Structural Biology Of Retrovirus Assembly

逆转录病毒组装的结构生物学

• 批准号: 7732831
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 58.88万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732831
• 关键词: Acquired Immunodeficiency Syndrome; Antiviral Agents; Area; C-terminal; Caliber; Capsid; Capsid Proteins; Cell Nucleus; Cells; Class; Complement; Cytoplasm; Data Set; Electrons; Endopeptidases; Enzymes; Exhibits; Fullerenes; Gagging; Genetic Polymorphism; Glycoproteins; Goals; HIV; Hand; Housing; Immune; Individual; Infectious Agent; Lead; Link; Measures; Membrane; Methods; Modeling; Molecular Conformation; Morphology; Nucleocapsid; Numbers; Patients; Peptide Hydrolases; Play; Property; Protease Inhibitor; RNA replication; Range; Research Project Grants; Resolution; Retinal Cone; Retroviridae; Role; Rous sarcoma virus; Shapes; Structure; Surface; Tomogram; Tube; Tubular formation; Vaccines; Viral; Virion; Virus; base; electron tomography; falls; gag Gene Products; novel; programs; reconstruction; size; structural biology; three dimensional structure; virus core

During FY08, we focussed primarily on two subprojects. (1) Capsid assembly and polymorphism of retroviruses and their implications for infectivity. Retroviruses capsids are unusual in that they are assembled inside the maturing virion, not in the cytoplasm or the nucleus of the infected cell. The capsid protein is incorporated into the provirion as part of the Gag polyprotein which forms a spherical shell lining the membrane of the provirion. After the provirion has budded off, the maturational protease is activated and dissects Gag into its matrix (MA), capsid (CA) and nucleocapsid (NC) moieties. Protease inhibitors were the first antiviral drugs to be used successfully against HIV. Of the Gag fragments, CA reassembles to form the shell of the virus core, housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity; however cores are highly polymorphic. In this context, we have been using cryo-electron tomography to visualize mature virions of Rous Sarcoma Virus, the prototypic alpha-retrovirus.The virions in our data set range from 105 to 175 nm in diameter. Their cores are highly polymorphic. We observed angular cores, including some that are distinctively coffin-shaped and for which we propose a novel fullerene geometry; cores with continuous curvature including, rarely, fullerene cones; and tubular cores. Angular cores are the most voluminous and densely packed; tubes and some curved cores contain less material, suggesting incomplete packaging. From the tomograms, we measured the surface areas of cores and hence their contents of CA subunits. From the virion diameters, we estimated their original complements of Gag. We find that RSV virions, like HIV, contain unassembled CA subunits and the fraction of CA that is assembled correlates with core type; angular cores incorporate 80% of the available subunits and open-ended tubes, 30%. The number of glycoprotein spikes is variable ( 0 to 118) and also correlates with core type; virions with angular cores average 82 spikes, whereas those with tubular cores, 14 spikes. These observations imply that initiation of capsid assembly, in which interactions of spike endodomains with the Gag layer play a role, is a critical determinant of core morphology.. (2) Retrovirus capsid geometry. Most capsid-containing viruses have capsids of uniquely defined sizes and shapes; of these, the most common structures observe icosahedral symmetry. In contrast, retrovirus capsids are highly polymorphic. Nevertheless, they may also be described as polyhedral foldings of a fullerene lattice on which the capsid protein (CA) is arrayed. Lacking the high order of symmetry that facilitates the reconstruction of icosahedral capsids from cryo-electron micrographs, the three-dimensional structures of individual retrovirus capsids may be determined by cryo-electron tomography, albeit at lower resolution. For example, In the case of HIV, the appropriate structures have been proposed to be fullerene cones. On the other hand, we have found that cones are rare for Rous sarcoma virus capsids. To investigate further their polymorphism (see (1) above). we developed computational and graphical methods to construct polyhedral models that match in size and shape, RSV capsids observed within intact virions. They fall into several shape classes, including tubes, "lozenges", and "coffins". The extent to which a capsid departs from icosahedral symmetry reflects the irregularity of the distribution of pentamers, which are always 12 in number for a closed polyhedral capsid. The number of distinct polyhedra grows rapidly with increasing quotas of hexamers, and ranks in the millions for RSV capsids, which have 150 to 300 hexamers. Unlike the capsid proteins of icosahedral viruses that assume a minimal number of quasi-equivalent conformations equal to the triangulation number (T), retroviral CAs exhibit a near-continuum of quasi-equivalent conformations a property that may be attributed to the flexible hinge linking the N- and C-terminal domains.

