Ivan Sadowski

Despite decades of research, HIV-1 remains an ever-prevalent burden on global human health. For many, antiretroviral therapy (ART) is capable of controlling viral replication. However, ART is not a cure and must be administered life-long with discontinuation resulting in viral rebound. The persistence of HIV-1 is the result of a fraction of provirus establishing latency early upon infection of T cells. This population of transcriptionally silent provirus are undetectable to the immune system, unaffected by ART, and are capable of spurious reactivation and clonal proliferation. It has been hypothesized that latency modulating compounds may produce a sterilizing cure for HIV-1. In this thesis, I identify and characterize transcriptional regulators of HIV-1 and examine the ability of small molecules that target these factors to affect viral latency. RBE3 and RBE1 are two of the most highly conserved cis-elements in the HIV-1 LTR. Binding of these elements by the RBF-2 complex, composed of TFII-I and a USF1/USF2 heterodimer, is essential for reactivation of latent provirus. In my work, TFII-I recruits TRIM24 to the LTR resulting in stimulated transcriptional elongation. Furthermore, inhibition of the TRIM24 bromodomain by IACS-9571 reversed proviral latency, an effect that was associated with increased TRIM24 LTR occupancy and facilitation of RNAPII elongation. Thus, TRIM24 bromodomain inhibitors such as IACS-9571 represent a novel class of HIV-1 LRAs that are of interest to the “shock and kill” strategy of viral elimination.Several LTR bound sequence-specific transcriptional activators are regulated by the Mediator complex kinase, CDK8/19. Here, I show that chemical inhibition of CDK8/19 kinase activity enforces the establishment of HIV-1 latency and hinders viral reactivation in response to latency reversal agents. Similar to kinase inhibition, genetic ablation of CDK8 promoted latency while also suppressing reactivation to several agonists. As such, CDK8/19 kinase activity is an attractive latency promoting target that may contribute to the development of a “block and lock” functional cure. Collectively, in this thesis I have identified novel regulators of proviral expression for which existing drugs can target to either promote or antagonize HIV-1 latency thus exhibiting therapeutic potential.

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Although antiretroviral therapies have improved the outlook of the HIV epidemic, they do not provide a cure. The major barrier to development of a cure lies in the virus’s ability to become transcriptionally silent as chromosomally integrated provirus. The presence of latently infected cells that harbor transcriptionally repressed viral genomes, gives rise to cellular reservoirs that are impenetrable by current therapies. Therefore, devising ways to selectively target these latent reservoirs is imperative for the long-term management of the disease. This thesis focuses on the shock phase of a proposed cure strategy known as “shock and kill,” which aims to induce latent HIV-1 reservoirs that could then be purged via a boosted immune response, specific targeting of infected cells, or by viral-induced apoptosis. Accordingly, research over the past decade has resulted in identification of small molecules capable of inducing HIV-1 latent reservoirs, by reactivation of viral transcription. Molecules with this capability, known as latency-reversing agents (LRAs). Thus far, none of the LRAs examined in clinical trials have reduced the size of persistent HIV-1 infection. Therefore, new classes of LRAs must be identified. To this end, I identified five novel LRAs, that are capable of reversing HIV latency without affecting the general T cell activation state. These compounds exhibit synergy for reactivation of latent provirus with other LRAs, in particular, ingenol-3-angelate. One compound, designated PH02, was efficient at reactivating viral transcription in several in vitro cell lines bearing HIV-1 reporters at different integration sites. Furthermore, this compound was capable of reversing latency in resting CD4+ T lymphocytes from patients on antiretroviral therapy. The combination of PH02 and ingenol-3-angelate produces a strong synergistic effect of reactivation, as demonstrated by a quantitative viral outgrowth assay on CD4+ T lymphocytes from HIV-1-infected individuals. A comparison of similar efforts from other groups is provided, with the goal of illustrating the diversity of molecular scaffolds that can produce HIV-1 latency reversing activity. I expect these results will contribute to a deeper understanding of mechanisms regulating HIV-1 latency but also will provide insight towards design of optimized structures for development of highly effective LRAs capable of forcing a purge of the persistent HIV-1 infection. 

