Cellular mechanisms that establish HIV-1 latency in CD4+ T cells and the potential for their manipulation as a therapeutic strategy
Gagne, Matthew James
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Human Immunodeficiency Virus 1 (HIV-1) remains a significant public health concern due to the lack of a cure. In spite of anti-retroviral therapies, HIV-1 persists within infected cells as integrated transcriptionally silent proviruses. Re-activation after therapy interruption results in new HIV-1 replication. Attempts to clear this reservoir through the use of latency reversing agents by targeting cellular mechanisms that maintain HIV-1 in a latent state have been unsuccessful. In addition, subsets of latently infected cells exist within the reservoir that display differential capacities for provirus induction. In order to understand the nature of the reservoir and manipulate it therapeutically, more knowledge is needed regarding factors that bias a virus towards latency or replication at the time of infection. Because multiple mechanisms that regulate HIV-1 transcription, including chromatin remodeling, transcription factor activation and polymerase pausing, are regulated by the T cell receptor (TCR), I hypothesized that signaling at the time of infection determines proviral fate. I transduced Jurkat cell lines and primary CD4+ T cells with chimeric antigen receptors (CARs) that mimicked signaling from the TCR. These CARs spanned a 3-log range of binding affinities for their ligand, providing a tunable model. High levels of TCR stimulation during infection biased cells towards productive replication and the formation of an inducible latent reservoir. Examination of the mechanisms downstream from TCR signaling revealed that robust cellular activation led to a release of the repressor Negative Elongation Factor from the paused RNA Polymerase II, facilitating transcriptional elongation. Because signaling determined the presence of repressive factors, I sought to manipulate the balance between latency and expression through recruitment of repressors to the HIV-1 provirus using a nuclease-deficient CRISPR Associated Protein 9 fused to a Krüppel Associated Box Domain. I screened a pool of guide RNAs that mediated transcriptional repression of HIV-1. Our lab discovered that guides bound to the HIV-1 Long Terminal Repeat prevented viral re-activation in an integrated cell model of HIV-1 latency. The research presented here confirms my hypothesis that signals during infection have prolonged effects on latency reversal. I provide evidence that manipulation of these mechanisms represent therapeutic targets for cure efforts.
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