Date of Award
2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Microbiology and Molecular Genetics
First Advisor
Elizabeth Bonney
Abstract
The Human Immunodeficiency Virus (HIV) causes a lifelong infection that may be managed through the administration of antiretroviral drugs, both reducing viral load to undetectable levels and allowing patients to live relatively normal lives. Interruption of antiretroviral therapy (ART) leads to resurgence in viremia, resulting from reactivation of long-lived infected memory T cells making up the latent reservoir of HIV in the body. Clonal expansion of T cells, along with an estimated half-life of about 44 months for the latent reservoir, make simply “waiting it out” an unviable curative method for HIV infection. Latency is widely recognized as the largest barrier to the development of a cure for HIV, and methods of probing the dynamics of latent infection are becoming increasingly important to understanding how latency is established and maintained. Latency is a key part of the long-lived nature of HIV. Models designed for probing latency have been proposed and developed for decades, with a variety of single cell “omics” studies being employed to generate transcriptional profiles for latently infected cells to differentiate them from their uninfected neighbors. At the same time, dual-fluorescent and muti-fluorescent reporter systems have proven invaluable in producing real time reports on the state of latency in infected cells. Developed by the Benjamin Chen lab at Mount Sinai, Enhanced HIV-Induced Lineage Tracing (EHILT) is a novel multi-fluorescent system that utilizes a Cre-lox genetic switch that reports on both the history of infection as well as the state of virus production and has been used both in vitro and in vivo to study the kinetics of latency establishment. The primary aim of this study is to develop an improvement upon EHILT called EHILTv2, addressing several concerns within the original system. Using fluorescent microscopy and a newly-developed data processing pipeline, we demonstrate that most of the changes proposed in EHILTv2 increase its usefulness in tracking and studying HIV latency, including the use of a modified degradFP protein to clarify the results of EHILTv2. We also developed two cyan fluorescent protein (CFP) variants of EHILTv2 that increase the flexibility of EHILT to be used with other reporter systems. We did find, however, that one of the original concerns, spectral bleedthrough of dsRed into the EGFP channel (which prompted replacement of dsRed with mCherry in EHILTv2), may have resulted from user error or technical issues during the testing of the original EHILT system. We further found that the newly introduced mCherry exhibits bleedthrough into a different channel used for reporting viral gene expression. Despite these shortcomings, EHILTv2 on its own successfully allows for confident differentiation between uninfected, actively infected, and latently infected cells in culture, and these results together mark a large improvement over EHILTv1, while posing a few final but necessary updates for a further updated EHILTv3.
Language
en
Number of Pages
76 p.
Recommended Citation
Smith, Keegan, "Development of an improved fluorescent reporter system for HIV latency" (2025). Graduate College Dissertations and Theses. 2077.
https://scholarworks.uvm.edu/graddis/2077