Mechanisms of HIV-1 RNA Methylation in Regulating Viral Replication

项目摘要/摘要 我们的目标是研究N6-甲基腺苷(M6A)修饰血管内皮细胞的功能和机制。 病毒感染过程中的HIV-1RNA。细胞RNA内部m6A修饰是一种新的机制 转录后对基因表达的控制，这是由三组 宿主蛋白，包括甲基转移酶(编写者)、去甲基酶(擦除器)和m6A选择性- 结合蛋白(阅读器)。阅读器与m6A修饰的细胞RNA的结合显著影响 翻译过程中RNA功能的各个方面。最近的三份出版物，其中一份来自我们的 他强调了HIV-1RNA的m6A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。为了澄清 这种差异，至关重要的是定义m6A修饰HIV-1RNA的机制 调节原代CD4T细胞中的病毒复制。 我们在HIV-1RNA中发现了由HIV-1阅读器结合的m6A修饰的多个区域。 被感染的原代CD4T细胞。我们发现，读者的表达水平显著 调节靶细胞中的HIV-1感染。病毒产生细胞中写入器或擦除器的击倒 显著影响HIV-1 Gag蛋白的合成，提示M6A修饰对HIV-1Gag蛋白合成的重要性 HIV-1RNA在调节病毒基因表达中的作用有趣的是，我们观察到艾滋病毒-1感染 上调原代CD4T细胞内源性阅读器的表达。我们的中央 假设HIV-1RNA的可逆m6A修饰调控病毒在CD4T细胞中的复制。 通过影响HIV-1RNA的结构、稳定性、剪接和/或运输而影响细胞。我们将对此进行测试 使用跨学科方法在三个具体目标上进行假设。目的1.鉴定M6A 目的：研究m6A基因修饰对HIV-1RNA结构和功能的影响。 与阅读器蛋白的相互作用；目的2.研究m6A修饰对细胞周期的影响 HIV-1RNA在受感染的原代CD4T细胞中的稳定性、剪接和运输；以及目标3 探讨HIV-1感染和促炎细胞因子对HIV1基因表达的影响 对读取器、写入器和擦除器进行本地化，并定义底层机制。 总体影响：完成我们提议的研究将确定HIV-1RNA M6A修饰调控CD4T细胞中的病毒感染。HIV-1核糖核酸M6A的研究 修饰和与宿主蛋白的相互作用代表了HIV-1RNA生物学的一个新领域，即 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.