的高精度测量相结合。这种构型非常适合于大分子的分析。这种对HDAC复合体的全面多学科分析将提供对宿主和病毒过程的洞察，这些过程决定了细胞的命运和感染的最终结果。

DESCRIPTION (provided by applicant): With a genetic capacity of only 104 bp, HIV has triggered the devastating acquired immunodeficiency syndrome (AIDS) epidemic. In large part, the refined mechanisms that control the fate of cells invaded by the pathogen remain unknown. To understand the disease at the molecular level and develop therapies we are developing new approaches to reveal the nature of these temporal and spatial protein interactions. These methods entail a rapid cryolysis-based strategy that uses a single fluorescent tag (GFP) to both visualize viral proteins in live cells and capture their interacting partners during the course of infection. Our preliminary work on HIV-1 host protein interactions demonstrates that chromatin remodeling complexes are among other cellular functions modulated by the virus. This finding suggests that viruses have developed mechanisms to control host gene expression by usurping these complexes. Indeed, recent reports have shown that histone deacetylases may mediate the HIV latency. We propose to initiate a comprehensive analysis of the known eleven histone deacetylases during the course of HIV-1 infection. To gain insights into the location, composition, structure, and function of these multi-protein complexes, we will merge novel proteomic approaches with molecular biology, virology, and biochemistry methodologies. Using human cell lines expressing HDAC fused to GFP, we will visualize and isolate these chimeras, first in uninfected cells, and then upon HIV-1 infection. The state-of-the-art mass spectrometric configuration in my laboratory is the first of its kind, combining speed of analysis with the robustness of MALDI (matrix assisted laser desorption ionization), the enrichment in the linear ion trap, and the high accuracy measurement in the Orbitrap. This configuration is perfectly suited for the analysis of macromolecules. This comprehensive multidisciplinary analysis of HDAC complexes will provide insights into the host and viral processes that determine the fate of a cell and the ultimate outcome of infection.