GENETIC STUDIES OF ADENO ASSOCIATED VIRUS

腺相关病毒的基因研究

• 批准号: 2331962
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 24.83万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2331962
• 关键词: DNA directed DNA polymerase; DNA replication; DNA replication origin; Herpesviridae; adeno associated virus group; affinity chromatography; capsid; genetic regulatory element; helper virus; host organism interaction; nucleic acid repetitive sequence; protein purification; virus DNA; virus genetics; virus integration; virus protein; virus replication; virus virus interaction

Adeno-associated virus (AAV) is a human DNA virus which contains a linear, single-stranded, DNA genome, approximately 5kb long. One of the unusual aspects of AAV is the mechanism of DNA replication, which involves only leading strand DNA synthesis. Viral replication relies on the AAV coded rep gene and cellular replication enzymes. Therefore, studies of AAV DNA replication should be useful for identifying the cellular enzymes that are involved uniquely in leading strand synthesis. During DNA replication, the virus uses a 145 bp hairpinned terminal repeat to prime DNA synthesis, and then regenerates the primer by a process called terminal resolution. In addition to being the origins for DNA replication, the terminal repeats also contain sequences that are essential for viral packaging and for integration of the viral genome into host chromosomes. In our previous work we purified the AAV Rep68 and 78 proteins and demonstrated that they are the site specific endonucleases that initiate terminal resolution. We also showed that they have an intrinsic DNA helicase activity. During the course of this work we developed several biochemical assays to study the interaction of Rep with the terminal repeats. These include the terminal resolution assay and the site specific nicking assay (or trs endonuclease reaction). More recently, we have also developed a complete in vitro DNA replication assay. Using these as well as conventional binding assays we demonstrated that there are at least three separate sequences within the terminal repeat that are required for substrate recognition by the Rep protein: the secondary structure element, a linear Rep binding sequence, and a specific sequence required for nicking by the enzyme, the terminal resolution site (trs). Finally, we and others have shown that the Rep proteins have multiple roles during the viral life cycle. In addition to being essential for DNA replication, Rep68 and 78 are also required for transactivation of AAV transcription, repression of AAV and heterologous gene expression, and the repression of oncogenic transformation by some viral and cellular oncogenes. This suggests that the Rep proteins probably interact with a variety of cellular proteins. Our specific aims are: 1) To characterize in detail the three elements of the terminal repeat required for origin function. In the process we also hope to identify sequences in the terminal repeat that are required for integration and packaging. 2) To purify and identify the cellular DNA replication enzymes that are required for AAV DNA replication. 3) To identify the cellular enzymes that interact with the AAV Rep protein.

腺相关病毒（AAV）是一种人类 DNA 病毒，含有 线性、单链 DNA 基因组，长约 5kb。中的一个 AAV 的不同寻常之处在于 DNA 复制机制， 仅涉及前导链 DNA 合成。病毒复制依赖于 AAV 编码rep 基因和细胞复制酶。所以， AAV DNA 复制的研究应该有助于识别 独特地参与前导链合成的细胞酶。 在 DNA 复制过程中，病毒使用 145 bp 的发夹末端 重复启动DNA合成，然后通过a重新生成引物 过程称为终端解析。除了作为起源 DNA复制时，末端重复序列还包含 对于病毒包装和病毒基因组整合至关重要 进入宿主染色体。 在我们之前的工作中，我们纯化了 AAV Rep68 和 78 蛋白并 证明它们是启动的位点特异性核酸内切酶 终端分辨率。我们还表明它们具有内在的 DNA 解旋酶活性。在这项工作的过程中，我们开发了几个 研究 Rep 与末端相互作用的生化测定 重复。这些包括最终分辨率测定和位点 特异性切口测定（或trs核酸内切酶反应）。最近，我们 还开发了完整的体外 DNA 复制测定。使用 这些以及传统的结合测定，我们证明了 末端重复内至少有三个独立的序列 Rep 蛋白识别底物所需：次级 结构元素、线性Rep结合序列和特定序列 酶切割所需的末端解析位点 (trs)。 最后，我们和其他人已经证明 Rep 蛋白具有多种 在病毒生命周期中的作用。除了对 DNA 至关重要之外 复制、Rep68 和 78 也是 AAV 反式激活所必需的 转录、抑制 AAV 和异源基因表达，以及 某些病毒和细胞对致癌转化的抑制 癌基因。这表明 Rep 蛋白可能与 多种细胞蛋白。 我们的具体目标是： 1）详细描述这三个要素 原点功能所需的终端重复。在这个过程中我们 还希望识别末端重复中所需的序列 用于集成和封装。 2) 细胞DNA的纯化和鉴定 AAV DNA 复制所需的复制酶。 3) 至 鉴定与 AAV Rep 蛋白相互作用的细胞酶。