Project 3: Investigation of capsid-host factor interactions using minimal, soluble capsid lattice assemblies

P3.摘要 成熟的HIV-1衣壳是一个有序的蛋白质外壳，包含病毒基因组和其他病毒或宿主 感染目标细胞所必需的因子。它的结构和稳定性对感染至关重要--它必须保持在 最少部分组装，直到它到达核外围，以及各种宿主因子，无论是有益的还是 抑制病毒，识别仅存在于组装核心中的特定衣壳模式。可能有数百人 宿主因子结合HIV-1衣壳，但我们对宿主因子-衣壳相互作用的了解在分子上 水平受到限制，部分原因是缺乏可溶的衣壳结构，这些结构概括了 重组衣壳结合宿主因子的组装状态和纯化过程中面临的挑战。要克服这些障碍 障碍，我们建议设计和建立一个代表所有独特的可溶性衣壳组件的曲目 衣壳-晶格界面。这将通过利用以前发表的Intra-Hexer来实现 通过引入SpyCatcher/SpyTag系统来创建稳定的异肽键 在六人组之间。有了这些新的衣壳组件，我们将使用成熟的生物物理和 利用生化技术绘制结合模式图并确定这些衣壳的高分辨率结构 具有一系列宿主因子的复杂组件，包括TRIM5α、TRIMCyp、MXB、Fez1和CypA，大多数 我们已经对其进行了表达和纯化优化。拟议的工作将使我们能够克服主要的 推动对HIV病毒生命中衣壳相关关键步骤的机械性理解的实验障碍 周而复始。

P3. Abstract The mature HIV-1 capsid is an ordered protein shell that houses the viral genome and other viral or host factors necessary for infection of a target cell. Its structure and stability are critical to infection—it must stay at least partially assembled until it reaches the nuclear periphery, and a variety of host factors, either beneficial or inhibitory to the virus, recognize specific capsid patterns only present in assembled cores. Potentially hundreds of host factors bind the HIV-1 capsid, but our knowledge of host factor-capsid interactions at the molecular level has been limited, in part caused by a lack of soluble capsid constructs that recapitulate lattice interfaces in the assembled state and by challenges in purifying recombinant capsid-binding host factors. To overcome these obstacles, we propose to design and build a repertoire of soluble capsid assemblies that represent all unique capsid-lattice interfaces. This will be achieved by making use of previously published intra-hexamer engineered disulfides and by incorporating the SpyCatcher/SpyTag system to create stable isopeptide bonds between hexamers. With these new capsid assemblies in hand, we will use well-established biophysical and biochemical techniques to map binding modes and determine high-resolution structures of these capsid assemblies in complex with a range of host factors including TRIM5α, TRIMCyp, MxB, Fez1, and CypA, most of which we have optimized for expression and purification. The proposed work will allow us to overcome major experimental hurdles to advance mechanistic understandings of the capsid-related key steps of the HIV life cycle.