Experimental Models of Viral Genome Evolution

描述（由申请人提供）：提出了一种实验系统，用于 研究病毒进化对基因组重大变化的反应：缺失， 基因重排、基因添加和调节的大规模变化。许多 这些基因组的变化将导致病毒适应性的大幅下降。让这些 修改后的病毒进化回高适应性，就像病毒一样。 在进化抗药性方面非常灵活这项工作将使用 噬菌体T7，其适合于大范围的基因组改变， 支持分子遗传学和生物化学是广泛的 足以发展出一个进化的预测框架， 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中的调控进化; 2）确定 基因缺失的病毒是否能进化到 适应度水平; 3）观察具有新基因的病毒基因组的进化。 基因组的改变将被执行，改变的病毒将生长 广泛地允许通过进化进行改进，而进化的基因组 将通过测序、绘制有益变化和定点突变来分析 突变来测试不同突变的影响。合并研究将 了解病毒基因组进化的机制， 在各种基因组修饰中保持高适应性。工作 在这里开发的将与活的减毒的 疫苗，抗菌药物的改进（噬菌体疗法）， 并评估任何被设计用来执行 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.