HIV-1 Capsid Protein/Cyclophilin-A Complex : A Structural Study

描述(申请人提供)：人类免疫缺陷病毒1型(HIV-1)在其生命周期中利用大量宿主细胞蛋白。其中比较突出的蛋白质是人类亲环素A(CypA)，病毒利用它来实现最佳的传染性和病毒复制。CypA有许多假定的功能，但我们对其在HIV-1生命周期中的确切作用的了解仍处于初级阶段。针对PA-06-388的声明，在当前的R21/R33申请中，建议使用溶液核磁共振和生物物理模拟相结合的方法来确定与亲环素A结合的HIV-1衣壳蛋白(CA)的详细三维结构。由于其固有的灵活性、寡聚倾向以及在溶液中的单体-二聚体平衡，与CypA结合的野生型全长CA的结构研究在结晶学和核磁共振光谱方面的尝试失败了。因此，在本研究中，我们将使用双突变CA来研究CypA/CA复合体，该CA在溶液中以单体形式存在，并保留了野生型CA的所有关键性质，包括组装活性。利用3D/4D-核磁共振波谱、剩余偶极耦合测量和生物物理模拟，我们将确定双突变CA及其与人CypA络合物的详细溶液结构。了解CypA/CA复合体的详细结构将为我们加强了解CypA在病毒生命周期中的确切作用提供结构生物学基础。这种结构也可能有助于开发具有治疗潜力的CypA-CA相互作用的新抑制剂。公共卫生相关性：这项研究旨在利用溶液核磁共振和生物物理模拟相结合的方法来确定人亲环素A与HIV-1病毒突变衣壳蛋白结合的详细三维结构。

DESCRIPTION (provided by applicant): The human immunodeficiency virus type 1 (HIV-1) exploits a large number of host cellular proteins in its life cycle. Among the more prominent proteins is the human cyclophilin A (CypA) which is utilized by the virus for optimal infectivity and viral replication. A number of putative functions have been postulated for CypA but our understanding of its precise role within HIV-1 life cycle is still rudimentary. In response to the PA-06-388 announcement, a combined solution NMR and biophysical simulation investigation is proposed in the current R21/R33 application to determine the detailed three dimensional structure of the HIV-1 capsid protein (CA) bound to cyclophilin A. Because of its inherent flexibility, tendency to oligomerize, and its monomer-dimer equilibrium in solution, a structural study of the wild type full-length CA bound to CypA has frustrated attempts by crystallography and NMR spectroscopy. Thus in this investigation we will study the CypA/CA complex using a double mutant CA that exists as a monomer in solution, and retains all the critical properties of the wild type CA including assembly activity. Utilizing 3D/4D-NMR spectroscopy, residual dipolar coupling measurements and biophysical simulations, we will determine the detailed solution structure of the double mutant CA, and of its complex with human CypA. A knowledge of the detailed structure of the CypA/CA complex will provide a structural biology basis in enhancing our understanding of the precise role of CypA within the life cycle of the virus. Such a structure is also likely to contribute to the development of new inhibitors of CypA-CA interaction with therapeutic potential. PUBLIC HEALTH RELEVANCE: This investigation is aimed at the determination of the detailed three dimensional structure of the human Cyclophilin A bound to a mutant capsid protein of HIV-1 virus using a combined solution NMR and biophysical simulation approach.