Functional role of RNA structure and m6A modification in viral genomes

项目摘要/摘要 RNA病毒编码篡改细胞新陈代谢和基因调控所需的信息，并使 它们自身以两种方式复制：在它们的RNA基因组的线性序列中和在复杂的更高顺序中 结构。尽管结构RNA元件普遍存在于病毒基因组中，并具有复杂的 对于病毒生命周期的所有阶段的调控作用，人们对RNA结构的程度知之甚少 在病毒基因组中发生，或者关键结构如何在机械上发挥作用。转录后RNA基因组 化学修饰，如N6-甲基腺苷(M6A)，是多种病毒感染的调节器，可以 对RNA结构有深远的影响，或受到其影响。然而，RNA结构-和 M6A介导的病毒感染性调节仍然没有特征，因为我们缺乏全面的结构 对RNA基因组的理解和对m6A修饰的研究一直不精确。在.期间 在这项提议的指导阶段，我将获得病毒学、核糖核酸表观遗传学和核糖核酸化学生物学方面的新培训 以确定RNA元件和m6A修饰如何调节病毒复制和感染。在《目标1》中，我将 通过以下方式表征RNA结构与m6A转录后RNA化学修饰的关系 创建登革病毒(DENV)的高分辨率RNA结构模型和m6A修饰位点图 呼吸道肠道病毒EV-D68RNA基因组。目前还没有广泛有效的疫苗或疗法可用 预防或治疗这两种单链、阳性RNA病毒引起的严重感染 不同的病毒家族。通过目标2，我将确定m6A相关RNA基因组的功能重要性 用基于细胞培养的病毒功能分析研究DENV和EV-D68病毒生命周期阶段的结构。最后， 在目标3中，我将定义DENV和EV-D68中与m6A相关的RNA结构中的结构-功能关系 通过评估RNA结构、m6A修饰和蛋白质结合的动态变化来研究基因组。一起， 这些研究将揭示RNA结构和m6A修饰之间的相互关系所扮演的复杂角色 在调节各种RNA病毒的生命周期中发挥作用，并将识别可能 可用于设计抗DENV和抗EV-D68疗法和疫苗接种战略。我有过 组建了一个由导师和合作者组成的专家团队，并计划参加研讨会、研讨会和 会议将导致必要的培训，以实现这里提出的研究目标，并 作为一名独立研究科学家过渡到成功的职业生涯。中国优秀的培训环境 Week和Horner实验室，以及我的合作者卡梅伦博士的实践培训，将提供 我有坚实的基础来建立一个成功的独立研究项目。

PROJECT SUMMARY/ ABSTRACT RNA viruses encode the information required to usurp cellular metabolism and gene regulation and to enable their own replication in two ways: in the linear sequence of their RNA genomes and in complex higher order structures. Although structured RNA elements are pervasive throughout viral genomes and have complex regulatory effects on all stages of the virus life cycle, little is known about the extent to which RNA structures occur across viral genomes or how critical structures function mechanistically. Post-transcriptional RNA genome chemical modifications such as N6-methyladenosine (m6A) are regulators of infection in diverse viruses and can have profound impacts on, or be impacted by, RNA structure. However, the link between RNA structure- and m6A-mediated regulation of viral infectivity remains uncharacterized because we lack a comprehensive structural understanding of RNA genomes and studies mapping m6A modifications have been imprecise. During the mentored phase of this proposal, I will gain new training in virology, RNA epigenetics, and RNA chemical biology to define how RNA elements and m6A modifications regulate viral replication and infection. In Aim 1, I will characterize RNA structure interrelationships with the m6A post-transcriptional RNA chemical modification by creating high-resolution RNA structure models and m6A modification site maps for the dengue virus (DENV) and the respiratory enterovirus EV-D68 RNA genomes. No broadly effective vaccines or therapeutics are available to prevent or treat the serious infections caused by these two single-stranded, positive-sense RNA viruses from distinct virus families. Through Aim 2, I will establish the functional importance of m6A-related RNA genome structures in DENV and EV-D68 viral life cycle stages using cell culture-based virus functional assays. Finally, in Aim 3, I will define structure-function relationships in m6A-related RNA structures in DENV and EV-D68 genomes by evaluating dynamic changes in RNA structure, m6A modification, and protein binding. Together, these studies will reveal the complex roles that interrelationships between RNA structure and m6A modification play in regulating the life cycles of diverse RNA viruses and will identify novel RNA regulatory motifs that might be exploited in the design of anti-DENV and anti-EV-D68 therapeutics and vaccination strategies. I have assembled an expert team of mentors and collaborators and plan to attend workshops, seminars, and conferences that will result in the training necessary to achieve the research goals proposed here and to transition into a successful career as an independent research scientist. The excellent training environments in the Weeks and Horner laboratories, along with hands-on training from my collaborator Dr. Cameron, will provide me with a solid foundation on which to build a successful independent research program.