Multiplexed approaches to investigate cellular mechanisms underlying HIV-1 transcriptional competence and viral replication 公开
Shah, Raven (Fall 2021)
Published
Abstract
Human immunodeficiency virus type 1 (HIV-1) remains a global public health burden with approximately 38 million individuals living with the disease in 2019 and 1.7 million new annual infections. There is currently no effective HIV-1 cure or vaccine. HIV is a retrovirus that inserts its genome into the target host chromatin, thus exploiting the cellular transcriptional machinery for replication. Investigating features of chromatin architecture at HIV sites of integration can provide insight into the molecular mechanisms governing HIV gene expression. Previous studies have demonstrated that HIV preferentially integrates into the introns of transcriptionally active genes, and productive HIV proviruses are associated with active epigenetic markers. The work presented in this thesis expands upon preliminary studies, undertaking an integrative approach to profile the relationship between HIV-1 transcription and chromatin state, map the local cellular and viral transcriptome(s) at HIV-host gene boundaries, and monitor viral transcriptional dynamics using multiplexed fluorescence imaging.
In Chapter II, we present an innovative and highly sensitive methodology that we “coin” Multiplexed immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), to simultaneously label viral nucleic acid(s) and protein(s) to visualize viral replication kinetics at single-cell resolution across a broad spectrum of viruses. Chapters III-V further demonstrate the power of our imaging modality, detailing how this technology can be used to study virus-host factor interactions (Chapter III), visualize how small molecules can impact viral transcription (Chapter IV), and measure virus replication kinetics with high spatiotemporal resolution (Chapter V). In Chapter VI, we apply a multiomics sequencing approach to profile chromatin and gene expression at proviral sites of integration using HIV-inducible cellular models. We seek to understand how lentiviral integration and activation of HIV-1 transcription can alter cellular chromatin structure and the local transcriptional environment. Our study provides the first in-depth integrative investigation of HIV-1 chromatin ultrastructure and viral transcription at provirus-host gene boundaries using high-resolution chromatin mapping techniques (ATAC-seq and Hi-C) and long-read Nanopore RNA-sequencing. Our presented findings may have translational implications providing insight into mechanisms influencing HIV-1 transcriptional competency, as well as providing a platform for applying cutting-edge sequencing and in-situ imaging technologies to study viral replication.
Table of Contents
ABSTRACT
PUBLICATIONS
CONFERENCE PRESENTATIONS
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
LIST OF ABBREVIATIONS
CHAPTER I: INTRODUCTION TO HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1), HIV-1 TRANSCRIPTIONAL REGULATION, & NUCLEAR ORGANIZATION
1-A: HIV-1 Disease Pathogenesis & Viral Replication
1-B: HIV-1 Genome, Virion Organization, & Viral Proteins
1-C: Current Antiviral Therapies
1-D: HIV-1 Proviral Integration
1-D-a: Cellular & Viral Factors that Compose the Pre-Integration Complex
1-D-b: Determinants of Integration Site Selection
1-E: HIV-1 Latency & Proviral Gene Expression
1-E-a: Establishment of the Latent Reservoir
1-E-b: HIV-1 Transcriptional Regulation
1-E-c: Therapeutic Strategies to Target the Latent Reservoir
1-E-d: Molecular & Cellular Toolbox for Mechanistic Latency Studies
1-F: Nuclear Chromatin Organization & Relevance to Viral Transcription
1-F-a: Current “Dogma” of Nuclear Organization
1-F-b: Molecular Toolbox to Investigate Chromatin Organization & Dynamics
1-F-c: Association between Chromatin Organization & Virus Replication
1-G: Dissertation Direction
CHAPTER II: SINGLE-CELL MULTIPLEXED FLUORESCENCE IMAGING TO VISUALIZE VIRAL NUCLEIC ACID & PROTEINS TO MONITOR VIRAL REPLICATION
SUMMARY
ABSTRACT
INTRODUCTION
STEP-BY-STEP PROTOCOL FOR MULTIPLEXED IMAGING
REPRESENTATIVE RESULTS
DISCUSSION
ACKNOWLEDGMENTS:
CHAPTER III: APPLICATIONS OF SINGLE-MOLECULE FISH TO STUDY VIRUS-HOST INTERACTIONS INFLUENCING TRANSCRIPTION FROM UNINTEGRATED HIV-1 DNA
INTRODUCTION
MATERIALS/METHODS
RESULTS
DISCUSSION
CHAPTER IV: RALTEGRAVIR TREATMENT LEADS TO AN INCREASE IN HIV-1 ANTISENSE (-) RNA EXPRESSION
INTRODUCTION
MATERIALS/METHODS
RESULTS
DISUCSSION
CHAPTER V: MULTIPLEXED FLUORESCENCE-BASED SINGLE-CELL IMAGING TO CAPTURE EARLY REPLICATION EVENTS OF SARS-CoV-2 INFECTION.
INTRODUCTION
MATERIALS/METHODS
RESULTS
DISUCSSION
CHAPTER VI: MULTIOMICS PROFILING REVEALS A UNIQUE CHROMATIN SIGNATURE ASSOCIATED WITH ACTIVE HIV-1 PROVIRUSES
ABSTRACT
SIGNIFICANCE STATEMENT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS/METHODS
ACKNOWLEDGEMENTS
SUPPLEMENTAL DATA
CHAPTER VII: DISCUSSION & FUTURE DIRECTIONS
VII-A: Probing Long-Range Chromatin Interactions between HIV-1 & Cellular DNA
VII-B: Multiomics Single-Cell Profiling of Primary HIV-Infected Cells
VII-C: Motif Analysis at HIV-1 Integration Sites Derived from ART-Suppressed Clinical Isolates
VII-D: Putative Cellular Pathways Involved in HIV-1 Transcriptional Activation & Chromatin Reorganization
VII-E: Concluding remarks
REFERENCES
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