Mechanisms of HIV-1 RNA Methylation in Regulating Viral Replication

项目总结/摘要 这项新的R 01提案的目标是研究N6- 甲基腺苷（m6 A）修饰的HIV-1 RNA在病毒感染。内部M6 A 细胞RNA的修饰是一种新出现的转录后调控机制， 基因表达，其由三组宿主蛋白协同调节，包括 腺苷甲基转移酶（writers）、m6 A脱甲基酶（erasers）和m6 A选择性- 结合蛋白（阅读器）。m6 A修饰的细胞RNA与读取器YTH结构域的结合 家族蛋白（YTHDF 1、2和3）可显著影响RNA功能的各个方面 在翻译过程中。然而，尚不清楚HIV-1 RNA是否含有m6 A修饰， 该修饰是否调节CD 4 + T细胞中的病毒复制。 我们最近在HIV-1基因组RNA（gRNA）中发现了多个m6 A修饰区域。 在HIV-1感染的细胞中被阅读器（YTHDF 1 -3蛋白）结合。我们的数据显示， 阅读者的表达水平可显著调节HIV-1感染后靶细胞的表达水平， 细胞此外，在病毒生产细胞中敲除m6 A写入器或擦除器显著地降低了病毒的产量。 影响HIV-1 Gag蛋白合成和病毒释放，表明m6 A的重要性 HIV-1 RNA在病毒生产中的修饰。因此，我们假设，m6 A修饰 HIV-1 RNA通过m6 A阅读器、写入器和橡皮擦来调节细胞中的病毒复制， 这可能对HIV-1生命周期的不同阶段产生消极或积极的影响。我们将 主要使用原代CD 4 + T细胞在三个特定目标中检验总体假设。目标1.到 研究HIV-1 RNA m6 A修饰在病毒感染中的作用以及m6 A的机制 阅读器介导的病毒抑制;目的2.研究m6 A的作用和机制 调节HIV-1感染的作家;目标3。探讨了在体外培养条件下， m6 A擦除器调节HIV-1感染。 总体影响：完成这些拟议的研究将揭示新的作用， HIV-1 RNA的m6 A修饰在调节细胞中病毒感染中的机制。研究 HIV-1 RNA的m6 A修饰及其与宿主蛋白的相互作用代表了一个新的研究领域。 HIV-1 RNA生物学，可以促进针对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.