Human embryonic stem cells (hESCs) have great potential for use in regenerative medicine due to their indefinite capacity for self-renewal and their ability to differentiate into cell types derived from all three embryonic germ layers. An important step toward advancing their use in cell-based therapies is development of methods for directed differentiation into specific lineages. Understanding the signaling pathways that underlie hESC differentiation is critical if we are to achieve the full potential of these cells.

EphrinB-ephB receptor interactions are a common regulator of multiple somatic stem cells, including neural stem cells (NSCs) and hematopoietic stem cells (HSCs). EphrinB2, in particular, has been identified as a molecular stem cell marker shared in mouse embryonic stem cells (ESCs), NSCs, and HSCs. Its receptor, ephB4, has also been implicated in playing a role in ESC differentiation. Despite evidence that the ephrinB2-ephB4 axis may be involved in ESC fate, this axis has not been carefully studied in hESCs due to a lack of highly specific reagents to interrogate ephrinB2-ephB4 interactions. Here, we characterized the role of ephrinB2 in hESC fate.

First, we developed a novel method to specifically tag ephrinB2 hESCs. We showed that we could efficiently pseudotype a lentiviral vector with the Nipah virus (NiV) envelope that could efficiently target ephrinB2+ cells in vitro. These NiV pseudotyped particles specifically transduced a sub-population of hESCs. Next, we sought to further characterize these ephrinB2+ hESCs by FACS-sorting the NiV-transduced GFP+ cells and examining their properties in culture. Passaging of the sorted cells revealed that ephrinB2 does not mark for a stable population of cells, but may be an intrinsic marker of heterogeneity. The ephrinB2+ hESCs demonstrated the abilities to self-renew in vitro, to differentiate into the three germ layers in vitro, and to form teratomas in vivo.

Lastly, to further examine the role of ephrinB2 in hESC fate, we generated a hESC line stably expressing an shRNA against ephrinB2 (shEFNB2). Real-time PCR analysis and microarray analysis of embryoid bodies (EBs) derived from shEFNB2 cells demonstrated a severe deficiency in neuro-ectoderm gene expression and an up-regulation of genes involved in mesendoderm specification. Further, functional mesoderm-directed differentiation assays revealed that shEFNB2 hESCs have an increased propensity to differentiate into one specific sub-type of mesenchymal cells. In sum, the findings of this dissertation suggest that the heterogeneity of ephrinB2 expression and perturbation of ephrinB2 signaling may both be manipulated to enhance directed differentiation of hESCs in vitro.

Nipah virus is a founding member of the newly described Henipavirus genus within the Paramyxoviridae family. The henipaviruses first came to light via spillover from their fruit bat reservoir, causing high mortality and instigating great concern. The cause of this high mortality, unusual among the paramyxoviruses, is unknown. Here, we investigate the molecular mechanisms underlying the functions of the Nipah virus matrix protein (NiV-M), which has the central role in virus assembly and budding, yet remains poorly understood. First, we developed new methods to modify the virus and the host to facilitate functional investigations. For the virus, we established and improved a reverse genetics system for Nipah virus to allow efficient rescue of replication-competent viruses from cDNA, which can be modified with mutations or insertion of useful reporter genes. For the host, we used the CRISPR/Cas gene-editing technology to knock-in a destabilization domain tag on all copies of an essential gene, thus allowing inducible knockdown of the tagged protein upon removal of the stabilizing compound from cell culture. We then investigated NiV-M function with two complementary approaches: mutagenesis of conserved residues and motifs in NiV-M, some of which mediated interactions with host factors, and comprehensive identification of cellular factors and pathways interacting with NiV-M. In doing so, we identified post-translational modifications, palmitoylation and ubiquitination, that regulate NiV-M function, and specifically found that ubiquitination of a conserved lysine within a nuclear localization sequence regulated nuclear-cytoplasmic trafficking of NiV-M. The conserved interaction of NiV-M and other paramyxovirus matrix proteins with nuclear import/export factors, vesicular trafficking pathways, and factors involved in actin cytoskeletal dynamics confirmed the importance of intracellular trafficking pathways for matrix function. Finally, we extended our study of matrix function beyond matrix itself by describing the Nipah virus C protein-mediated recruitment of the cellular ESCRT complex, which was required for efficient NiV-M-driven budding of Nipah virus.

Over the past 34 years astonishing global efforts have transformed HIV/AIDS from a clinical death sentence into a manageable long-term illness. With approximately 35 million people now living with HIV, our eyes move toward managing and containing HIV infection as a chronic condition on a global scale. Achieving this goal will require the advent of new treatment strategies that target a variety of highly conserved viral features and a more complex analytical framework to assess resistance and transmission potential during treatment as well as to evaluate the potential clinical pathologies of resistant variants.

