DESCRIPTION (provided by applicant): Quantification of HIV-1 Splicing Phenotypes and the Role of RNA Structure and Splice Regulator Elements I am interested in the role of RNA secondary structure and splicing regulators as determinants of alternative splicing patterns in the expression of HIV-1. I have adapted the Primer ID method to create a tool that quantifies HIV RNA splicing patterns using deep sequencing. This is a straightforward method to compare the relative abundance of different spliced transcript within each size class of HIV-1 transcripts (i.e. the relative amounts of the different transcripts within the 4 kb and 1.8 kb groups of transcripts). I will use a more traditional method (S1 nuclease mapping) to compare total spliced versus unspliced and 4 kb to 1.8 kb groups of transcripts. I am interested in the effects of determinants likely to affect splicing patterns, particularly splice regulatory elements and RNA secondary structure. I plan to look at deletions across the genome that non-specifically disrupt secondary structure and potentially affect splicing patterns, asking what are the global constraints on viral RNA structure for controlling splicing patterns. I will use this assay in collaboration with other investigators, particularly Dr. Blanton Tolbert and Dr. Alice Telesnitsky, to define the splicing phenotype of regulatory site mutations in the context of the entire viral genome. I have also developed a tool to identify cryptic splice sites that may arise when structure or regulatory elements are perturbed. I believe the ability to rapidly quantify the complex splicing patterns seen in HIV-1 gene expression will add to the current understanding of RNA structure and function.

PUBLIC HEALTH RELEVANCE: HIV-1 RNA splicing is a complex and incompletely understood process. Previous attempts to quantify splicing patterns have used artificially contrived systems that are complicated and labor intensive. This application presents a new tool that combines deep sequencing with Primer ID to give a straightforward, inexpensive, and relatively rapid quantification of HIV-1 splice patterns in the context of viral infection.