Next-Generation Sequencing Techniques to Study HIV-1 Transcription and RNA Structure

Abstract

HIV-1 remains a global health challenge. New sequencing techniques and bioinformatic approaches are being developed to study key aspects of HIV-1 biology. I leveraged and developed innovative deep-sequencing approaches to advance our understanding of HIV-1 RNA structure and latency. In Chapter 1, I will review the HIV-1 replication cycle. Special focus will be devoted to the roles that HIV-1 RNA structures play during replication. I will also review HIV-1 splicing and the current understating of its regulation. Understanding the splice pattern of HIV-1 is crucial to both projects, as it affects the interpretation of RNAseq data. I will then review the latest research on HIV-1 latency, which has been the focus of intense research. Measuring RNA structure by chemical probing and deep sequencing is a complex technique that must be developed and adapted for each experimental system. In Chapter 2, the technical development of Dimethyl Sulfate (DMS)- Mutational Profiling and Sequencing (MaPseq) for HIV-1 infected cells and HIV-1 virions will be described. Few studies analyze intracellular RNA structure and no previous technique can measure alternate RNA structure in cells. To address these knowledge gaps, I utilized a novel alternate RNA structure detecting algorithm in conjunction with DMS-MaPseq. In Chapter 3, the use of this system to study the Rev response element (RRE) within cells, to identify novel RNA structures that regulate splicing, and to measure overall HIV-1 RNA structure heterogeneity will be presented and discussed. The mechanisms that regulate HIV-1 transcriptional latency and reactivation remain incompletely understood. In Chapter 4, I will discuss our study that developed an enrichment-based RNAseq technique to study HIV-1 reversal of latency in response to a number of drug candidates. We were able to detect, measure, and quantify coverage across the HIV-1 genome despite the extreme rarity of HIV-1 RNA. Finally, in Chapter 5, I will discuss how these techniques can be further used to advance the study of basic HIV-1 biology and how the techniques can be used to help develop better latency reversing agents. I will also speculate on the role that RNA structure may contribute to HIV-1 latency.