权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Cellular Interactions of VSV Nucleocapsids

VSV 核衣壳的细胞相互作用

基本信息

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
项目类别：
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

项目摘要

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.

细胞的结构元素从来不是随机分布的。细胞质和细胞核都是分为不同的功能区。区域化的机制对于理解正常的细胞生理学以及疾病状态如癌症或神经疾病。病毒会在其宿主细胞内的区域组织的优势，导致增强的病毒复制，致病性细胞质的区域化问题对于细胞质的形成是一个特别重要的问题。负链RNA病毒的核蛋白核心（核衣壳），因为其作用多样性核衣壳在病毒复制周期中起作用。特别是，它们作为病毒RNA合成模板的作用发生在细胞质的区域，这些区域与它们被整合到后代中的位点不同病毒体从宿主细胞膜出芽。核衣壳太大，不能在细胞中自由扩散。细胞质中，因此必须有特定的运输机制，以确保其适当的分布。拟议实验研究了原型负链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运动，解决这些假设。第一种方法，我们称之为边界到边界的分配方法，量化了稳态分布。第二个涉及改进活细胞成像方法，确定粒子运动的动力学，特别适合于分析肌动蛋白依赖性运动。使用这些方法，我们的数据表明，核衣壳被运输到细胞周边的两个肌动蛋白丝和微管。然而，我们的数据表明，肌动蛋白丝是更重要的，微管到达病毒装配的最终位点。此外，核衣壳的分布细胞质似乎与分泌途径的膜偶联。这些新的分析工具将用于目标1，以确定不同肌球蛋白马达在建立肌球蛋白分布中的作用。细胞质中的核衣壳和掺入病毒体。具体目标2是确定依赖性核衣壳分布在分泌途径的细胞膜上。这些实验将集中在参与膜分选和转运的GTP结合蛋白，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了膜结合的核衣壳。在Aim 3中，将确定核衣壳在极化上皮细胞和神经元中分布的机制，因为这些代表了参与VSV自然感染的细胞类型。拟议的实验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。它们还基于新颖的概念和分析方法，在细胞生物学中的普遍适用性。