Contributing to this goal, this dissertation focuses on evaluating novel treatment strategies from two complementary and important perspectives. First, we apply analytical frameworks adapted from biochemistry to more comprehensively asses the therapeutic expectations of emerging immunotherapeutics and novel inhibitor targets. These extensible methods provide a more accurate description of antiviral activity at levels that will be necessary to suppress viral replication and prevent resistance than more traditional potency-based comparisons. Additionally, the mathematical foundation of these methods connects high clinical expectation to more detailed biochemical mechanisms that present specific criteria to aid in the rational design of more effective therapies.

This analytical framework can, alternatively, be used to understand how a virus responds to the presence of a drug, which will be necessary for assessing how HIV and its pathology might evolve, on a global scale, in the face of widespread treatment. To this end, quantifying infection efficiency and understanding how HIV virion interact with their target cells through extensible mathematical models reveal the impacts that treatment can have on the most fundamental properties of HIV infection. Understanding treatment from the perspective of the virus can aid the design of more potent therapies that pose unsurmountable barriers to resistance and might specifically target transmissibility.

Our results present a unique analysis of the activity of broadly neutralizing HIV antibodies that provides a new dimension to evaluating the clinical expectations of novel immunotherapies in the context of long-term management. This analysis is also used to further distinguish the cytotoxic and antiviral activities of a novel HIV inhibitor class: the disulfide isomerase inhibitors, where we reveal the significant potential of this class as well as specific criteria for the development of stronger and less toxic analogs to boost the diversity of available HIV treatments. We then extend this analytical method to assess the inherent infectious properties of HIV in response to treatment, to evaluate resistance in the more clinical context of target-cell tropism, receptor usage and infectivity.

Managing HIV infection as a long-term condition on a global scale will require more sophisticated efforts in developing and assessing novel treatments in terms of both inhibitor activity and direct viral responses. The methods and experimental strategies presented here are an essential first step to describing the activity of inhibitors and the activity of HIV, itself. Our results illuminate novel mechanistic features that can aid the development of novel treatments specifically suited to contain and control HIV.

HIV-1 affinity for CD4 and CCR5 is associated with differential pathogenicity. Therefore, we pioneered a dually inducible cell line based system that could quantitatively and comprehensively characterize viral entry efficiency as a co-dependent function of CD4 and CCR5 expression levels. This receptor affinity profiling system (Affinofile) has revealed biologically relevant phenotypes in Envelopes (Envs) with differential CD4/CCR5 usage efficiencies, providing a more refined understanding of receptor and coreceptor affinities, which can have an impact on the development and use of HIV-1 therapeutics.

To facilitate a more rapid and refined analysis of CD4 and CCR5 usage efficiencies with even greater sensitivity, we engineered a reporter Affinofile system containing a tat-rev dependent eGFP-Gaussia luciferase Reporter (GGR). Using this GGR Affinofile system, we (1) characterized the phenotypic and biological consequences for Envs with defined mutations that modulate CD4 or CCR5 binding, (2) determined Transmitter/Founder (T/F) Envs have differential CD4/CCR5 usage compared to Chronic Env, (3) revealed phenotypically distinct CD4/CCR5 usage patterns among the prevalent HIV-1 subtypes (A,B, C and D), and (4) uncovered that mutations that confer resistance to Broadly Neutralizing Antibodies (BNAbs) often compromised the efficiency of CD4/CCR5 usage and entry.

Analysis of mutations known to only modulate CCR5 (K421D, S142N) binding demonstrated that CD4 and CCR5 usage is an inter-related process as mutations that affect CD4 binding influenced the efficiency of CCR5 usage and vice versa. The relative entry efficiencies defined in GGR Affinofile system were also reflected in their entry efficiencies into primary CD4+ T cell subsets. In addition, we show that transmitter/founder and chronic envelopes have distinct entry efficiencies that yield characteristic vector metrics. Next, we analyzed over 28 pseudotyped HIV-1 viruses from four different subtypes, and noted that subtype C envelopes could be distinguished from the other subtypes based on their greater efficiency of CD4/CCR5 usage which was reflected in their vector metrics (increased vector angle and mean infectivity). Lastly, envelopes with engineered mutations known to confer resistance to BNAbs, VRC01 and PG6/PG19, invariably resulted in a decreased CD4/CCR5 usage efficiency.

Our results suggest that our GGR Affinofile system can quantify and reveal biologically relevant differences in CD4/CCR5 usage patterns in Envs that reflect their genetic-epidemiological differences, pathogenicity, cell tropism, and even fitness cost as a result of resistant-mutations to BNAbs.

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