Host Factors That Restrict HIV mRNA With Retained Introns

限制 HIV mRNA 保留内含子的宿主因素

• 批准号: 10480987
• 负责人: MARIE-LOUISE HAMMARSKJOLD
• 金额: $ 28.26万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-13 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480987
• 关键词: Address; Affect; Alternative Splicing; Binding; Binding Proteins; CRISPR screen; CRISPR/Cas technology; CUG repeat; Cell Nucleus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Confocal Microscopy; Cytoplasm; Disease; Elements; Endogenous Retroviruses; Equine Infectious Anemia Virus; Future; Genes; HIV; Human T-lymphotropic virus 1; Immunofluorescence Immunologic; Integration Host Factors; Introns; Knock-out; Knowledge; Lead; Mammalian Cell; Mason-Pfizer monkey virus; Mediating; Messenger RNA; Monitor; Mouse Mammary Tumor Virus; Nuclear; Ovine pulmonary adenocarcinoma virus; Play; Post-Transcriptional Regulation; Proteins; RNA; RNA Splicing; RNA replication; Retroviridae; Role; Spliceosomes; Translating; Translations; Virus; cellular targeting; experimental study; genetic regulatory protein; genome-wide; knock-down; mRNA Expression; member; prevent; protein complex; protein function; rev Protein; small hairpin RNA; targeted treatment

Abstract In preliminary experiments, we have performed a genome wide CRISPR/Cas9 screen to identify host cell proteins involved in the nuclear retention of unspliced HIV RNA in the absence of Rev. This screen identified several proteins in the “Nineteen” Complex (NTC) and Muscleblind 3 (MBNL3) as prime candidates for factors involved in retention. The NTC is an integral component of the spliceosome, whereas MBNL3 has been shown to function in alternative splicing and retention of CUG-repeat RNAs. The current collective knowledge about NTC proteins and MBNL3 suggest that these two classes of proteins are likely to mediate retention by distinct mechanisms, since MBNL3 is not part of the NTC. Here, we propose to further study the role of MBNL3, as well as components of the NTC to further validate the roles of these proteins in retention. In two specific aims, we propose several different experiments to further analyze how MBNL3 and NTC proteins affect expression and replication of HIV and other retroviruses and to start to address the mechanisms involved in retention by these factors. Aim #1: To analyze and compare the effects of perturbation of NTC and MBNL3 on expression and replication of HIV and other retroviruses. In this aim, we will determine the effects of "knockout" (KO) or "knockdown" (KD) of specific retention factors on the different classes of HIV RNA and replication of HIV with no or low Rev activity. We will also determine if KO or KD of these proteins overcomes the block that exists to export of unspliced MPMV RNA and endogenous HERV-K RNA in SupT1 cells. Aim #2. To further analyze and compare how MBNL3 and NTC proteins function in RNA retention. In this aim, we will determine if NTC proteins and MBNL3 bind directly to HIV RNA and whether these proteins interact with each other. We will also determine the localization of these proteins in cells and analyze whether expression of HIV alters this localization.

抽象的 在初步实验中，我们进行了全基因组 CRISPR/Cas9 筛选来识别宿主 在没有 Rev 的情况下，细胞蛋白参与未剪接的 HIV RNA 的核保留。此筛选 确定了“十九”复合体 (NTC) 和 Muscleblind 3 (MBNL3) 中的几种蛋白质作为主要候选蛋白 涉及保留的因素。 NTC 是剪接体的一个组成部分，而 MBNL3 具有 已被证明在 CUG 重复 RNA 的选择性剪接和保留中发挥作用。 目前关于 NTC 蛋白和 MBNL3 的集体知识表明，这两类蛋白 由于 MBNL3 不是 NTC 的一部分，因此蛋白质可能通过不同的机制介导保留。在这里，我们 建议进一步研究MBNL3的作用以及NTC的组成部分，以进一步验证 这些蛋白质被保留。为了两个具体目标，我们提出了几个不同的实验来进一步分析 MBNL3 和 NTC 蛋白如何影响 HIV 和其他逆转录病毒的表达和复制，并开始 解决这些因素影响保留的机制。 目标#1：分析和比较 NTC 和 MBNL3 的扰动对表达的影响 以及艾滋病毒和其他逆转录病毒的复制。为了这个目标，我们将确定“淘汰”（KO）或 不同类别的 HIV RNA 上的特定保留因子的“敲低”(KD) 和 HIV 的复制 无或低转速活动。我们还将确定这些蛋白质的 KO 或 KD 是否克服了存在的障碍 SupT1 细胞中未剪接的 MPMV RNA 和内源 HERV-K RNA 的输出。 目标#2。进一步分析和比较 MBNL3 和 NTC 蛋白在 RNA 中的功能 保留。为此，我们将确定 NTC 蛋白和 MBNL3 是否直接与 HIV RNA 结合，以及是否 这些蛋白质相互作用。我们还将确定这些蛋白质在细胞中的定位 分析 HIV 的表达是否会改变这种定位。