Host Factors That Restrict HIV mRNA With Retained Introns

限制 HIV mRNA 保留内含子的宿主因素

基本信息

批准号：
10553285
负责人：
MARIE-LOUISE HAMMARSKJOLD
金额：
$ 16.15万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-13 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10553285
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 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 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

项目摘要

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。这个屏幕确定了“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 NTC 是剪接体的一个组成部分，而 MBNL3 则具有。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，这两类蛋白蛋白质可能通过不同的机制介导保留，因为 MBNL3 不是 NTC 的一部分。建议进一步研究MBNL3的作用以及NTC的组成部分，以进一步验证为了进一步分析这些蛋白质的两个具体目标，我们提出了几个不同的实验。 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 HIV 和其他逆转录病毒的复制为此目的，我们将确定“敲除”（KO）或的效果。不同类别的 HIV RNA 上的特定保留因子的“敲低”(KD) 和 HIV 的复制我们还将确定这些蛋白质的 KO 或 KD 是否克服了存在的阻碍。在SupT1细胞中输出未剪接的MPMV RNA和内源性HERV-K RNA。目标#2：进一步分析和比较 MBNL3 和 NTC 蛋白在 RNA 中的功能。为此，我们将确定 NTC 蛋白和 MBNL3 是否直接与 HIV RNA 结合，以及是否与 HIV RNA 结合。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。