Mechanisms of HIV-1 RNA Methylation in Regulating Viral Replication

项目摘要/摘要 这个新的R01提案的目标是研究N6- 病毒感染中HIV-1RNA的甲基腺苷(M6A)修饰。内置M6A 细胞RNA修饰是一种新的转录后调控机制 基因表达受三组宿主蛋白的协调调节，包括 腺苷甲基转移酶(编写者)、m6A去甲基酶(擦除器)和m6A选择性- 结合蛋白(阅读器)。Yth结构域阅读器与m6A修饰细胞RNA的结合 家族蛋白(YTHDF1、2和3)可以显著影响RNA功能的各个方面 在翻译过程中。然而，目前尚不清楚HIV-1RNA是否含有m6A修饰和 该修饰是否调节了CD4+T细胞中的病毒复制。 我们最近在HIV-1基因组RNA(GRNA)中发现了m6A修饰的多个区域 由HIV-1感染细胞中的读取器(YTHDF1-3蛋白)结合。我们的数据显示， 阅读器的表达水平可以显著调节HIV-1进入靶区后的感染 细胞。此外，病毒产生细胞中m6A编写器或擦除器的敲除显著 影响HIV-1 Gag蛋白合成和病毒释放，提示M6A的重要性 HIV-1RNA在病毒生产中的修饰。因此，我们假设M6A的修改 HIV-1RNA通过激活m6A读取器、写入器和擦除器来调节细胞中的病毒复制， 这可能会对HIV-1生命周期的不同阶段产生负面或积极的影响。我们会 主要使用原代CD4+T细胞在三个具体目标上测试整个假设。目标1.目标 检测HIV-1RNA m6A修饰在病毒感染中的作用及其机制 读者介导的病毒抑制；目的2.探讨m6A的作用和机制 作者在调控HIV-1感染中的作用；以及目标3。调查 M6A在调节HIV-1感染中的作用。 总体影响：完成这些拟议的研究将揭示新的角色和 HIV-1RNA m6A修饰调控细胞内病毒感染的机制正在研究的 HIV-1RNA的M6A修饰及其与宿主蛋白的相互作用代表着一个新的领域 HIV-1 RNA生物学，可促进针对HIV-1感染的治疗开发。

Project Summary/Abstract The goal of this new R01 proposal is to investigate the novel role and mechanisms of N6- methyladenosine (m6A) modification of HIV-1 RNA in viral infection. The internal m6A modification of cellular RNA is a newly emerging mechanism of post-transcriptional control of gene expression, which is coordinately regulated by three groups of host proteins, including adenosine methyltransferases (writers), m6A demethylases (erasers), and m6A-selective- binding proteins (readers). Binding of m6A-modified cellular RNA by the readers, YTH domain family proteins (YTHDF1, 2, and 3), can significantly affect various aspects of RNA functions during translation. However, it is unknown whether HIV-1 RNA contains m6A modification and whether the modification regulates viral replication in CD4+ T-cells. We recently identified multiple regions of m6A modification in HIV-1 genomic RNA (gRNA) bound by the readers (YTHDF1-3 proteins) in HIV-1-infected cells. Our data showed that the expression levels of the readers can significantly modulated HIV-1 postentry infection in target cells. Moreover, knockdown of the m6A writers or an eraser in virus producer cells significantly affected HIV-1 Gag protein synthesis and viral release, suggesting the importance of m6A modification of HIV-1 RNA in viral production. Thus, we hypothesize that m6A modification of HIV-1 RNA regulates viral replication in cells by engaging the m6A readers, writers, and erasers, which can have negative or positive impacts on different stages of the HIV-1 lifecycle. We will test the overall hypothesis in three specific aims mainly using primary CD4+ T-cells. Aim 1. To examine the role of HIV-1 RNA m6A modification in viral infection and the mechanisms of m6A reader-mediated viral inhibition; Aim 2. To investigate the role and mechanisms of the m6A writers in regulating HIV-1 infection; and Aim 3. To investigate the role and mechanisms of the m6A erasers in regulating HIV-1 infection. Overall impact: Accomplishing these proposed studies will reveal the novel role and mechanisms of m6A modification of HIV-1 RNA in regulating viral infection in cells. Studying the m6A modification of HIV-1 RNA and its interactions with host proteins represents a new area of HIV-1 RNA biology, which can facilitate therapeutic development against HIV-1 infection.