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Current therapies to treat patients infected with HIV-1 do not represent a cure. The virus persists as a latent chromosomally integrated provirus in unstimulated helper T-cells that is unaffected by current drugs and can become reactivated upon stimulation of the T-cell receptor. Much research is currently focused on understanding mechanisms that control HIV-1 latency and devising ways to eliminate latent viral populations. In this thesis, I characterize a protein, YY1 that is involved in establishing HIV-1 latency, examine the role of a histone methyltransferase inhibitor on activation of the HIV-1 LTR, and identify lariat peptides that recognize TFII-I to examine the role of an interaction between USF1 and TFII-I on HIV-1 transcription.The transcription factor YY1 has been shown to promote repressive chromatin modifications by the recruitment of histone deacetylases (HDACs). In this thesis, I identify a novel binding site for YY1 on the HIV-1 LTR, near the highly conserved RBEIII element immediately upstream of the enhancer, and show that YY1 dissociates from the LTR in vivo upon T-cell activation. Overexpression of YY1 causes an increase in HIV-1 expression, which illustrates the importance of this factor for establishment of latency. I also show that an inhibitor of the histone methlytransferase SUV39H1, chaetocin, causes induction of latent HIV-1 expression, with minimal cell toxicity and without T-cell activation. The effect of chaetocin is amplified synergistically in combination with histone deacetylase (HDAC) inhibitors. These results indicate that drugs with properties similar to chaetocin may provide a therapy to purge cells of latent HIV-1, possibly in combination with other chromatin remodeling drugs. In a parallel objective, I sought to identify cyclic lariat peptide inhibitors of TFII-I, an additional factor shown to be involved in expression of HIV-1 transcription. In these studies, I identified a lariat peptide that binds the R4 domain of TFII-I using a yeast two-hybrid assay. Expression of this peptide in cells was found to activate the HIV-1 LTR 2-fold. This result demonstrates that lariat peptide inhibitors might offer an alternative to small molecule compounds as potential therapies targeting the latent reservoir.

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HIV/AIDS is undoubtedly one of mankind’s most pressing health concerns.Currently, there are ~34 million people infected worldwide, with ~3 million newinfections and ~2 million deaths every year. Despite 30 years of research andthe development of potent antiretroviral drugs, a cure for HIV-1 remains elusive.This is largely due to viral latency, a phenomenon that makes lifelong HAARTtherapy essential.While proviral DNA is usually transcribed, the integrated HIV-1 LTR promotermay also exist in a transcriptionally inactive latent state. Current models indicatethat latency results primarily from the progressive epigenetic silencing ofotherwise active infections. However, the majority of latency models utilizesingle reporters and selection/culturing to establish latency; therefore, theycannot differentiate between direct and progressive silencing. We hypothesizethat direct LTR-silent infections are underappreciated because current modelsmay poorly represent the entire spectrum of HIV-1 latency.In this thesis we aim to characterize a novel double-labeled Red-Green-HIV-1vector (RGH) to comprehensively study latency. Our results show that, contraryto current dogma, the majority of RGH infections in Jurkat T cells are directlysilenced. Moreover, direct silent infections are observed in several cell types andare transcriptionally competent, as known HIV-1 agonists can efficiently reactivatethem. We observe that direct silencing occurs at all sites of viralintegration and that cellular NFκB levels at the time of infection mediate directLTR-silencing. Additionally, we aim to characterize the cellular transcriptionfactor RBF-2 with respect to RGH latency and basal transcription. Our resultsshow that RBF-2 binds two conserved sites on the HIV-1 LTR, and that thisorganization is necessary for mediating proper transcriptional activation.Consequently, this interaction also modulates RGH latency, as RBF-2 mutantsdisplayed higher levels of latency relative to wild type.Collectively, our results shed new light on the previously underappreciated andimmeasurable contribution of direct silent infections to HIV-1 latency.Considering these infections make up the majority of total infections in vitro,direct silencing is likely a major component of the latent reservoir in vivo. Fullyunderstanding the entire spectrum of latency, including both direct andprogressive mechanisms, will undoubtedly aid HIV-1 eradication strategies.

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No abstract available.

Clinical diagnostic genotyping has the potential to predict an individual’s response to a prescribed drug, and could thus dramatically improve drug efficacy and reduce adverse drug interactions. However, widespread implementation of clinical diagnostic genotyping is currently prevented by a lack of fast, simple clinical genotyping platforms. This thesis describes the development of a new genotyping technique based on nanopore force spectroscopy (NFS) which may fulfill this need, and serves as a feasibility study for further development towards a commercial instrument. The thesis begins by describing NFS, which is a novel, general technique used to detect bio-molecules and characterize their physical interactions with one another. NFS is applied to base-calling by forming a duplex between an engineered single-stranded DNA probe and a DNA sample, and then measuring the dissociation rate under an applied force. The dissociation rate is shown to be extremely sensitive to duplex sequence homology: tests using purified synthetic DNA fourteen bases long demonstrate that even a single base mismatch can increase the dissociation rate over 100-fold. This high specificity, combined with the sensitivity of nanopore detection, allows a base-call to be made from as few as 100 single molecule dissociation events involving the target. Based on these results, it is estimated that with further development, NFS genotyping could be possible from purified, unlabeled genomic DNA in less than 1 hour, without requiring PCR amplification. These characteristics would make NFS extremely attractive as a clinical diagnostic genotyping technology. Further development is still required to produce an instrument capable of testing genomic DNA. However, based on the success of tests so far, this thesis concludes that further development of such an instrument is clearly warranted, especially given its potential impact on human health.

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