Collaborative Research: Dynamics of RNA dependent RNA polymerases

合作研究：RNA 依赖性 RNA 聚合酶的动力学

• 批准号: 1614940
• 负责人: David Grunwald
• 金额: $ 24万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1614940&HistoricalAwards=false
• 关键词: Collaborative; Research; Dynamics; RNA; dependent

The goal of this project is to understand the molecular mechanisms of early stages of replication of a virus with an RNA genome. Viruses with similar RNA genomes include potent human and animal pathogens, including Ebola, measles, and vesicular stomatitis virus (VSV). VSV is the model being studied in this project. To read and copy their RNA genomes, VSV and related RNA viruses make a special RNA polymerase that makes RNA copies of the viral RNA genomes, and therefore are called "RNA dependent RNA Polymerases" or RdRPs. This project will examine the mechanism by which RdRPs read and copy the viral genome template in infected cells which is critical for the replication of viruses. The results from this study will improve scientific understanding of how RdRPs work and identify potential target mechanisms for inhibition of viral infections. In addition to furthering knowledge about RNA virus replication, this study will train minority undergraduate students by incorporating them within teams doing research at the University of Utah and UMass Medical School at Worcester. The proposed activities will also support development of Biophysics courses that will benefit undergraduate interdisciplinary education and encourage participation of K-12 children in science by communicating aspects of the scientific results through creation of virus dance projects for children.This project will use single molecule live cell imaging to visualize transcription events from single VSV genome templates in infected cells and utilize fluorescence correlation spectroscopy to measure the concentration of free RdRPs during transcription in vivo. When stretched, the genome template of VSV is more than 4 microns in size and RdRPs can only initiate transcription on its 3' end. While 50 RdRPs are tightly bound to this template, how they initiate and sustain transcription is not clear. The live cell imaging experiments will determine the number of active RdRPs per genome and their cooperativity. Measurements of the concentration of free RdRPs not bound to the genome will verify if a dissociation and 3' binding mechanism can support viral transcription in vivo. This project will also use reconstituted templates in vitro to visualize the mechanism by which RdRPs redistribute on the genome template. We have observed single RdRP sliding on purified genome templates in vitro. In this project we will further characterize the sliding mechanism of RdRPs on genome templates. By measuring the dynamics of RdRPs in vivo as well as in vitro this project will determine the mechanism of transcription by RdRPs during VSV RNA virus infection.The project was funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.

该项目的目标是了解具有RNA基因组的病毒复制早期阶段的分子机制。具有相似RNA基因组的病毒包括有效的人类和动物病原体，包括埃博拉病毒、麻疹病毒和水疱性口炎病毒（VSV）。VSV是本项目正在研究的模型。为了读取和复制它们的RNA基因组，VSV和相关的RNA病毒产生一种特殊的RNA聚合酶，该聚合酶产生病毒RNA基因组的RNA拷贝，因此被称为“RNA依赖性RNA聚合酶”或RdRP。该项目将研究RdRPs在感染细胞中读取和复制病毒基因组模板的机制，这对病毒的复制至关重要。这项研究的结果将提高对RdRP如何工作的科学理解，并确定抑制病毒感染的潜在靶机制。除了进一步了解RNA病毒复制，这项研究将通过将少数民族本科生纳入在犹他州大学和伍斯特马萨诸塞大学医学院进行研究的团队来培训他们。拟议的活动还将支持生物物理学课程的开发，这将有利于本科生的跨学科教育，并鼓励K-通过为儿童创建病毒舞蹈项目，交流科学成果的各个方面，使12名儿童参与科学。该项目将使用单分子活细胞成像技术，在受感染细胞中可视化来自单个VSV基因组模板的转录事件，并利用荧光相关性在一个实施方案中，RdRP可以通过光谱法来测量体内转录期间游离RdRP的浓度。当拉伸时，VSV的基因组模板的大小超过4微米，并且RdRPs只能在其3'端启动转录。虽然50个RdRP与该模板紧密结合，但它们如何启动和维持转录尚不清楚。活细胞成像实验将确定每个基因组的活性RdRP的数量及其协同性。测量未与基因组结合的游离RdRP的浓度将验证解离和3'结合机制是否可以支持体内病毒转录。该项目还将使用体外重组模板来可视化RdRPs在基因组模板上重新分布的机制。我们已经观察到单个RdRP在体外纯化的基因组模板上滑动。在这个项目中，我们将进一步表征RdRPs在基因组模板上的滑动机制。本项目通过测定RdRPs在体内和体外的动态变化，来确定VSV RNA病毒感染时RdRPs的转录机制，该项目由分子和细胞生物科学部遗传机制计划资助。