AAV, helper virus and host cell interactions

AAV、辅助病毒和宿主细胞相互作用

• 批准号: 6752120
• 负责人: JAMES P TREMPE
• 金额: $ 29.74万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752120
• 关键词: Adenoviridae; adeno associated virus group; affinity chromatography; binding sites; cell line; chromatin; genetic promoter element; genetic transcription; genetic translation; helper virus; host organism interaction; immunoprecipitation; messenger RNA; mutant; polymerase chain reaction; protein binding; purines; transcription factor; transfection /expression vector; virus protein; virus replication; virus virus interaction

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a common, nonpathogenic, helper-dependent human parvovirus. AAV has generated much enthusiasm in recent years because of its potential as a human gene therapy vector. Although vector development is progressing rapidly, there is a lack of understanding of how AAV amplification, gene expression and site-specific integration affect the helper virus and host cell. All three processes are dependent upon de novo expression of, and regulation by, viral replication (Rep) proteins, Rep78 and Rep68. Interestingly, exogenous expression of these proteins has profound effects on the host cell and helper virus. To understand the interactions between AAV and its environment, we have begun a study of the inhibitory effects of AAV on Adenovirus (Ad) replication and gene expression. We present evidence that AAV and its encoded Rep proteins inhibit Ad replication. These inhibitions are due, in part, to inhibition of early gene transcription. The Rep protein-mediated alterations on transcription are due to effects on the Ad early gene promoters as well as on E1A-mediated trans-activation of early gene expression. The following specific aims are designed to elucidate the mechanistic roles played by AAV and its Rep proteins in an Ad co-infection; (1) Characterize early interactions between AAV and Ad in a co-infection, (2) Define the Rep and E1A protein domains and Ad promoter elements involved in Rep regulation of Ad gene expression, (3) Characterize the biochemical mechanisms of Rep-mediated inhibition, and activation, of gene expression. This novel, model system of host-virus interaction benefits from the wealth of knowledge in the literature on Ad biology and the abundance of available Ad reagents for study. Determining how AAV interacts with its most efficient helper virus, we will obtain valuable insights into how AAV interacts with host cells as well. These studies will also have clear ramifications for further AAV vector development, especially with regard to improvement in AAV vector packaging systems.

描述（由申请人提供）：腺相关病毒（AAV）是一种常见的、非致病性的、依赖帮助者的人细小病毒。近年来，AAV因其作为人类基因治疗载体的潜力而引起了人们的极大热情。虽然载体开发进展迅速，但对AAV扩增、基因表达和位点特异性整合如何影响辅助病毒和宿主细胞缺乏了解。这三个过程都依赖于病毒复制（Rep）蛋白Rep78和Rep68的从头表达和调控。有趣的是，这些蛋白的外源表达对宿主细胞和辅助病毒有深远的影响。为了了解AAV与其环境之间的相互作用，我们已经开始研究AAV对腺病毒（Ad）复制和基因表达的抑制作用。我们提出证据证明AAV及其编码的Rep蛋白抑制Ad复制。这些抑制部分是由于早期基因转录的抑制。Rep蛋白介导的转录改变是由于对Ad早期基因启动子以及e1a介导的早期基因表达反式激活的影响。以下具体目的旨在阐明AAV及其Rep蛋白在Ad合并感染中的机制作用；(1)确定AAV和Ad在合并感染中的早期相互作用；(2)确定Rep和E1A蛋白结构域以及参与Rep调控Ad基因表达的Ad启动子元件；(3)确定Rep介导的基因表达抑制和激活的生化机制。