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.