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. We will develop sensitive and multiplexed assays (HyPR-MS) to elucidate the protein interactomes of up to 20 conserved HIV- 1 mRNA splice variants. We will develop a branched DNA fluorescence in situ hybridization technique (SV-FISH) to examine how changes in the expression levels of individual splice variant protein interactors alter their localization. Finally, we will determine which of the splice variants and protein interactors are critical to HIV replication and infectivity using siRNA and protein knock-down and overexpression studies. These powerful new tools will be used here 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.