Mechanisms of the AAV2 Rep motor protein

腺相关病毒2型是一种明显的非致病性人类病毒，可以感染细胞， 细胞周期独立的方式。AAV的特性已经被广泛地利用， 这些努力表明AAV是最有前途的治疗性基因转移载体之一。在 除了AAV的生产性生命周期（其需要辅助病毒的存在， 腺病毒或疱疹病毒）AAV已经进化出整合其基因组位点- 特别是人类19号染色体，以建立潜伏感染。我们的长期目标是 为了阐明AAV 2复制、位点特异性整合和 包装. AAV的小基因组包含两个开放阅读框（REP和CAP）。的Rep 蛋白质协调病毒生活方式的所有方面，包括DNA复制、位点特异性 集成、从集成状态和病毒包装中拯救。对这种蛋白质的研究 一直局限于生化表征和缺乏结构信息，阻止了一个 代表介导的过程的机制方面的全面描述。最近我们 已经解决了AAV 2 Rep的核心解旋酶结构域的结构。 存在于所有四种Rep蛋白中，它可能在Rep功能的所有方面起重要作用。 在本申请中，我们提出了一种协作和多方面的方法（结合结构， 生物物理和生物化学研究），旨在了解电机的结构基础 在不同异构体的情况下Rep的活性。具体来说，我们将调查 Rep 40 ATP依赖性运动活动的结构决定因素（目的1）;我们将补充 并通过生物化学研究扩展结构信息，旨在帮助开发一种模型， Rep的运动活动机制（目标2）;并且，使用Rep 68，我们将研究 运动活动，在这种情况下，是针对复制DMA解旋。(Aim 3）。

Adeno-associated virus type 2 is an apparently non-pathogenic human virus that can infect cells in a cell cycle-independent fashion. The characteristics of AAV have been exploited in extensive efforts that have shown AAV to be among the most promising therapeutic gene transfer vehicles. In addition to the productive life cycle of AAV (that requires the present of a helper virus such as adenovirus or herpes viruses) AAV has evolved the unique ability to integrate its genome site- specifically into human chromosome 19 in order to establish a latent infection. Our long-term goal is to elucidate the molecular mechanisms underlying AAV2 replication, site-specific integration and packaging. The small genome of AAV contains two open reading frames (REP and CAP). The Rep proteins orchestrate all aspects of the viral life style including DMA replication, site-specific integration, rescue from the integrated state and virus packaging. The study of this protein has been limited to biochemical characterizations and the lack of structural information has prevented a comprehensive depiction of the mechanistic aspects of Rep-mediated processes. Recently, we have solved the structure of the core helicase domain of AAV2 Rep. As this motor domain is present in all four Rep proteins it is likely to play an essential role in all aspects of Rep function. In this application we propose a collaborative and multifaceted approach (combining structural, biophysical and biochemical studies) aimed at understanding the structural basis for the motor activity of Rep within the context of the different isomers. Specifically, we will investigate the structural determinants of the Rep40 ATP-dependent motor activity (Aim 1); we will complement and extend the structural information by biochemical studies designed to help develop a model for the mechanism of Rep's motor activity (Aim 2); and, using Rep68, we will study the utilization of the motor activity that, in this context, is directed at replicative DMA unwinding. (Aim 3).