Retrovirus Assembly and Maturation

了解病毒颗粒组装和成熟的分子机制至关重要，原因如下。首先，它可以作为更广泛的复杂大分子组装问题的模型，其特殊的优点是，相关结构对细胞来说是外来的，但在细胞成分的帮助下，将在细胞环境中组装。其次，它为抗病毒治疗提供了许多潜在的靶点，由于我们缺乏开发治疗策略所需的详细知识，这些靶点迄今尚未被开发。_____在这个项目中，我们继续对HIV-1颗粒的组装和成熟进行分析。具体来说，我们已经描述了HIV-1 Gag蛋白的特性，这是HIV-1病毒颗粒的基本组成部分。Gag在哺乳动物细胞中组装成含有数千个Gag分子的未成熟病毒颗粒。同样，从细菌中纯化的重组HIV-1 Gag蛋白可以在体外特定系统中组装成病毒样颗粒（vlp）；这表明蛋白质与单链核酸（NA）相互作用后，会进行相当规则的蛋白质-蛋白质相互作用，形成曲率半径相当均匀的大致球形结构。Gag分子在体外和细胞内组装的颗粒中的排列是相同的，并且许多Gag突变体在这两个系统中具有类似的作用。我们在体外和哺乳动物细胞中研究了这种蛋白质的组装，其中Gag明显比体外稀释得多，并且被无数细胞成分包围。虽然组装颗粒的结构在很大程度上已经确定，但对于Gag分子从自由的可溶性蛋白质转化为准备组装的分子，然后转化为最终组装结构的组成部分的步骤，人们知之甚少。我们正在解决以下问题：HIV-1 Gag分子的结构是什么，这种结构是如何使它与其他Gag分子和核酸一起组装成不成熟的逆转录病毒颗粒的？如果溶液中的Gag分子结构与未成熟病毒粒子中的结构不同，那么是什么控制了这种差异？如何通过与核酸结合来触发粒子组装？来自不同逆转录病毒属的Gag蛋白和病毒粒子是如何相互相似的，它们又是如何不同的？这些差异会影响装配的控制吗？_____我们利用体外系统的简单性来分析伴随HIV-1 Gag组装的分子变化。利用各种各样的实验方法，包括凝胶过滤，静态和准弹性光散射，沉降速度分析，小角度中子散射，以及分子模型，我们已经表征了HIV-1 Gag蛋白的构象。我们发现，当Gag分子靠近时，它们经历了一个主要的构象变化，为组装做好了准备。所有的数据表明，HIV-1 Gag蛋白在溶液中折叠，其末端在三维空间中彼此靠近，与未成熟颗粒中的棒状形状形成鲜明对比。我们最近关注Gag的SP1结构域的构象变化，Gag是衣壳（CA）和核衣壳（NC）结构域之间的一个短“连接体”。我们的数据表明，SP1假设一个α -螺旋构象，当两个或更多的分子包含SP1被带到并置。发生这种变化是因为SP1可以形成两向螺旋；几个这样的螺旋结合产生螺旋束，其中疏水残基面向内，与溶剂隔绝。我们提出SP1中的这种变化明显导致CA结构域的变化，并为Gag-Gag相互作用暴露新的接口，这些相互作用需要将Gag转化为“可组装”的蛋白质。这些数据还表明SP1结构域与CA结构域之间的结合相互作用参与了未成熟颗粒的组装。与Eric Freed博士（HIV动力学和复制项目）合作，我们正在测试含有sp1的小螺旋束结合成熟抑制剂（如bevirimat）的可能性；如果是这样的话，它们将对体外筛选HIV-1的新抑制剂非常有用。_____我们正在合作详细研究小鼠白血病病毒（MLV，一种伽玛逆转录病毒）的未成熟颗粒的结构和组装。MLV颗粒的最终结构与HIV-1有显著差异。这些差异可能反映了这样一个事实，即MLV Gag的二聚化倾向远弱于HIV-1 Gag，不含SP1，并且似乎通过与HIV-1不同的途径转化为组装准备状态。与HIV-1 Gag不同，MLV Gag是溶液中的棒状结构，其尺寸与VLP大致相同。短的脯氨酸在基体和p12结构域中的运行，以及靠近CA末端的“电线”，有助于MLV Gag结构的刚性。对这些差异的分析将揭示粒子组装背后的机械原理。

Understanding the molecular mechanisms underlying virus particle assembly and maturation is of critical importance for several reasons. First, it can serve as a model for the broader problem of complex macromolecular assembly, with the particular virtue that the relevant structures are foreign to the cell but will be assembled in the cellular context, with the help of cellular constituents. Second, it offers many potential targets for antiviral therapy, which have not been exploited to date because we lack the detailed knowledge needed for the development of therapeutic strategies. _____In this project, we have continued our analysis of the assembly and maturation of HIV-1 particles. Specifically, we have characterized the properties of the HIV-1 Gag protein, the fundamental building block of the HIV-1 virus particle. Gag assembles in mammalian cells into the immature virus particle, containing several thousand Gag molecules. Similarly, recombinant HIV-1 Gag protein, purified from bacteria, can assemble into virus-like particles (VLPs) in a defined system in vitro; this shows that the protein will, upon interaction with a single-stranded nucleic acid (NA), engage in rather regular protein-protein interactions, forming a roughly spherical structure with a rather uniform radius of curvature. The arrangement of Gag molecules is the same in particles assembled in vitro and in cells, and many mutants of Gag have analogous effects in the two systems. We study assembly of this protein both in vitro and in mammalian cells, where Gag is obviously far more dilute than in vitro and is surrounded by myriad cellular constituents. While the structures in the assembled particle have been defined to a considerable degree, little is known about the steps by which a Gag molecule is converted