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.