Structure Analysis of Viral Assembly Mechanisms

描述（由申请人提供）：本研究项目的总体目标是探索在感染性病毒组装过程中协调大分子相互作用的基本原理。自1981年以来，已有超过2500万人死于艾滋病。尽管艾滋病的病原体HIV-1已被广泛研究，但其复制周期的许多阶段仍然知之甚少。一个重要但尚未明确的阶段是逆转录病毒的组装和成熟，这将是我们下一个资助周期的研究重点。在病毒复制的后期，新翻译的逆转录病毒Gag蛋白组装成球形的未成熟衣壳，然后从细胞中释放出来。伴随着释放，逆转录病毒经历一个成熟过程，在这个过程中Gag被病毒蛋白酶切割成几个更小的结构域（MA， CA和NC），引发剧烈的形态变化，使颗粒具有传染性。在成熟的病毒粒子内，MA仍然与病毒包膜相关，而CA组装成一个成熟的衣壳，包围由RNA基因组和NC形成的核糖核蛋白复合物。未成熟和成熟衣壳缺乏全局对称性阻碍了逆转录病毒的高分辨率结构研究。然而，广泛的生物化学和遗传学研究导致了Gag和CA模型系统的发展，这些模型系统表现出全局对称性。在低分辨率电镜结构的基础上，提出了未成熟和成熟衣壳的模型。该项目的总体目标是利用电子冷冻显微镜，图像分析和分子建模来获得这些模型系统的更高分辨率结构。我们将通过描述一个有序的、新颖的MuLV Gag模型系统来定义Gag-Gag相互作用对未成熟衣壳形成的重要作用。我们将把全长CA （HIV-1和MuLV）的两个模型系统的分辨率扩展到10 A以上，并研究小的球形CA组件，以检查假定的五聚体CA的结构。这些研究将提供对未成熟和成熟晶格中的分子相互作用的更深入了解，这将对设计新的治疗策略很重要。

DESCRIPTION (provided by applicant): The overall goal of this research program is to explore the principles underlying the coordinated macromolecular interactions that culminate in the assembly of infectious viruses. Since 1981, more than 25 million people have died of AIDS. Although HIV-1, the causative agent of AIDS, has been extensively studied, many stages of its replication cycle remain poorly understood. One essential yet ill-defined stage is retroviral assembly and maturation, and this will be the focus of our research for the next funding cycle. During the late stages of viral replication, newly translated retroviral Gag proteins assemble into spherical immature capsids that are then released from the cell. Concomitant with release, retroviruses undergo a maturation process in which Gag is cleaved by the viral protease into several smaller domains (MA, CA, and NC), triggering dramatic morphological changes that render the particles infectious. Within the mature virion, MA remains associated with the viral envelope, while CA assembles into a mature capsid surrounding a ribonucleoprotein complex formed by the RNA genome and NC. The lack of global symmetry in the immature and mature capsids has thwarted high-resolution structural studies of retroviruses. However, extensive biochemical and genetic studies have led to the development of model systems for Gag and CA that manifest global symmetry. On the basis of low resolution EM structures, models have been proposed for the immature and mature capsids. The overall goal of this project is to use electron cryo-microscopy, image analysis and molecular modeling to derive higher resolution structures of these model systems. We will define the Gag-Gag interactions important for immature capsid formation by characterizing a well-ordered, novel model system for MuLV Gag. We will extend the resolution of two model systems of full-length CA (HIV-1 and MuLV) to beyond 10 A resolution and study small, spherical CA assemblies to examine the structure of putative pentamers CA. These studies will provide a deeper understanding of the molecular interactions in the immature and mature lattices, which will be important for the design of new therapeutic strategies.