Genetic Recombination in Brome Mosaic Virus: The Role of Intergenic Region in Junction Site Selection

本项目的长期目标是了解RNA病毒基因重组的机制。 通过使用雀麦花叶溴病毒（BMV）作为模型RNA病毒，在RNA和蛋白质水平上的同源和非同源事件之间的机制差异先前已被报道。 这些研究提出了一种拷贝选择机制，其中BMV复制酶复合物在RNA复制过程中在RNA模板之间发生了变化。为了确定信号序列在连接位点选择过程中的作用，从而确定导致有效重组热点的条件，本项目的重点是BMV RNA 3中的多功能基因间区域。 最近的实验证实了BMV RNA 3分子在基因间热点内的频繁同源交换。该项目的目标之一是利用分子遗传学方法绘制基因间区域内的重组活性序列。 它的第二个目标是确定这些序列如何相互关联，以便有效地指导连接位点。实现上述研究目标的具体目标包括研究亚基因组启动子在重组中的作用，包括1）核心区; 2）polyA区; 3）增强序列;和4）二级结构。这种新的实验方法将帮助我们获得有关同源RNA重组机制的新数据。这反过来将提供有关RNA病毒重组活性控制的信息。该模型研究的结果将适用于利用基因间（亚基因组）启动子序列的各种RNA病毒。

ABSTRACTThe long-term goal of this project is to understand the mechanism(s) of genetic recombination in RNA viruses. By using brome mosaic bromovirus (BMV) as a model RNA virus, mechanistic differences between homologous and nonhomologous events at the RNA and protein levels have been previously reported. These studies have suggested a copy choice mechanism, where the BMV replicase complex has changed among the RNA templates during RNA replication. To define the role of signal sequences during the junction sites selection and thus to determine the conditions leading to the efficient recombination hot spots, this project focuses on the multifunctional intergenic region in BMV RNA3. Frequent homologous exchanges between molecules of BMV RNA3 within the intergenic hot spot have been recently confirmed experimentally. One goal of this project is to map the recombinationally active sequences inside the intergenic region, by using molecular genetics approaches. Its second goal is to determine how these sequences relate to each other in order to direct the junction sites efficiently. Specific aims to achieve the above research goals include studies of the role of a subgenomic promoter in recombination, including 1) the core region; 2) the polyA tract; 3) enhancing sequences; and 4) secondary structures. This novel experimental approach will help us obtain new data regarding the mechanism(s) of homologous RNA recombination. This, in turn, will provide information concerning the control of recombination activities in RNA viruses. The results of this model research will be applicable to a variety of RNA viruses that utilize intergenic (subgenomic) promoter sequences.