Role of Subgenomic RNAs in Genetic Recombination of Brome Mosaic Bromovirus

知识价值。RNA病毒是最具挑战性的传染性病原体之一。有些RNA病毒会引起人类的严重疾病，而另一些RNA病毒则会对牲畜或农作物造成严重疾病。限制RNA病毒控制的主要问题之一是其遗传信息的快速变化导致新的病毒株不断出现。RNA重组涉及到病毒RNA片段的交换，RNA重组策略是RNA病毒确保其快速适应和进化的关键工具。与包括DNA病毒在内的基于DNA的生物体的基因重组相比，RNA病毒中的RNA重组尚未得到很好的研究。溴梅花叶病毒（BMV）系统是了解RNA重组基本机制的一个极好的精心设计的模型。在这个项目中，研究将集中在负责高效同源BMV RNA-RNA交叉的分子因子的表征上。该项目利用了先前BMV RNA分子上支持异常高重组频率的局部区域（定义为RNA3顺子间重组热点或“sgp”）以及最近发现的新型亚基因组（sg） BMV RNA（命名为“sgRNA3a”），以阐明sgp已知的多种功能中哪一种负责重组。该项目的实验设计依赖于对改良的BMV RNA3构建体进行各种体外和体内试验。该项目的智力价值在于其对sgRNA分子在模型RNA病毒系统中的作用的研究。这些研究将为科学家们提供有关RNA病毒调控有效基因重组的分子策略的新信息。这些研究的结果将有助于更全面地了解适用于其他RNA病毒的重组机制。项目的广泛影响。通过使用BMV模型系统，该项目将对我们关于病毒分子适应的知识做出重大贡献，并将有助于在DNA和RNA遗传学之间进行类比。BMV是一个更大的RNA病毒超级家族的成员，其分子策略在其他病毒中是常见的。这项研究将为学生提供在生物化学、分子生物学、植物生物学和病毒学等重要领域的实践训练。首席研究员教授分子病毒学课程，这项工作将培养年轻的病毒学家。此外，首席研究员还教授一门名为“RNA世界”的高级课程。首席研究员有长期的本科培训记录，包括那些来自代表性不足的群体。所有学生都将被鼓励在科学会议上展示他们的研究成果（例如，Sigma Xi，美国植物生物学家学会，美国病毒学学会）。首席研究员小组与芝加哥大学和波兰波兹南的生物有机化学研究所保持合作，并与几个国际病毒学小组保持积极联系。这些互动将增加学生对国际环境的接触。首席研究员正在努力通过翻译英文作品来传播科学（已将马特·里德利（Matt Ridley）的一本书《红皇后，人性的性与进化》翻译成波兰语）。总的来说，该项目的社会效益取决于我们对RNA病毒中RNA重组的理解的提高，这将为控制这些重要的感染因子提供新的潜在手段。它还将通过外展活动提高公众的科学素养。通过学习如何预测重组热点，科学家将能够设计更稳定的病毒载体作为重要的生物技术工具。

Intellectual Merit. RNA viruses are among the most challenging infectious agents. Some RNA viruses cause serious diseases in human beings, while other RNA viruses do so to livestock or to crop plants. One of the main problems limiting the control of RNA viruses is rapid change of their genetic information which leads to constant emergence of new viral strains. RNA recombination involves the exchange of viral RNA fragments and the strategies of RNA recombination are key tools needed for RNA viruses to secure their fast adaptation and evolution. In contrast to genetic recombination of DNA-based organisms including DNA viruses, RNA recombination in RNA viruses is not well studied. The Brome mosaic virus (BMV) system is an excellent well-elaborated model for the understanding of basic mechanisms of RNA recombination. In this project the research will focus on the characterization of the molecular factors that are responsible for efficient homologous BMV RNA-RNA crossovers. The project takes advantage of a previously localized region on the BMV RNA molecule that supports unusually high frequency of recombination (defined as the RNA3 intercistronic recombination hot-spot or "sgp") as well as a recently discovered novel subgenomic (sg) BMV RNA (named as "sgRNA3a") in order to elucidate which of the known multiple functions of the sgp is responsible for recombination. The experimental design of this project relies on testing of the modified BMV RNA3 constructs in a variety of both in vitro and in vivo assays. The intellectual merit of the project lies in its investigation of the roles of sgRNA molecules in a model RNA virus system. These studies will provide the scientists with new information about molecular strategies that are utilized by RNA viruses to condition the efficient genetic recombination. Results from these studies will contribute to a more integrated picture of recombination mechanisms that will be applicable to other RNA viruses.Broader impacts of the project. This project will contribute significantly to our knowledge about viral molecular adaptations, and will help to draw analogies between DNA and RNA genetics, by using the BMV model system. BMV is a member of a larger RNA virus super-family, and its molecular strategies are common among other viruses. This research will provide students with hands-on training in the important areas of biochemistry, molecular biology, plant biology, and virology. The principal investigator teaches a molecular virology course and this work will train young virologists. Also, the principal investigator teaches an advanced course entitled "The RNA World". The principal investigator has a long record of undergraduate training including those from underrepresented groups. All the students will be encouraged to present their research at scientific meetings (e.g., Sigma Xi, American Society of Plant Biologists, American Society for Virology). The principal investigator's group maintains collaborations with the University of Chicago and with an Institute of Bioorganic Chemistry in Poznan, Poland, and keeps active contacts with several international virology groups. These interactions will enhance the exposure of the students to the international environment. The principal investigator is making efforts to disseminate science by translation of English works (has translated into Polish language a book by Matt Ridley "Red Queen, sex and evolution of human nature"). In general, social benefits of this project rely upon improvement of our understanding of RNA recombination in RNA viruses which will provide new potential means for control of these important infectious agents. It will also raise the scientific literacy of the public via outreach activities. By learning how to predict recombination hot-spots scientists will be able to design more stable viral vectors as important biotechnology tools.