Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Ho, Ya-Chi

Abstract

Curing HIV-1 is challenging due to the presence and persistence of CD4+ T cells harboring HIV-1 proviruses. Understanding the epigenetic and transcriptional regulation that enables HIV-1 to persist despite antiretroviral therapy (ART) and immune pressure is critical to counteracting this latent reservoir. A first chapter consists of an edited published review and preview covering a relevant subset of known features of HIV-1 persistence and HIV-1-T cell interaction biology. The remaining three chapters are parallel projects to understand the multifaceted nature of HIV-1: the interrogation and longitudinal tracking of HIV-1 RNA+ cells from infected individuals, the identification HIV-1 silencing host factors using a genome-wide CRISPRi screen, and the investigation of changes in host accessibility, transcription, and chromatin architecture at the site of HIV-1 integration. Counteracting HIV-1 persistence in vivo requires an understanding of HIV-1-infected cells in vivo. This is challenging due to the rarity of HIV-1 infected cells, 1 to 100 per million in vivo, and the heterogeneity of CD4+ T cells, each encoding different memory, polarization, activation, exhaustion, and proliferation phenotypes. In chapter 2, to understand HIV-1 persistence despite these challenges, we leveraged Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by Sequencing (ECCITE-seq) to capture the transcriptome, T cell receptor (TCR) sequence, surface antigen expression, and HIV-1 RNA+ status from the same single cell. Using longitudinal samples from viremia through long term suppressive ART, we identified a previously unreported subset of GZMB+ effector memory T cells which preferentially harbor HIV-1 proviruses capable of producing p24. Additionally, we were able to track single T cell clones and identify specific characteristics of these clones that correlate with harboring HIV-1 using machine learning. we noted that many of these cells have disparate phenotypes and limited differences from non-HIV-1-RNA+ host cells, preventing specific targeting of HIV-1-harboring cells. These findings highlight novel subsets for therapeutic targeting. Immune clearance of HIV-1 requires HIV-1 protein expression. Understanding HIV-1 transcriptional and translational blocks is a major goal for an HIV-1 cure. We wanted to identify integration site independent HIV-1-transcriptional inhibitors; in chapter 3 we employed a CRISPR inhibition-based genome wide screen across four Jurkat cell lines with independent integration sites. Across these lines we found that the SAFB-family proteins, including SLTM, SAFB, and SAFB2 which modulate chromatin and regulate host transcription, inhibited HIV-1 transcriptional activity. We confirmed that knockdown of SLTM increased the HIV-1-GFP expression, the killing of HIV-1 infected cells ex vivo, and HIV-1-driven chromatin accessibility at the site of integration. This factor was not known to impact HIV-1 transcription, and its identification enables a novel latency reversal strategy. Importantly, inhibition of this factor did not alter global chromatin accessibility, suggesting it is specific to HIV-1. HIV-1 persists and clonally expands at specific genomic loci over time in different participants and distinct integration sites. While some of these specific integrations may be due to cancer-related gene expression, such as BACH2, other integrations have less apparent proliferation advantages, such as MKL2. In chapter 4, we hypothesized that it was not only the linear distance but also the 3D distal chromatin interactions that impacted HIV-1 persistence. Another human retrovirus, HTLV-1, uses a host protein to form proviral-host chromatin loops and regulate its own expression and that of host genes. We employed a bevy of genomic techniques to comprehensively identify the impact of HIV-1 on chromatin structure including chromatin accessibility, chromatin looping, enhancer-promoter connections, and ectopic transcription. We identified a local disruption of chromatin accessibility which is dependent on HIV-1 transcription using a combination of CRISPR activation, CRISPR inhibition, and ATAC-seq. We also identified HIV-1-host loops that occur in cis up to 500kb away from the site of integration using a novel HIV-4C-seq approach. Finally, we used H3K27ac HiChIP, which specifically captures enhancer-promoter interactions in 3D space based on the H3K27Ac histone modification, to examine the region around HIV-1 integration. We found that HIV-1 can disrupt local enhancer-promoter connections which may alter the regulation of genes in the same chromatin region HIV-1 is integrated in. Since enhancer-promoter connections are specifically modulated without impacting global chromatin structure, HIV-1 may have evolved a tailored mechanism to impact host gene expression.

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