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

P3.摘要 成熟的HIV-1衣壳是一种有序的蛋白质外壳，容纳病毒基因组和其他病毒或宿主 感染靶细胞所必需的因子。它的结构和稳定性对感染至关重要--它必须保持在 至少部分组装，直到它到达核周边，和各种宿主因素，无论是有益的， 抑制病毒，识别仅存在于组装核心中的特定衣壳模式。潜在的数百 的宿主因子结合HIV-1衣壳，但我们对宿主因子-衣壳相互作用在分子水平上的认识， 水平受到限制，部分原因是缺乏可溶性衣壳构建体，该构建体在细胞中重演晶格界面。 组装状态和纯化重组captain结合宿主因子的挑战。克服这些 障碍，我们建议设计和建立一个可溶性衣壳组件的库，代表所有独特的 帽-晶格界面这将通过利用先前发表的六聚体内 工程二硫化物，并通过纳入SpyCatcher/SpyTag系统，以创建稳定的异肽键 六聚体之间有了这些新的衣壳组装，我们将使用成熟的生物物理和 生物化学技术来绘制结合模式并确定这些衣壳的高分辨率结构 与一系列宿主因子（包括TRIM 5 α、TRIMCyp、MxB、Fez 1和CypA）复合的组装体， 我们已经优化了其表达和纯化。拟议的工作将使我们能够克服重大的 实验障碍，以促进对HIV生命中与captain相关的关键步骤的机械理解 周期

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.