Cellular Interactions of VSV Nucleocapsids

细胞的结构元素从来不是随机分布的。细胞质和细胞核都是 组织成不同的职能区域。区域化的机制对于理解很重要 正常的细胞生理和疾病状态，如癌症或神经系统疾病。病毒夺走 区域组织在其宿主细胞内的优势，从而增强病毒复制和 致病性。细胞质的区域化问题是一个特别重要的问题 负链RNA病毒的核蛋白核心(核衣壳)因其作用的多样性 核衣壳在病毒复制周期中发挥作用。特别是，它们作为病毒RNA合成的模板的作用 发生在细胞质的区域，不同于它们结合到后代中的位置 从寄主质膜萌发的病毒粒子。核衣壳太大，不能在体内自由扩散 因此，必须有特定的运输机制来确保它们的适当分布。建议数 实验解决了原型负链RNA病毒-水泡性口炎病毒的这些机制。 我们已经开发了两种新的细胞成像方法来量化细胞成分的移动到 解决这些假设。第一种方法，我们称之为边界到边界分配方法， 量化稳态分布。第二个涉及改进活细胞成像方法，以 确定特别适合分析肌动蛋白依赖的运动的粒子运动的动力学。 使用这些方法，我们的数据显示，核衣壳通过两种途径向细胞外围运输 肌动蛋白细丝和微管。然而，我们的数据表明，肌动蛋白细丝比 微管到达病毒组装的终极位置。此外，核衣壳在人体内的分布 细胞质似乎与分泌途径的膜相连。这些新的分析工具将 在目标1中被用来确定不同的肌球蛋白马达在建立 核衣壳存在于细胞质中，并与病毒粒子结合。具体目标2是确定依赖关系 核衣壳在分泌途径细胞膜上的分布。这些实验将集中在 参与膜分选和转运的GTP结合蛋白以及与之相关的宿主膜蛋白 通过一系列新的蛋白质组学实验确定了膜结合的核衣壳。在目标3中， 核衣壳在极化上皮细胞和神经元中的分布机制将被确定，因为 这些代表了VSV自然感染所涉及的细胞类型。拟议中的实验面临挑战 并试图改变目前对病毒衣壳与宿主相互作用机制的看法 细胞骨架和细胞膜。它们还基于新的概念和分析方法，应该 在细胞生物学中具有普遍适用性。

The structural elements of the cell are never randomly distributed. Both the cytoplasm and nucleus are organized into different functional regions. The mechanisms of regionalization are important for understanding normal cellular physiology as well as disease states such as cancer or neurological diseases. Viruses take advantage of the regional organization within their host cells, resulting in enhanced virus replication and pathogenicity. The question of regionalization of the cytoplasm is a particularly important one for the nucleoprotein core (nucleocapsid) of negative strand RNA viruses because of the diversity of roles nucleocapsids play in the virus replication cycle. In particular, their roles as templates for viral RNA synthesis occur in regions of the cytoplasm that are distinct from the sites at which they are incorporated into progeny virions by budding from the host plasma membrane. Nucleocapsids are too large to diffuse freely in the cytoplasm, so there must be specific transport mechanisms to ensure their proper distribution. The proposed experiments address these mechanisms for the prototype negative strand RNA virus, vesicular stomatitis virus. We have developed two new cellular imaging approaches to quantify the movement of cellular elements to address these hypotheses. The first approach, which we call the border-to-border distribution method, quantifies the steady state distribution. The second involves improvement of live cell imaging approaches to determine the kinetics of particle movement that are particularly well suited to analyze actin-dependent motion. Using these approaches, our data show that nucleocapsids are transported toward the cell periphery by both actin filaments and microtubules. However, our data indicate that actin filaments are more important than microtubules in reaching the ultimate sites of virus assembly. Furthermore, the distribution of nucleocapsids in the cytoplasm appears to be coupled to membranes of the secretory pathway. These new analytical tools will be used in Aim 1 to determine the role of different myosin motors in establishing the distribution of nucleocapsids in the cytoplasm and incorporation into virions. Specific Aim 2 is to determine the dependence of nucleocapsid distribution on cellular membranes of the secretory pathway. These experiments will focus on GTP-binding proteins involved in membrane sorting and transport, and host membrane proteins associated with membrane-bound nucleocapsids identified by a series of new proteomics experiments. In Aim 3 the mechanisms of nucleocapsid distribution in polarized epithelial cells and neurons will be determined, since these represent the cell types involved in the natural infection by VSV. The proposed experiments challenge and seek to shift the current thinking on the mechanisms of interaction of viral capsids with the host cytoskeleton and membranes. They are also based on novel concepts and analytical methods that should be of general applicability in cell biology.