Host Factors That Restrict HIV mRNA With Retained Introns

摘要 在初步实验中，我们已经进行了全基因组CRISPR/Cas9筛查来识别宿主 在没有Rev.这张屏幕的情况下，参与未剪接HIV RNA核保留的细胞蛋白 确定19个复合体(NTC)和Musclebled3(MBNL3)中的几个蛋白质为主要候选蛋白质 有关留存的因素。NTC是剪接体的组成部分，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 蛋白质可能通过不同的机制来调节滞留，因为MBNL3不是NTC的一部分。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 这些蛋白质处于滞留状态。在两个具体的目标上，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 通过这些因素阐述留存的相关机制。 目的1：分析比较NTC和MBNL3基因扰动对基因表达的影响 以及艾滋病毒和其他逆转录病毒的复制。在这一目标中，我们将确定“击倒”(KO)或 不同类别HIV RNA上特定保留因子的“击倒”(Kd)和HIV的复制 无REV活动或低REV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 在SupT1细胞中输出未剪接的MPMV RNA和内源性HERV-K RNA。 目的#2.进一步分析和比较MBNL3和NTC蛋白在RNA中的功能 留存。在这个目标中，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和 分析HIV的表达是否改变了这种定位。

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.