Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Genomics & Computational Biology

First Advisor

Frederic D. Bushman

Abstract

Over 35 million people are living with human immunodeficiency virus (HIV-1). The mechanisms causing integrated provirus to become latent, the diversity of spliced viral transcripts and the cellular response to infection are not fully characterized and hinder the eradication of HIV-1. We applied high-throughput sequencing to investigate the effects of host chromatin on proviral latency and variation of expression and splicing in both the host and virus during infection.

To evaluate the link between host chromatin and proviral latency, we compared genomic and epigenetic features to HIV-1 integration site data for latent and active provirus from five cell culture models. Latency was associated with chromosomal position within individual models. However, no shared mechanisms of latency were observed between cell culture models. These differences suggest that cell culture models may not completely reflect latency in patients.

We carried out two studies to explore mRNA populations during HIV infection. Single-molecule amplification and sequencing revealed that the clinical isolate HIV89.6 produces at least 109 different spliced mRNAs. Viral message populations differed between cell types, between human donors and longitudinally during infection. We then sequenced mRNA from control and HIV89.6-infected primary human T cells. Over 17 percent of cellular genes showed altered activity associated with infection. These gene expression patterns differed from HIV infection in cell lines but paralleled infections in primary cells. Infection with HIV89.6 increased intron retention in cellular genes and abundance of RNA from human endogenous retroviruses. We also quantified the frequency and location of chimeric HIV-host RNAs. These two studies together provided a detailed accounting of both HIV89.6 and host expression and alternative splicing.

A more cost-effective method of detecting viral load would aid patients with poor access to healthcare. We developed improved methods for assaying HIV-1 RNA using loop-mediated isothermal amplification based on primers targeting regions of the HIV-1 genome conserved across subtypes. Combined with lab-on-a-chip technology, these techniques allow quantitative measurements of viral load in a point-of-care device targeted to resource-limited settings.

This work disclosed novel HIV-host interactions and developed techniques and knowledge that will aid in the study and management of HIV-1 infection.

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