Experimental Models of Viral Genome Evolution

描述(申请人提供)：提出了一个实验系统，用于 研究病毒在基因组发生重大变化时的进化：缺失， 基因重排，基因增加，以及监管的大规模变化。许多 这些基因组的变化将导致病毒适合性的大幅下降。这些能不能 修改后的病毒进化回高适合度，就像病毒一样 在进化对药物的抗药性方面非常多才多艺？这项工作将使用 噬菌体T7，它能适应广泛的基因组改变和 对其支持的分子遗传学和生物化学是广泛的 足以为进化开发一个预测性框架，并解释 进化机制。三个具体目标是：1)表征 具有修饰的完整基因组的病毒的调节进化；2)确定 带有工程缺失的病毒是否可以进化到恢复原来的状态 适应度水平；3)观察带有新基因的病毒基因组的进化。 基因组的改变将被执行，改变的病毒将被培养 广泛地允许通过进化和进化的基因组进行改进 将通过测序、绘制有益变化图和现场定向进行分析 突变以测试不同突变的效果。合并后的研究将 了解病毒基因组进化和进化的机制 在各种基因组修改中保持高适应性。这项工作 这里开发的将与开发相关的带电、衰减 疫苗，改进噬菌体作为抗菌剂(噬菌体疗法)， 并评估任何病毒的长期后果 具体功能。

DESCRIPTION (provided by applicant): An experimental system is proposed for studying viral evolution in response to major changes in its genome: deletions, gene rearrangements, gene additions, and wholesale changes in regulation. Many of these genome changes will cause major drops in viral fitness. Can these modified viruses evolve back to high fitness, just as viruses have been extremely versatile in evolving resistance to drugs? The work will use the bacteriophage T7, which is amenable to a wide scope of genomic alterations and for which the supporting molecular genetics and biochemistry is extensive enough to develop a predictive framework for evolution and to interpret the evolutionary mechanisms. The three specific aims are: 1) Characterize regulatory evolution in viruses with modified, complete genomes; 2) Determine whether viruses with engineered deletions can evolve to recover original fitness levels; 3) Observe the evolution of viral genomes with new genes. Genome alterations will be performed, the altered viruses will be grown extensively to allow improvement through evolution, and the evolved genomes will be analyzed by sequencing, mapping beneficial changes, and site-directed mutation to test the effects of different mutations. The combined studies will yield an understanding of the mechanisms by which viral genomes evolve and maintain high fitness across a variety of genome modifications. The work developed here will have relevance to the development of live, attenuated vaccines, the improvement of phages as antibacterial agents (phage therapy), and to assess the long-term consequences of any viruses engineered to perform specific functions.