Cellular Interactions of VSV Nucleocapsids

VSV 核衣壳的细胞相互作用

• 批准号: 9512738
• 负责人: DOUGLAS S. LYLES
• 金额: $ 42.36万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-20 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9512738
• 关键词: Actins; Adaptor Signaling Protein; Address; Animal Model; Antiviral Agents; Biological Assay; Capsid; Cell Nucleus; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular biology; Characteristics; Chemicals; Computer software; Conflict (Psychology); Confocal Microscopy; Coupled; Cytoplasm; Cytoskeleton; Data; Dependence; Diffuse; Disease; Elements; Ensure; Epithelial Cells; Fluorescence Microscopy; GTP-Binding Proteins; Goals; Hela Cells; Image; Image Analysis; Individual; Infection; Kinetics; Lead; Literature; Malignant Neoplasms; Membrane; Membrane Proteins; Methods; Microfilaments; Microtubules; Molecular Motors; Motion; Motor; Movement; Mus; Myosin ATPase; Neuraxis; Neurons; Nucleocapsid; Nucleoproteins; Pathogenicity; Pathway interactions; Pharmacology; Physiologic pulse; Play; Process; Proteins; Proteomics; RNA Interference; RNA Viruses; RNA chemical synthesis; Role; Series; Shapes; Site; Skeleton; Sorting - Cell Movement; Supporting Cell; Thinking; Tubular formation; Vesicular stomatitis Indiana virus; Viral; Virion; Virus; Virus Assembly; Virus Replication; analytical method; analytical tool; base; cell type; cellular imaging; crosslink; experimental study; fluorescence imaging; genetic information; imaging approach; live cell imaging; nervous system disorder; novel; novel strategies; particle; prototype; viral RNA; virology

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.

细胞的结构元素永远不会随机分布。细胞质和核都是 组织成不同的功能区域。区域化的机制对于理解很重要 正常的细胞生理以及癌症或神经疾病等疾病。病毒服用 在其宿主细胞内的区域组织的优势，从而增强了病毒复制和 致病性。细胞质区域化的问题对于该问题是一个特别重要的问题。 由于作用的多样性 Nucleocapsids在病毒复制周期中发挥作用。特别是，它们作为病毒RNA合成模板的作用 发生在细胞质区域，与将它们合并到后代的位置不同 从宿主质膜萌芽的病毒体。 Nucleocapsids太大，无法在 细胞质，因此必须有特定的运输机制来确保其适当的分布。提议 实验解决了原型负链RNA病毒，囊泡炎病毒的这些机制。 我们开发了两种新的细胞成像方法，以量化细胞元件的运动 解决这些假设。我们称之为边界到边界分布方法的第一种方法， 量化稳态分布。第二个涉及改进活细胞成像方法 确定特别适合分析肌动蛋白依赖性运动的粒子运动动力学。 使用这些方法，我们的数据表明，核素是由两者传输到细胞周围的 肌动蛋白丝和微管。但是，我们的数据表明肌动蛋白丝比 微管达到病毒组装的最终部位。此外，核素的分布在 细胞质似乎与分泌途径的膜耦合。这些新的分析工具将 在目标1中使用以确定不同肌球蛋白电动机在建立分布中的作用 细胞质中的核蛋白质并掺入病毒体中。具体目标2是确定依赖性 分泌途径的细胞膜上的核蛋白分布。这些实验将重点放在 GTP结合蛋白参与膜分类和运输，以及相关的宿主膜蛋白 通过一系列新的蛋白质组学实验鉴定的膜结合的核素。在目标3中 将确定极化上皮细胞和神经元中核蛋白分布的机制，因为 这些代表VSV自然感染所涉及的细胞类型。提出的实验挑战 并寻求转移当前对病毒包包与宿主相互作用机制的思维 细胞骨架和膜。它们也基于应有的新颖概念和分析方法 细胞生物学中的一般适用性。