Role of Subgenomic RNAs in Genetic Recombination of Brome Mosaic Bromovirus

智力上的功绩。RNA病毒是最具挑战性的感染源之一。一些RNA病毒会给人类造成严重的疾病，而另一些RNA病毒会对牲畜或农作物造成严重的疾病。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，导致不断出现新的病毒株。RNA重组涉及病毒RNA片段的交换，而RNA重组策略是RNA病毒确保其快速适应和进化所需的关键工具。与包括DNA病毒在内的基于DNA的生物的遗传重组不同，RNA病毒中的RNA重组还没有得到很好的研究。雀麦花叶病毒(BMV)系统是理解RNA重组基本机制的一个很好的精心设计的模型。在本项目中，研究重点将集中在有效同源BMV RNA-RNA交换的分子因素的表征上。该项目利用BMV RNA分子上先前定位的支持异常高频率重组的区域(定义为RNA3顺反子间重组热点或“SGP”)以及最近发现的新的亚基因组(Sg)BMV RNA(称为“sgRNA3a”)，以阐明SGP的已知多项功能中的哪一项负责重组。该项目的实验设计依赖于在各种体外和体内测试中对修改后的BMV RNA3结构的测试。该项目的学术价值在于它研究了sgRNA分子在一个模型RNA病毒系统中的作用。这些研究将为科学家提供有关RNA病毒用来条件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重组机制，这将适用于其他RNA病毒。这个项目将对我们关于病毒分子适应的知识做出重大贡献，并将通过使用BMV模型系统，帮助在DNA和RNA遗传学之间进行类比。BMV是一个较大的RNA病毒超家族的成员，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培训。首席研究员教授一门分子病毒学课程，这项工作将培养年轻的病毒学家。此外，首席研究员还讲授一门名为“RNA世界”的高级课程。首席调查员有很长的本科培训记录，包括那些来自代表性不足的群体的培训。所有学生将被鼓励在科学会议(例如，美国植物生物学家学会、美国病毒学学会)上展示他们的研究成果。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组织保持积极联系。这些互动将增加学生对国际环境的接触。首席研究人员正在努力通过翻译英文作品来传播科学(已将马特·里德利的一本书翻译成波兰语：红皇后，性与人性的进化)。总的来说，这个项目的社会效益依赖于我们对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性病原体提供新的潜在手段。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.