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

决定RNA病毒感染结果的RNA调控控制尚不完全清楚。特别是，转录后RNA修饰n6 -甲基腺苷（m6A）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知之甚少，也不知道它如何调节扰乱细胞稳态的系统，如RNA病毒感染。该项目旨在确定m6A如何调节丙型肝炎病毒（HCV）的复制，HCV是黄病毒科的一种正链RNA病毒。我们的中心假设是m6A直接作用于HCV RNA基因组，在病毒生命周期的不同阶段调节RNA的命运。这项研究的基本原理是，通过了解m6A如何调节RNA病毒复制，我们可以操纵和靶向m6A靶向过程，设计新的和创新的方法来预防和治疗RNA病毒感染。在我们的初步数据的指导下，我们的假设将通过以下三个具体目标来验证：1)在单核苷酸分辨率下确定m6A在HCV RNA基因组中的位置，2)确定m6A调节HCV RNA复制的机制，3)确定m6A结合蛋白调节HCV组装的机制。在第一个目标中，我们将通过m6A单核苷酸分辨率交联、免疫沉淀和直接RNA测序，在单核苷酸分辨率下确定HCV RNA基因组上腺苷甲基化的动力学和位点。对于第二个目标，我们将使用m6A位点失活和m6A甲基转移酶和m6A去甲基化酶基因缺失的病毒突变体来确定m6A影响HCV RNA复制的机制。在第三个目标中，我们将通过使用光激活核糖核苷增强交联和免疫沉淀来确定m6a结合蛋白与HCV RNA基因组结合的位置，并通过鉴定这些蛋白的功能域来确定m6a结合蛋白差异调节HCV组装的机制。综上所述，该方法将确定m6A对HCV RNA基因组的直接修饰如何调节HCV的生命周期，从而鉴定和表征一种新的RNA调控病毒感染。最终，对m6A如何影响HCV感染的详细了解将揭示开发抗病毒治疗的新策略，以靶向RNA病毒利用其复制的RNA调节控制。

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.