Retrovirus Assembly and Maturation

由于几个原因，了解病毒颗粒组装和成熟的分子机制至关重要。首先，它可以作为更广泛的复杂大分子组装问题的模型，具有独特的优点，即相关结构对细胞来说是外来的，但将在细胞成分的帮助下在细胞环境中组装。其次，它为抗病毒治疗提供了许多潜在的靶点，但由于我们缺乏制定治疗策略所需的详细知识，迄今尚未开发这些靶点。在这个项目中，我们继续分析HIV-1颗粒的组装和成熟。具体地说，我们已经表征了HIV-1 Gag蛋白的特性，它是HIV-1病毒颗粒的基本构件。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蛋白在体外可以在确定的系统中组装成病毒样颗粒(VLP)；这表明该蛋白与单链核酸(NA)相互作用时，将进行相当规则的蛋白质-蛋白质相互作用，形成一个大致球形的结构，具有相当均匀的曲率半径。在体外组装和细胞内组装的颗粒中，GAG分子的排列是相同的，并且许多GAG突变体在这两个系统中具有相似的作用。我们研究了这种蛋白质在体外和哺乳动物细胞中的组装，在哺乳动物细胞中，GAG明显比体外稀释得多，并被无数的细胞成分包围。虽然已经在相当程度上定义了组装颗粒中的结构，但关于GAG分子如何从自由、可溶的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，这种结构如何使它能够与其他Gag分子和核酸共同组装成未成熟的逆转录病毒颗粒？如果Gag分子在溶液中的结构与在未成熟病毒粒子中的结构不同，是什么控制了这种差异？与核酸结合是如何触发粒子组装的？来自不同逆转录病毒属的Gag蛋白和病毒粒子彼此之间有何相似之处，又有何不同？这些差异是否影响组装的控制？_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。利用凝胶过滤、静态和准弹性光散射、沉降速度分析、小角中子散射以及分子模拟等多种实验方法，我们表征了HIV-1 Gag蛋白的构象。我们发现，当Gag分子靠近时，它们会经历一个重大的构象变化，为组装做准备。所有数据都表明，HIV-1 Gag蛋白在溶液中折叠，其末端在三维空间中紧靠在一起，与其在未成熟颗粒中的杆状形成鲜明对比。最近，我们重点研究了Gag的SP1结构域的构象变化，GAG是衣壳(CA)和核壳(NC)结构域之间的一个短“连接物”。我们的数据表明，当含有SP1的两个或多个分子并置时，SP1呈α螺旋构象。这种变化是因为SP1可以形成一个两亲性螺旋；几个这样的螺旋结合产生螺旋束，其中疏水残基向内，不受溶剂的影响。我们认为，SP1的这种变化显然会导致CA结构域的变化，并为Gag-Gag相互作用提供新的界面，这些界面是将Gag转化为“组装就绪”蛋白质所必需的。数据还表明，SP1结构域之间的缔合相互作用与CA结构域的缔合作用一起参与了未成熟颗粒的组装。与Eric Freed博士(HIV动力学和复制计划)合作，我们正在测试未成熟的病毒颗粒或含有SP1的小螺旋束与Bevirimat等成熟抑制剂结合的可能性；如果是这样的话，它们将对体外筛选新的HIV-1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，一种伽马逆转录病毒)未成熟颗粒的结构和组装。未成熟的MLV颗粒和成熟的MLV颗粒的最终结构都与HIV-1的结构有显著差异。这些差异可能反映了这样一个事实，即MLV Gag比HIV-1 Gag具有更弱的二聚化倾向，不包含SP1，并且似乎通过与HIV-1略有不同的途径转换到组装就绪状态。与HIV-1 Gag不同，MLV Gag是溶液中的杆状结构，其尺寸与VLP中的尺寸大致相同。在基质和p12结构域中有较短的脯氨酸，以及CA末端附近的“电线”，这有助于MLV Gag结构的刚性。对这些差异的分析将有助于阐明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 immature virus particles, or 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 both immature and mature MLV particles show significant differences from those 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.