2008 财年，我们主要关注两个子项目。 (1)逆转录病毒的衣壳组装和多态性及其对感染性的影响。逆转录病毒衣壳的不同寻常之处在于它们在成熟的病毒粒子内组装，而不是在受感染细胞的细胞质或细胞核中组装。衣壳蛋白作为 Gag 多蛋白的一部分掺入到病毒颗粒中，Gag 多蛋白形成衬在病毒颗粒膜上的球壳。病毒体出芽后，成熟蛋白酶被激活并将 Gag 分解为其基质 (MA)、衣壳 (CA) 和核衣壳 (NC) 部分。蛋白酶抑制剂是第一个成功用于对抗艾滋病毒的抗病毒药物。在 Gag 片段中，CA 重新组装形成病毒核心的外壳，容纳病毒 RNA 和复制酶。有证据表明，正确形成的核心对于传染性至关重要。然而，核心是高度多态的。在这种情况下，我们一直在使用冷冻电子断层扫描来可视化劳斯肉瘤病毒（原型α逆转录病毒）的成熟病毒体。 数据集直径范围为 105 至 175 nm。它们的核心是高度多态性的。我们观察到了有角的核心，包括一些独特的棺材形状，我们为此提出了一种新颖的富勒烯几何结构；具有连续曲率的核，很少包括富勒烯锥体；和管状核心。角形核心体积最大、密度最大；管子和一些弯曲的芯材含有较少的材料，表明包装不完整。根据断层图，我们测量了核心的表面积，从而测量了它们的 CA 亚基含量。根据病毒体直径，我们估计了它们的 Gag 原始互补体。我们发现 RSV 病毒颗粒与 HIV 一样，含有未组装的 CA 亚基，并且组装的 CA 比例与核心类型相关；角核包含 80% 的可用子单元，开放式管则占 30%。糖蛋白尖峰的数量是可变的（0 到 118）并且也与核心类型相关；具有角状核心的病毒粒子平均有 82 个尖峰，而具有管状核心的病毒粒子平均有 14 个尖峰。这些观察结果表明，衣壳组装的启动（其中刺突内域与 Gag 层的相互作用发挥作用）是核心形态的关键决定因素。 (2)逆转录病毒衣壳几何结构。大多数含衣壳病毒的衣壳具有独特的大小和形状；其中， 最常见的结构遵循二十面体对称性。相反，逆转录病毒衣壳具有高度多态性。然而，它们也可以被描述为富勒烯晶格的多面体折叠，衣壳蛋白（CA）排列在富勒烯晶格上。由于缺乏有利于从冷冻电子显微照片重建二十面体衣壳的高阶对称性，单个逆转录病毒衣壳的三维结构可以通过冷冻电子断层扫描来确定，尽管分辨率较低。例如，就艾滋病毒而言，合适的结构被认为是富勒烯锥体。另一方面，我们发现劳斯肉瘤病毒衣壳中的锥体很少见。进一步研究它们的多态性（见上文（1））。我们开发了计算和图形方法来构建多面体模型，其大小和形状与完整病毒颗粒内观察到的 RSV 衣壳相匹配。它们分为几个形状类别，包括管状、“菱形”和“棺材”。衣壳偏离二十面体对称性的程度反映了五聚体分布的不规则性，对于封闭的多面体衣壳，五聚体的数量始终为 12。随着六聚体配额的增加，不同多面体的数量迅速增长，RSV 衣壳的数量达到数百万个，其具有 150 至 300 个六聚体。与二十面体病毒的衣壳蛋白假设最少数量的准等效构象等于三角测量数（T）不同，逆转录病毒CA表现出近乎连续的准等效构象，这一特性可能归因于连接N端和C端结构域的柔性铰链。