Multiplexed approaches to investigate cellular mechanisms underlying HIV-1 transcriptional competence and viral replication Public

Shah, Raven (Fall 2021)

Permanent URL: https://etd.library.emory.edu/concern/etds/tt44pp087?locale=fr
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

 

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research Field
Mot-clé
Committee Chair / Thesis Advisor
Committee Members
Dernière modification

Primary PDF

Supplemental Files