AAV and the DNA Damage Machinery

AAV 和 DNA 损伤机制

• 批准号: 7022645
• 负责人: Matthew D. Weitzman
• 金额: $ 42.71万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7022645
• 关键词: Adenoviridae; DNA damage; DNA repair; adeno associated virus group; biological models; host organism interaction; microorganism culture; molecular /cellular imaging; phosphatidylinositol 3 kinase; protein kinase; virus DNA; virus genetics; virus protein; virus replication; virus virus interaction

DESCRIPTION (provided by applicant): Viruses have proven to be powerful tools to study fundamental cellular mechanisms. In this proposal we will focus on interactions of viruses with cellular DNA repair pathways. We have discovered that viral genomes can represent substrates for the cellular DNA repair machinery, and that viral infection can lead to activation of signaling pathways characteristic of the DNA damage response. We will use the human adeno-associated virus (AAV) as a simple model system to study interactions with cellular factors. AAV possesses a single-stranded DNA genome and a biphasic lifecycle that requires helper viruses for efficient productive infection. We have uncovered a novel cellular DNA damage response that is activated by AAV co-infection with adenovirus, involving signaling by PI3-like kinases through cellular repair proteins. We will investigate the nature of the damage response, the viral elements that trigger it, the cellular kinases involved, and its impact on virus replication. The virus lifecycle is subject to remarkable spatial and temporal regulation, with the formation of structures within the nucleus where viral replication occurs. We will develop live cell imaging approaches to visualize AAV infection and will use these to (1) detect the conversion of incoming single- stranded DNA genomes to a duplex form, (2) study the dynamics of viral replication and (3) track interactions of cellular repair factors with viral replication centers. Our studies have demonstrated that viruses provide model systems to study the cellular responses to DNA damage. The experiments proposed in this grant will address mechanistic aspects of the recognition of the AAV genome by cellular repair proteins and how this cellular response affects virus infection. We will use a combination of genetic, biochemical and cytological approaches to study these host responses to viral infection. Recombinant forms of AAV provide one of the simplest and most promising vectors for gene therapy applications. Understanding how the cell recognizes, responds and processes viral DNA, is important for any gene therapy approach that delivers genetic material in viral vectors. Therefore, in addition to providing insights into the fundamental process of recognition of damaged DNA, these studies will also have implications for genetic approaches to combat disease.

描述（由申请人提供）：事实证明，病毒是研究基本细胞机制的强大工具。在此提案中，我们将重点关注病毒与细胞DNA修复途径的相互作用。我们已经发现，病毒基因组可以代表细胞DNA修复机制的底物，并且病毒感染可以导致DNA损伤反应的信号传导途径的激活。我们将使用人类腺相关病毒（AAV）作为一个简单的模型系统来研究与细胞因子的相互作用。 AAV具有单链DNA基因组和双相生命周期，需要辅助病毒才能有效生产感染。我们发现了一种新型的细胞DNA损伤反应，该反应通过AAV共感染与腺病毒一起激活，涉及PI3样激酶通过细胞修复蛋白的信号传导。我们将研究损伤反应的性质，触发它的病毒元素，所涉及的细胞激酶及其对病毒复制的影响。病毒生命周期受到明显的空间和时间调节的约束，并在发生病毒复制的核内形成结构。我们将开发活细胞成像方法来可视化AAV感染，并将其用于（1）检测传入的单链DNA基因组向双链形式的转化，（2）研究病毒复制的动力学，（3）跟踪细胞修复因子与病毒复制中心的相互作用。我们的研究表明，病毒提供了研究对DNA损伤的细胞反应的模型系统。该赠款中提出的实验将解决通过细胞修复蛋白识别AAV基因组的机械方面，以及该细胞反应如何影响病毒感染。我们将使用遗传，生化和细胞学方法的结合来研究这些宿主对病毒感染的反应。 AAV的重组形式为基因治疗应用提供了最简单，最有希望的载体之一。了解细胞如何识别，反应和过程病毒DNA对于任何传递病毒载体遗传物质的基因治疗方法都很重要。因此，除了提供识别受损DNA的基本过程的见解外，这些研究还将对打击疾病的遗传方法有影响。