Functional role of RNA structure and m6A modification in viral genomes

RNA结构和m6A修饰在病毒基因组中的功能作用

• 批准号: 10301540
• 负责人: Mark A Boerneke
• 金额: $ 12.1万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10301540
• 关键词: Affinity; Binding; Binding Proteins; Bioinformatics; Biological; Biological Assay; Biological Models; Biology; Cell Culture Techniques; Cells; Chemicals; Child; Complex; Consensus; Dengue; Dengue Virus; Disease; Educational workshop; Elements; Enterovirus; Enterovirus 68; Environment; Epigenetic Process; Family; Foundations; Gene Expression Regulation; Genetic Transcription; Genome; Goals; Health; Higher Order Chromatin Structure; Human; Immunologic Factors; Infection; Instruction; Laboratories; Life Cycle Stages; Link; Lung diseases; Maps; Mass Spectrum Analysis; Mediating; Mentors; Metabolism; Modification; Mutation; Nucleotides; Phase; Play; Positioning Attribute; Protein Biosynthesis; Proteins; RNA; RNA Conformation; RNA Viruses; RNA-Protein Interaction; Regulation; Research; Resolution; Role; Scientist; Site; Solid; Structural Models; Structure; Structure-Activity Relationship; Techniques; Therapeutic; Training; Vaccine Therapy; Vaccines; Viral; Viral Genome; Virion; Virus; Virus Diseases; Virus Replication; Work; acute flaccid myelitis; base; career; design; disorder control; genome-wide; improved; mortality; mosquito-borne; mutant; nervous system disorder; next generation; novel; prevent; programs; respiratory; symposium; vaccination strategy; viral RNA; virology; virus culture

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.

项目总结/摘要 RNA病毒编码所需的信息，以篡夺细胞代谢和基因调控， 它们以两种方式复制：RNA基因组的线性序列和复杂的高级序列 结构.尽管结构化RNA元件在整个病毒基因组中是普遍存在的，并且具有复杂的生物学特性。 尽管RNA对病毒生命周期的所有阶段都有调节作用，但人们对RNA结构的程度知之甚少。 发生在病毒基因组中或关键结构如何机械地发挥作用。转录后RNA基因组 化学修饰如N6-甲基腺苷（m6 A）是多种病毒感染的调节剂， 对RNA结构有着深远的影响，或者受到RNA结构的影响。然而，RNA结构和 m6 A介导的病毒感染性调节仍然没有特征，因为我们缺乏一个全面的结构， 对RNA基因组的理解和绘制m6 A修饰的研究一直不精确。期间 在这个建议的指导阶段，我将获得病毒学，RNA表观遗传学和RNA化学生物学的新培训 确定RNA元件和m6 A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m6 A转录后RNA化学修饰的相互关系： 创建登革热病毒（DENV）的高分辨率RNA结构模型和m6 A修饰位点图， 呼吸道肠道病毒EV-D 68 RNA基因组。没有广泛有效的疫苗或治疗方法 预防或治疗这两种单链正义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m6 A相关RNA基因组的功能重要性 DENV和EV-D 68病毒生命周期阶段的结构，使用基于细胞培养的病毒功能测定。最后， 在目标3中，我将定义DENV和EV-D 68中m6 A相关RNA结构的结构-功能关系 通过评估RNA结构，m6 A修饰和蛋白质结合的动态变化来研究基因组。在一起， 这些研究将揭示RNA结构和m6 A修饰之间相互关系的复杂作用， 在调节不同RNA病毒的生命周期中发挥作用，并将确定新的RNA调节基序， 在抗DENV和抗EV-D 68治疗剂和疫苗接种策略的设计中被利用。我有 组建了一个由导师和合作者组成的专家团队，并计划参加讲习班、研讨会和 这些会议将导致必要的培训，以实现这里提出的研究目标， 过渡到一个成功的职业生涯作为一个独立的研究科学家。优秀的培训环境， 威克斯和霍纳实验室，沿着我的合作者卡梅隆博士的实践培训，将提供 我有一个坚实的基础，在此基础上建立一个成功的独立研究计划。