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.

在08财年期间，我们主要专注于两个次要注射器。 （1）逆转录病毒的衣壳组装和多态性及其对感染性的影响。逆转录病毒的胶囊是不寻常的，因为它们在成熟的病毒体中组装而不是在细胞质或感染细胞的细胞核中。作为GAG多蛋白的一部分，将衣壳蛋白纳入了Propirion，该蛋白形成了Provirion膜的球形壳。在证明启用后，成熟蛋白酶被激活，并将插科打入其基质（MA），衣壳（CA）和Nucleocapsid（NC）部分。蛋白酶抑制剂是成功用于HIV的第一个抗病毒药物。在GAG片段中，重新组装形成病毒核的外壳，饲养病毒RNA和复制酶。有证据表明，正确形成的核心对于感染至关重要。但是核心是高度多态的。在这种情况下，我们一直在使用冷冻电子层析成像来可视化肌肉瘤病毒的成熟病毒，这是原型α-转子病毒。我们 数据集的直径为105至175 nm。它们的核心高度多态。我们观察到角核，包括一些棺材形的岩体，并为此提出了一种新颖的富勒烯几何形状。具有连续曲率的岩心，包括很少，富勒烯锥；和管状核。角芯是最庞大，堆积最密集的核心。试管和某些弯曲的核心包含较少的材料，表明包装不完整。从断层图中，我们测量了核心的表面积，因此是CA亚基的内容物。从病毒素直径中，我们估计了它们的原始插科打,，我们发现RSV病毒体（例如HIV）包含未组装的Ca亚基，而组装的CA的比例与核心类型相关。角核含有80％的可用亚基和开放式管，30％。糖蛋白峰值的数量是可变的（0至118），并且与核心类型相关。具有角核的病毒体平均82个尖峰，而具有管状核心的峰值为14个尖峰。这些观察结果表明，山皮组装的启动，其中尖峰内分域与插孔层的相互作用起着作用，是核心形态的关键决定因素。 （2）逆转录病毒衣壳几何形状。大多数含衣壳的病毒具有独特定义的大小和形状的衣壳。其中， 最常见的结构观察到二十面体对称性。相反，逆转录病毒衣壳高度多态。然而，它们也可以描述为富勒烯晶格的多面体折叠，在该晶格上，衣壳蛋白（CA）在该圈子上阵列。缺乏促进来自冷冻电子显微照片的二十面体皮的高度对称性的高阶，但可以通过低分辨率分辨率来确定单个逆转录病毒衣壳的三维结构。例如，在艾滋病毒的情况下，已经提出适当的结构为富勒烯锥。另一方面，我们发现圆锥体很少见肉瘤病毒衣壳。为了进一步研究它们的多态性（请参见上面的（1））。我们开发了计算和图形方法来构建与大小和形状相匹配的多面体模型，在完整的病毒体中观察到的RSV衣壳。他们属于几个形状的课程，包括管，“ lozenges”和“棺材”。衣壳从二十面体对称性偏离的程度反映了五聚体的分布的不规则性，对于封闭的多面体衣壳而言，它们的数量始终为12。随着六聚体的配额的增加，不同的多面体的数量迅速增长，并且在数百万的RSV Capsids中排名为150至300个六聚体。与二十面体病毒的衣壳蛋白不同，假设的准等效构象与等于三角剖分数（t）的数量最少，逆转录病毒CAS表现出近乎等效构象的近似值，这些特性可能归因于N-链接n-链接n-和c-末端域的柔性链接。