Mechanisms of HIV-1 RNA Methylation in Regulating Viral Replication

项目概要/摘要 我们的目的是研究N6-甲基腺苷（m6 A）修饰的功能和机制， HIV-1 RNA在病毒感染期间。细胞RNA的内部m6 A修饰是一种新的机制 基因表达的转录后控制，这是由三组协调调节， 宿主蛋白，包括甲基转移酶（写入器）、脱甲基酶（擦除器）和m6 A选择性- 结合蛋白（阅读器）。阅读器对m6 A修饰的细胞RNA的结合显著影响 RNA在翻译过程中的各种功能。最近的三份出版物，包括我们的 强调了HIV-1 RNA的m6 A修饰在调节病毒复制中的关键作用。 然而，这些研究报告了一些不同的结果，并提出了不同的机制。澄清 为了消除这种差异，必须确定m6 A修饰HIV-1 RNA的机制， 调节原代CD 4 + T细胞中的病毒复制。 我们鉴定了HIV-1 RNA中m6 A修饰的多个区域，这些区域被HIV-1中的阅读器结合， 感染的原代CD 4 + T细胞。我们发现，阅读者的表达水平显著高于阅读者。 调节靶细胞中的HIV-1感染。在产生病毒的细胞中敲除写入器或擦除器 显着影响HIV-1 Gag蛋白的合成，表明m6 A修饰的重要性， HIV-1 RNA对病毒基因表达的调控作用有趣的是，我们观察到HIV-1感染 上调原代CD 4 + T细胞中内源性阅读者的表达。我们的中央 假设HIV-1 RNA的可逆m6 A修饰调节CD 4 + T细胞中的病毒复制， 通过影响HIV-1 RNA的结构、稳定性、剪接和/或运输来影响细胞。我们将测试这个 假设在三个具体目标，使用跨学科的方法。目标1.为了识别M6 A 研究m6 A修饰对HIV-1 RNA结构的影响， 与阅读器蛋白的相互作用;目的2.为了研究m6 A修饰对细胞增殖的影响， HIV-1 RNA在感染的原代CD 4 + T细胞中的稳定性、剪接和运输;以及目的3.到 研究HIV-1感染和促炎细胞因子对表达的影响， 本地化的阅读器，作家，和橡皮擦，并定义的基本机制。 总体影响：完成我们提出的研究将确定HIV-1 RNA m6 A修饰调节CD 4 + T细胞中的病毒感染。HIV-1 RNA m6 A的研究 修饰和与宿主蛋白的相互作用代表了HIV-1 RNA生物学的一个新领域， 可以促进针对HIV-1感染的治疗开发。

Project Summary/Abstract Our goal is to investigate the functions and mechanisms of N6-methyladenosine (m6A) modification of HIV-1 RNA during viral infection. The internal m6A modification of cellular RNAs is a novel mechanism of post-transcriptional control of gene expression, which is coordinately regulated by three groups of host proteins, including methyltransferases (writers), demethylases (erasers), and m6A-selective- binding proteins (readers). Binding of m6A-modified cellular RNA by the readers significantly affects various aspects of RNA functions during translation. Three recent publications, including one from our group, highlighted the critical role of m6A modification of HIV-1 RNA in regulating viral replication. However, these studies reported some different results and suggested distinct mechanisms. To clarify the discrepancy, it is essential to define the mechanisms by which m6A modification of HIV-1 RNA regulates viral replication in primary CD4+ T-cells. We identified multiple regions of m6A modification in HIV-1 RNA bound by the readers in HIV-1- infected primary CD4+ T-cells. We found that the expression levels of the readers significantly modulated HIV-1 infection in target cells. Knockdown of the writers or erasers in virus-producing cells significantly affected HIV-1 Gag protein synthesis, suggesting the importance of m6A modification of HIV-1 RNA in regulating viral gene expression. Interestingly, we observed that HIV-1 infection upregulated the expression of endogenous readers in primary CD4+ T-cells. Our central hypothesize is that reversible m6A modification of HIV-1 RNA regulates viral replication in CD4+ T- cells by affecting the structure, stability, splicing, and/or trafficking of HIV-1 RNA. We will test this hypothesis in three specific aims using interdisciplinary approaches. Aim 1. To identify the m6A residues in HIV-1 RNA and to investigate the effects of m6A modification on HIV-1 RNA structure and interactions with the reader proteins; Aim 2. To investigate the effects of m6A modification on the stability, splicing and trafficking of HIV-1 RNA in infected primary CD4+ T-cells; and Aim 3. To investigate the effects of HIV-1 infection and proinflammatory cytokines on the expression and localization of the readers, writers, and erasers and to define the underlying mechanisms. Overall impact: Accomplishing our proposed studies will define the mechanisms by which HIV-1 RNA m6A modification regulates viral infection in CD4+ T-cells. Investigations of the HIV-1 RNA m6A modification and interactions with host proteins represent a new area of HIV-1 RNA biology, which can facilitate therapeutic development against HIV-1 infection.