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.