Deciphering HIV-1 alternatively spliced transcript functionality with HyPR-MS

About This Grant

Project Summary/Abstract The complexity of the HIV-1 transcriptome has been progressively revealed throughout recent decades using increasingly advanced RNA sequencing technologies. However, knowledge of the RNA primary sequence alone has not been sufficient to determine the importance or function of each of the over 40 highly conserved HIV-1 splice variants, which code for nine known proteins and polyproteins. RNA-protein interactions are fundamental to RNA fate and function. From transcription to cellular localization to translation of the gene product, and many steps in between, proteins interact with RNA to regulate gene expression and, in the case of HIV-1, viral replication. Notwithstanding the high significance of these splice variants in the HIV-1 life cycle, technologies for interrogating the functions, interactions, and cellular localizations of individual splice variants are woefully lacking. We propose to develop and validate a suite of powerful new tools to interrogate the functions, interactions, and cellular localizations of individual splice variants of HIV-1. For Aim 1, we will develop sensitive and multiplexed assays (HyPR-MS) to elucidate the protein interactomes of up to 20 conserved HIV-1 mRNA splice variants and advance mechanistic studies of newly identified viral RNA regulatory co-factors. In Aim 2, we will determine which HIV-1 splice variants are most critical to HIV-1 replication using virological assays and measure single- cell viral RNA abundance and subcellular localization using a multiplexed branched DNA fluorescence in situ hybridization technique (SV-FISH). These powerful new tools and strategies will be used to elucidate previously unobtainable information about HIV- 1 replication. Once developed, these same novel technologies will comprise a powerful new toolset that can be applied to splice variant investigations in other viral and cellular systems.