HIV-1 Antisense RNA Production and Latency Increases when the LEDGF/p75 and Integrase Interaction is Inhibited Open Access

Wahoski, Claudia (Spring 2022)

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Human Immunodeficiency Virus type 1 (HIV-1) is a parenterally transmitted retrovirus that infects CD4+ T cells and can lead to acquired immunodeficiency syndrome. While treatment exists to manage actively replicating virus, a major barrier to curing HIV-1 is latent virus harbored in long-lasting CD4+ T cells that are transcriptionally quiescent, leading to immune evasion. However, the mechanisms of latency are incompletely understood. The HIV-1 RNA genome is reverse transcribed into DNA that is integrated into the host genome by integrase, acting in concert with host factors, including lens epithelium-derived growth factor (LEDGF)/p75. LEDGF/p75 is important for viral DNA integration into active, gene-dense regions of the host genome. Development and testing in clinical trials of new HIV-1 therapies include allosteric integrase inhibitors (ALLINIs) that target LEDGF/p75’s interaction with integrase. ALLINI treatment has been shown to increase latency in vitro. HIV-1 antisense RNA (asRNA), an RNA transcript originating from transcription in the 3’ long terminal repeat of HIV-1, has been shown to be involved in inducing and maintaining HIV-1 latency. Unpublished data from the Sarafianos lab shows that asRNA production increases in ALLINI-treated cells, specifically using the ALLINI known as BI-D. This research aims to further explore the relationship between asRNA production in ALLINI-treated cells, and the involvement in latency. We used a dual fluorescent HIV-1 reporter (Hi-Fate Tomato) to identify three different subpopulations of cells, latent, active, and uninfected cells differentiated by two fluorescent proteins using flow cytometry. We assessed the asRNA production in each subpopulation when left untreated, or treated with BI-D. We found that the Hi-Fate Tomato reporter produces detectable asRNA that can be used in subsequent asRNA research. After cell sorting, we found that treatment with BI-D does not lead to differences in asRNA in the three subpopulations of infected cells, but definitive conclusions cannot be made with the current data due to a single replicate being performed. This research provides the foundation for further work that can investigate asRNA production in ALLINI-treated cells. The implications of the results can contribute to further understanding the mechanisms of latency that can lead to curing HIV-1.

Table of Contents

Introduction       1

Human Immunodeficiency Virus Type 1                                                    1

HIV-1 Latency                             2 

Lens Epithelium-Derived Growth Factor p/75 and Allosteric Integrase Inhibitors      4

HIV-1 antisense RNA       5

Dual Fluorescent HIV-1 Reporters       7

Rationale       8

Materials and Methods      10

Cell Lines, HIV-1 Reporters, and Virus Like Particles      10 

Allosteric Integrase Inhibitor – BI-D      11

Nucleic Acid Probes      11

HIV-1 RNA Staining             11

Spectral Flow Cytometry      13

Fluorescence Activated Cell Sorting      14

Imaging, Image Analysis, and Statistics      15

Results      17

Hi-Fate Tomato Dual Fluorescent HIV-1 Reporter Produces asRNA      17

Spectral Flow Cytometry Analysis of Hi-Fate Tomato Populations      21

asRNA Production in Subpopulations of HIV-1 Transduced Cells      23

Discussion      26

Acknowledgements      33

References      34

Appendix      42

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