from a free, soluble protein to a molecule ready to assemble and then to a constituent of the final, assembled structure. We are addressing the following questions: What is the structure of an HIV-1 Gag molecule, and how does this structure enable it to coassemble, with other Gag molecules and with nucleic acid, into immature retrovirus particles? If the structure of a Gag molecule in solution is different from that in an immature virion, what controls this difference? How is particle assembly triggered by binding to nucleic acid? How are Gag proteins and virions from different retroviral genera similar to each other and how do they differ? Do these differences affect the control of assembly? _____We have exploited the simplicity of the in vitro system to analyze the molecular changes accompanying HIV-1 Gag assembly. Using a wide variety of experimental approaches, including gel filtration, static and quasi-elastic light-scattering, sedimentation velocity analysis, and small-angle neutron scattering, as well as molecular modeling, we have characterized the conformation of the HIV-1 Gag protein. We found that when Gag molecules are brought into close proximity, they undergo a major conformational change that prepares them for assembly. All of the data indicate that HIV-1 Gag protein is folded over in solution, with its ends near each other in three-dimensional space, in striking contrast to its rod-like shape in immature particles. We have recently focused on conformational changes in the SP1 domain of Gag, a short "linker" between the capsid (CA) and nucleocapsid (NC) domains. Our data show that SP1 assumes an alpha-helical conformation when two or more molecules containing SP1 are brought into juxtaposition. This change occurs because SP1 can form an amphipathic helix; association of several such helices produces helical bundles in which hydrophobic residues face inward, shielded from the solvent. We proposed that this change in SP1 apparently leads to changes in the CA domain and exposes new interfaces for the Gag-Gag interactions that are needed to convert Gag to an "assembly-ready" protein. The data also suggest that the associative interactions between SP1 domains participate, along with those of CA domains, in immature particle assembly. In collaboration with Dr. Eric Freed (HIV Dynamics and Replication Program), we are testing the possibility that small SP1-containing helical bundles will bind maturation inhibitors such as bevirimat; if so, they would be extremely useful for in vitro screens for new inhibitors of HIV-1. _____We are collaborating in a detailed study of the structure and assembly of immature particles of murine leukemia virus (MLV, a gammaretrovirus). The final structure of the MLV particle shows significant differences from that of HIV-1. These differences may reflect the fact that MLV Gag has a far weaker tendency to dimerize than HIV-1 Gag, does not contain SP1, and appears to be converted to an assembly-ready state by a somewhat different pathway from HIV-1. Unlike HIV-1 Gag, MLV Gag is a rod in solution, with approximately the same dimensions as it has in a VLP. Short proline runs in the matrix and p12 domains, and the "electric wire" near the end of CA, contribute to rigidity of the MLV Gag structure. Analysis of these differences will shed light on the mechanistic principles underlying particle assembly.