Host Factors That Restrict HIV mRNA With Retained Introns

摘要 在初步实验中，我们进行了全基因组CRISPR/Cas9筛选，以鉴定宿主 在没有Rev的情况下，参与未剪接的HIV RNA的核保留的细胞蛋白。 在“Nineteen”Complex（NTC）和Muscleblind 3（MBNL 3）中鉴定出几种蛋白质作为主要候选物 保留的因素。NTC是剪接体的组成部分，而MBNL 3具有 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MBNL 3的集体知识表明，这两类 由于MBNL 3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。这里我们 建议进一步研究MBNL 3的作用，以及NTC的组成部分，以进一步验证 这些蛋白质被保留下来。在两个具体的目标，我们提出了几个不同的实验，以进一步分析 MBNL 3和NTC蛋白如何影响HIV和其他逆转录病毒的表达和复制， 通过这些因素解决保留所涉及的机制。 目的#1：分析和比较NTC和MBNL 3的扰动对表达的影响 以及HIV和其他逆转录病毒的复制。在这个目标中，我们将确定“敲除”（KO）或 不同类型HIV RNA上特异性保留因子的“敲低”（KD）和HIV复制， 无或低Rev活动。我们还将确定这些蛋白质的KO或KD是否克服了存在的阻断， SupT 1细胞中未剪接的MPMV RNA和内源性HERV-K RNA的输出。 目标2。为了进一步分析和比较MBNL 3和NTC蛋白在RNA中的功能， 潴留在这个目标中，我们将确定NTC蛋白和MBNL 3是否直接与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.