Cellular Interactions of VSV Nucleocapsids

VSV 核衣壳的细胞相互作用

基本信息

批准号：
10170210
负责人：
DOUGLAS S. LYLES
金额：
$ 42.08万
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 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10170210
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 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 Structure 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都不是随机分布的。细胞质和细胞核都是划分为不同的功能区域。区域化机制对于理解很重要正常细胞生理学以及癌症或神经系统疾病等疾病状态。病毒采取利用宿主细胞内的区域组织的优势，从而增强病毒复制和致病性。细胞质的区域化问题对于细胞质的研究来说是一个特别重要的问题。由于作用的多样性，负链RNA病毒的核蛋白核心（核衣壳）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 合成模板的作用发生在与它们并入后代的位点不同的细胞质区域病毒粒子从宿主质膜上出芽。核衣壳太大而不能在细胞内自由扩散细胞质，因此必须有特定的运输机制来保证它们的正确分布。拟议的实验针对原型负链 RNA 病毒（水泡性口炎病毒）解决了这些机制。我们开发了两种新的细胞成像方法来量化细胞元素的运动解决这些假设。第一种方法，我们称之为边界到边界分布方法，量化稳态分布。第二个涉及活细胞成像方法的改进确定颗粒运动的动力学，特别适合分析肌动蛋白依赖性运动。使用这些方法，我们的数据表明，核衣壳通过两种方式运输到细胞外围：肌动蛋白丝和微管。然而，我们的数据表明肌动蛋白丝比微管到达病毒组装的最终位点。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并掺入病毒粒子。具体目标 2 是确定依赖性分泌途径细胞膜上核衣壳的分布。这些实验将集中于 GTP 结合蛋白参与膜分选和运输，以及与宿主膜蛋白相关通过一系列新的蛋白质组学实验鉴定出膜结合的核衣壳。在目标 3 中极化上皮细胞和神经元中核衣壳分布的机制将被确定，因为这些代表参与 VSV 自然感染的细胞类型。提出的实验挑战并寻求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，这些概念和分析方法应该具有在细胞生物学中的普遍适用性。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Vesicular stomatitis virus nucleocapsids diffuse through cytoplasm by hopping from trap to trap in random directions.

水疱性口炎病毒核衣壳通过随机方向从一个陷阱跳跃到另一个陷阱而在细胞质中扩散。

DOI：
10.1038/s41598-020-66942-6
发表时间：
2020
期刊：
Scientific reports
影响因子：
4.6
作者：
Holzwarth,George;Bhandari,Arnav;Tommervik,Lucas;Macosko,JedC;Ornelles,DavidA;Lyles,DouglasS
通讯作者：
Lyles,DouglasS