Structural Biology Of Retrovirus Assembly

在2008财年，我们主要关注两个子项目。 (1)逆转录病毒衣壳装配和多态性及其对感染性的影响。逆转录病毒衣壳的不寻常之处在于它们组装在成熟的病毒粒子内，而不是在感染细胞的细胞质或细胞核中。衣壳蛋白作为Gag多聚蛋白的一部分掺入前病毒体中，Gag多聚蛋白形成衬在前病毒体膜上的球形壳。在前病毒出芽后，成熟蛋白酶被激活并将Gag分解成其基质（MA）、衣壳（CA）和核衣壳（NC）部分。蛋白酶抑制剂是第一种成功用于抗HIV的抗病毒药物。在Gag片段中，CA重组形成病毒核心的外壳，容纳病毒RNA和复制酶。有证据表明，一个正确形成的核心是必不可少的传染性，但核心是高度多态性。在这种情况下，我们已经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。 数据集的直径范围为105至175 nm。它们的核心是高度多态的。我们观察到角核，包括一些独特的棺材形状，我们提出了一种新的富勒烯几何形状;连续曲率的核心，包括，很少，富勒烯锥;和管状核心。角形芯体体积最大，包装最密;管状芯体和一些弯曲芯体所含物质较少，表明包装不完整。从断层图像中，我们测量了核心的表面积，从而测量了它们的CA亚基含量。根据病毒粒子直径，我们估计了它们的Gag原始互补序列。我们发现，RSV病毒粒子，像HIV，含有未组装的CA亚基和CA的分数组装与核心类型相关;角形核心纳入80%的可用亚基和开放式管，30%。糖蛋白刺突的数量是可变的（0至118），也与核心类型相关;具有角形核心的病毒粒子平均82个刺突，而具有管状核心的病毒粒子平均14个刺突。这些观察结果表明，衣壳组装的起始是核心形态的关键决定因素，其中刺突内域与Gag层的相互作用起作用。 (2)逆转录病毒衣壳几何形状。大多数含有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。相反，逆转录病毒衣壳是高度多态的。然而，它们也可以被描述为富勒烯晶格的多面体折叠，其上排列着衣壳蛋白（CA）。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，在HIV的情况下，已经提出了合适的结构是富勒烯锥。另一方面，我们已经发现，锥是罕见的劳斯肉瘤病毒衣壳。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大小和形状匹配的多面体模型，在完整的病毒体中观察到RSV衣壳。它们分为几种形状类别，包括管状，“菱形”和“棺材”。衣壳偏离二十面体对称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，五聚体的数量总是12。不同的多面体的数量随着六聚体配额的增加而迅速增长，并且对于具有150至300个六聚体的RSV衣壳，其数量在数百万个中。与二十面体病毒的衣壳蛋白不同，其假定最小数量的准等价构象等于三角数（T），逆转录病毒CA表现出准等价构象的近连续体，这一性质可能归因于连接N-和C-末端结构域的柔性铰链。

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.