HIV is a worldwide epidemic, remaining the ever-present specter among all matters regarding blood or other bodily fluids, such as sexual intercourse, healthcare, blood transfusions, or even surgery, particularly in resource-poor areas. While antiretroviral drug therapy is highly successful in suppressing the virus and preventing transmission, it cannot cure the infection due to latency – the ability of HIV to go into a silent state within cells; once drug therapy is stopped, reactivating latent virus will cause a surge of the viral load within days or weeks. Current drug therapies do not act on this latent reservoir, necessitating lifelong adherence to medication, which is both extremely costly and comes with negative side effects. The mainstay of research into HIV latency deals with teasing apart the mechanisms involved in establishing and maintaining latency, in the hopes of finding drug compounds which can efficiently reverse latency and thus purge the body of hidden HIV. In searching for cellular pathways involved in latency, our lab has employed a genome-wide negative-selection screen, which indicated that the ubiquitin-proteasome system plays a role in maintaining latency. This led us to discover that proteasome inhibitors act as bifunctional antagonists of HIV, both reversing the latent state and reducing infectivity of virions. We went on to identify the specific ubiquitin-proteasome pathway that is involved in maintaining latency. We then investigated the activity of a small-molecule inhibitor of one component of the ubiquitin-proteasome system, showing its ability to reactivate latent HIV in both a primary cell model and in cells taken from aviremic HIV+ patients. These results are a proof-of-concept that inhibition of a specific ubiquitin-proteasome pathway can allow for specific reversal of HIV latency.

Regulation of gene expression is pivotal to cell behavior. It is achieved predominantly by transcription factor proteins binding to specific DNA sequences (sites) in gene promoters. Identification of these short, degenerate sites is therefore an important problem in biology. The major drawbacks of the probabilistic machine learning methods in vogue are the use of arbitrary thresholds and the lack of biophysical interpretations of statistical quantities. We have developed two machine learning methods and linked them to the biophysics of transcription factor binding by incorporating simple physical interactions. These methods estimate site binding energy, recognizing that it determines a site's function and evolutionary fitness. They use the occupancy probability of a transcription factor on a DNA sequence as the discriminant function because it has a straightforward physical interpretation, forms a bridge between binding energy and evolutionary fitness, and has a natural threshold for classifying sequences into sites that allows establishing the threshold in a principled manner. Our methods incorporate additional characteristics of sites to enhance their identification. The first method, based on a hidden Markov model (HMM), identifies self-overlapping sites by combining the effects of their alternative binding modes. It learns the threshold by training emission probabilities using unaligned sequences containing known sites and estimating transition probabilities to reflect site density in all promoters in a genome. While identifying sites, it adjusts parameters to model site density changing with the distance from the transcription start site. Moreover, it provides guidance for designing padding sequences in experiments involving self-overlapping sites. Our second method, the Phylogeny-based Quadratic Programming Method of Energy Matrix Estimation (PhyloQPMEME), integrates evolutionary conservation to reduce false positives while identifying sites. It learns the threshold by solving an iterative quadratic programming problem to optimize the distribution of correlated binding energies of neutrally evolving orthologous sequences while restricting the values of binding energies of known sites and their orthologs. We have used the NF-κB transcription factor family as a case study for both methods and gained new insights into its biology.

The main parts of my thesis are studies aimed at investigating circadian regulation of innate immunity using Drosophila as a model system. In unrelated work, I also participated in a collaborative study showing circadian regulation of microRNAs (miRNAs) in Drosophila. We sought to determine if the innate immune response is under circadian regulation and whether this impacts overall health status. To this end, Drosophila was infected with the human opportunistic pathogenic bacteria Pseudomonas aeruginosa as a model system. The results show that the survival rates of wild-type flies vary as a function of when during the day they are infected, peaking in the middle of the night. Also the kinetics of bacterial growth and the expression of a limited number of innate immunity genes correlate with time-of-day effects on survival. Our findings suggest that medical intervention strategies incorporating chronobiological considerations could enhance the innate immune response, boosting the efficacy of combating pathogenic infections. This study also led us to a second study where we characterized the innate immune response in the Drosophila head. We showed that the innate immunity pathway in the head is similar to the well described pathway in the body. Furthermore, the pericerebral fat body in the head or neurons are sufficient to combat bacterial infections, independent of the abdominal fat body. Our findings suggest that the pericerebral fat body may provide a fast and local immune response in the head, improving the survival outcome of Drosophila. A minor aspect of my thesis work was unrelated to host defense. In this study, we used Drosophila to investigate the possibility that circadian clocks regulate the expression of miRNAs. From the analysis of microarray data, we found two miRNAs (dme-miR-263a and -263b) that exhibit robust daily changes in abundance in adult heads of wild-type flies that are abolished in the cyc01 mutant. Our results suggest that cycling miRNAs contribute to daily changes in mRNA and/or protein levels in Drosophila.

The analysis of lymphocytes from immunodeficient patients has provided valuable insights into understanding the development and function of the immune system. In particular, studies of patients with Hyper IgM syndrome, a rare immunodeficiency resulting from defects in both CD40L in activated CD4+ T cells and components of the CD40 signaling pathway in B cells have been crucial for the elucidation of molecular pathways involved in the activation, proliferation and differentiation of B lymphocytes. Previously our lab generated EBV transformed lymphoblastic cells with inducible LMP1 from a female patient diagnosed with non-X-linked HIGM (Pt1-LCLtet cells, Pt1). Analysis of these cells revealed a CD40-specific activation defect associated with low c-Rel levels. To broaden our understanding of the Pt1 phenotype we analyzed c-Rel expression and found that low c-Rel levels were the outcome of depressed transcriptional activation of the c-rel promoter. In-vivo analysis revealed that low transcription was coincident with increased p65 activity at the c-rel promoter although this was insufficient to induce normal c-Rel expression. Further investigations of the Pt1-LCLtet cells uncovered a proliferation and survival defect characterized by reduced c-Myc transcription and increased levels of caspase-4. This defect was complemented after c-Rel overexpression, supporting a causative role for c-Rel deficiency in the observed phenotype of the Pt1-LCLtet cells. The LCLtet cells were further utilized as a model system for the independent analysis of LMP1 and CD40 signaling. These studies revealed several outcomes of LMP1 signaling in the biology and function of CD40. Specifically, LMP1 signaling induced a unique pattern of CD40 phosphorylation as well as the expression of several novel CD40 isoforms. Moreover, when CD40 and LMP1 were activated simultaneously a synergistic effect was observed towards p65 and p38 phosphorylation suggesting a functional interaction that might occur during early stages of EBV infection or malignant transformation where both LMP1 and CD40 are co-expressed in the infected cell. Collectively, our results demonstrate that a majority of the defects displayed in the Pt1-LCLtet cells are a result of reduced c-Rel expression. Importantly, these results ascribe c-Rel a novel function as a negative regulator of caspase-4 expression and a putative key player in the control of cell survival in EBV-immortalized B cells.