Defining the role of the RNA modification N6-methyladenosine in the hepatitis C virus lifecycle

定义 RNA 修饰 N6-甲基腺苷在丙型肝炎病毒生命周期中的作用

• 批准号: 9157887
• 负责人: Stacy Michelle Horner
• 金额: $ 51.78万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9157887
• 关键词: Address; Adenosine; Affect; Antiviral Agents; Antiviral Therapy; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological; Biology; Data; Development; Disease; Family; Family member; Flaviviridae; Gene Expression; Genetic; Genome; Goals; Hepatitis C; Hepatitis C virus; Hepatitis C-Like Viruses; Homeostasis; Human; Immune; Immunoprecipitation; Individual; Infection; Infectious hepatitides; Investigation; Kinetics; Lead; Life Cycle Stages; Lipids; Mediating; Messenger RNA; Methylation; Methyltransferase; Modeling; Modification; Molecular; Morbidity - disease rate; Nucleotides; Outcome; Pathway interactions; Physiology; Post-Transcriptional RNA Processing; Prevention approach; Process; Production; Proteins; Public Health; RNA; RNA Virus Infections; RNA Viruses; RNA methylation; RNA replication; RNA-Binding Proteins; Research; Resolution; Ribonucleosides; Role; Site; Staging; Structure; System; Tertiary Protein Structure; Testing; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; base; crosslink; design; genetic approach; hepatoma cell; improved; innovation; insight; methylation pattern; mortality; mutant; novel; novel strategies; transcriptome sequencing; viral RNA

The RNA regulatory controls that dictate the outcome of RNA virus infection are not fully understood. In particular, the post-transcriptional RNA modification N6-methyladenosine (m6A) is a potent and dynamic modulator of RNA function. However, little is known about the biological significance of this modification or how it regulates systems that perturb cellular homeostasis, such as RNA virus infection. This project aims to determine how m6A regulates the replication of hepatitis C virus (HCV), a positive strand RNA virus of the Flaviviridae family. Our central hypothesis is that m6A acts directly on the HCV RNA genome to regulate the fate of the RNA for the different stages of the viral life cycle. The rationale for the proposed research is that by understanding how m6A regulates RNA virus replication, we can manipulate and target m6A-targeted processes to design new and innovative approaches for the prevention and treatment of RNA virus infection. Guided by our preliminary data, our hypothesis will be tested by pursuing these three specific aims: 1) Identify the sites of m6A within the HCV RNA genome at single nucleotide resolution, 2) define the mechanism by which m6A regulates HCV RNA replication, 3) determine the mechanism by which m6A-binding proteins regulate HCV assembly. In the first aim, we will define the kinetics and sites of adenosine methylation on the HCV RNA genome at single nucleotide resolution by using m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and direct RNA sequencing. For the second aim, we will use viral mutants with inactivated m6A sites and genetic depletion of the m6A methyltransferases and m6A-demethylase to identify the mechanism by which m6A affects HCV RNA replication. In the third aim, we will determine the mechanism by which the m6A-binding proteins differentially regulate HCV assembly by defining where they bind to the HCV RNA genome using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and by identifying the functional domains of these proteins. Taken together, this approach will define how direct modification of the HCV RNA genome by m6A regulates the life cycle of HCV, thereby identifying and characterizing a novel RNA regulatory control to viral infection. Ultimately, a detailed understanding of how m6A affects HCV infection will uncover novel strategies to develop antiviral therapies to target this RNA regulatory control that is exploited by RNA viruses for their replication.

决定RNA病毒感染结果的RNA调节控制尚未完全了解。特别地，转录后RNA修饰N6-甲基腺苷（m6 A）是RNA功能的有效和动态调节剂。然而，人们对这种修饰的生物学意义以及它如何调节扰乱细胞稳态的系统（例如RNA病毒感染）知之甚少。该项目旨在确定m6 A如何调节丙型肝炎病毒（HCV）的复制，HCV是黄病毒科的正链RNA病毒。我们的中心假设是m6 A直接作用于HCV RNA基因组，以调节RNA在病毒生命周期不同阶段的命运。这项研究的基本原理是，通过了解m6 A如何调节RNA病毒复制，我们可以操纵和靶向m6 A靶向过程，以设计新的创新方法来预防和治疗RNA病毒感染。在我们的初步数据的指导下，我们的假设将通过追求以下三个具体目标进行验证：1）确定单核苷酸分辨率的HCV RNA基因组中的m6 A位点，2）定义m6 A调节HCV RNA复制的机制，3）确定m6 A结合蛋白调节HCV组装的机制。在第一个目标中，我们将通过使用m6 A单个核苷酸分辨率交联和免疫沉淀以及直接RNA测序来定义HCV RNA基因组上单核苷酸分辨率的腺苷甲基化的动力学和位点。对于第二个目标，我们将使用失活的m6 A位点和m6 A甲基转移酶和m6 A-脱甲基酶的基因缺失的病毒突变体，以确定m6 A影响HCV RNA复制的机制。在第三个目标中，我们将确定的机制，其中的m6 A结合蛋白差异调节HCV装配定义在哪里他们结合到HCV RNA基因组使用光活化核糖核苷增强交联和免疫沉淀，并通过确定这些蛋白质的功能域。总之，这种方法将定义如何通过m6 A直接修饰HCV RNA基因组调节HCV的生命周期，从而确定和表征一种新的RNA调节控制病毒感染。最终，对m6 A如何影响HCV感染的详细了解将揭示开发抗病毒疗法的新策略，以靶向RNA病毒复制所利用的RNA调控。