Common, complex diseases are not caused by a single gene mutation but rather have genetic and environmental components. As a subset, autoimmune diseases also possess robust gene expression signatures that have both genetic and environmental contributions. We explored gene expression signatures in type 2 diabetes, coronary artery disease, and their precursor state metabolic syndrome, and identified overlapping gene expression signatures exhibiting greater resemblance to each other than to an autoimmune disease. These signatures are consistent with activation of the innate immune response. Genetic variations contribute to familiality and we sought to determine if large-scale genomic variants, copy number variants (CNVs), are associated with common, complex diseases. To do so, we first developed methods to identify CNVs from SNP-based arrays. We analyzed genomic variation in type 1 diabetes and identified CNVs that were differentially present in patients with or at high risk for type 1 diabetes versus control. Thus, we conclude that both gene expression profiles and genomic variants are easily detected clinical markers that may be useful predictors of disease liability and serve to identify new classes of therapeutic targets.

Coronaviruses belong to a family of enveloped, positive-strand RNA viruses that cause a multitude of devastating diseases in both animals and humans. Like other positive-strand RNA viruses, coronaviruses utilize several common mechanisms to support critical steps in their life cycle. Two of these mechanisms are inducing cellular membrane rearrangements to support viral RNA synthesis and utilizing polyprotein processing to regulate protein expression and function. A well-established model used to study virus replication and the life cycle of coronaviruses is murine hepatitis virus (MHV). For MHV, gene 1 nonstructural proteins (nsps), which are proteolytically processed from the replicase polyprotein into their mature form by three virus-encoded proteases, are hypothesized to mediate many aspects of the virus life cycle, including modification of intracellular membranes, formation of replication complexes, and viral RNA synthesis. Nevertheless, much is still unknown about the requirements of protein processing and the roles individual proteins play during the coronavirus life cycle. To aid in our understanding of coronavirus replication, the research presented in this dissertation addressed the requirements of virus papain-like protease (PLP) processing on nsps 1 through 4 and the roles that these individual proteins play in the virus life cycle. The results of these studies provide new information about how coronaviruses regulate protein processing, nsps functions, viral RNA synthesis, and virus-induced membrane modifications. These results are also expected to contribute to the rational design of therapeutics aimed at preventing and inhibiting coronavirus-induced diseases.

Natural killer T (NKT) cells are innate-like lymphocytes that develop and mature in the thymus. From there, they home to the peripheral lymphoid tissues where, in some cases, they can finalise the maturation program. NKT cells function by close cell-cell interactions with antigen presenting cells and upon activation release copious amounts of proinflammatory and immunoregulatory cytokines and chemokines. NKT cells acquire their functions during development. I address two questions in this dissertation: (a) the role of negative selection in sculpting a functional NKT cell repertoire; and (b) the role of interleukin (IL)-15 in NKT cell effector differentiation and homeostasis. My studies generated mouse models for overt or impaired negative selection and revealed direct evidence for negative selection in sculpting the semi-invariant T cell receptor repertoire of NKT cells. Nonetheless, the effect of negative selection, as probed using currently known NKT cell agonists, was subtle in that it had modest effects on NKT cell function. The most significant advance I made is related to an understanding of cytokine signals that induce NKT cell effector differentiation and how these signals are integrated to affect this outcome. I discovered that IL-15 plays a pivotal role in signalling NKT cell survival through the induction of Bcl-2 family member Bcl-xL. Rescue of NKT cell development by enforced Bcl-xL expression in IL-15 deficient mouse thymocytes exposed an additional role of IL-15 in inducing terminal maturation and effector differentiation of NKT cells. Gene expression analysis indicated that IL-15 regulated Tbx21 (T-bet) expression and that of T-bet-regulated genes, including those that mediate effector functions of NKT cells. Because T-bet was previously shown to regulate terminal maturation and effector differentiation of NKT cells, I conclude that IL-15 mediates its functions within NKT cells at least in part through regulated expression of T-bet. Thus, my findings have revealed important new insights into signals that regulate the development of functional NKT cells.

Several of the most broadly neutralizing HIV antibodies (bnAbs) contain unique genetic or structural elements, including long heavy chain complementarity determining region 3 (HCDR3) loops and extensive somatic hypermutation. Two exceptionally broad, potently neutralizing HIV-specific antibodies, PG9 and PG16, encode HCDR3 loops that are among the longest of any antigen-specific antibody described to date. Passive immunization with two other bnAbs that encode long HCDR3s, 4E10 and 2F5, is able to protect against HIV infection. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV, however it is unclear at present how to induce such antibodies. There has been speculation that antibodies with long HCDR3s are generated primarily through the accumulation of somatic hypermutation-associated insertions. These short insertion events are rare, and design of an immunogen that efficiently induces many such insertions in a single antibody sequence is likely to be extremely difficult. Through the use of high-throughput antibody sequencing, I have identified genetic evidence that these long HCDR3 antibodies are typically formed at the original recombination event, not through accumulation of somatic hypermutation-induced insertions. Antibodies with long HCDR3s were found in all tested individuals, and long HCDR3 antibodies typically use a restricted subset of D and J gene segments, resulting in the incorporation of highly conserved genetic elements in the majority of such antibody sequences. This work provides an important first step toward the realization of a vaccine that efficiently induces broadly neutralizing HIV antibodies with long HCDR3s by identifying a conserved genetic target through which B cells encoding antibodies with long HCDR3s may be induced selectively through vaccination.

A common feature of nonenveloped virus replication is the requirement for structural capsid rearrangements to facilitate productive cell entry. Using mammalian orthoreovirus as a model, my work has focused on defining molecular features that determine the stability of viral outer-capsid structures and influence their interplay with host-cell mediators of infection. The initial event in reovirus disassembly is the proteolytic processing of outer-capsid protein σ3 by host endosomal cathepsin proteases. I identified a pair of residues in σ3 that act in opposition to control the rate of σ3 proteolysis, in turn influencing reovirus sensitivity to protease inhibitors. Moreover, I found that accelerated σ3 proteolysis correlates with diminished particle stability. Surprisingly, viruses with accelerated disassembly kinetics replicate more rapidly in the organs of infected mice, causing increased morbidity and mortality. Furthermore, reovirus variants with accelerated disassembly kinetics induce exaggerated myocarditis in infected animals, suggesting protease utilization may influence reovirus tissue tropism. Finally, I found that diminishing reovirus outer-capsid stability results in enhanced transmission between littermates. Together, this work furthers an understanding of the molecular mechanisms that determine reovirus protease sensitivity and establishes outer capsid stability as a determinant of nonenveloped virus pathogenesis and host-to-host spread.

My thesis project explores the NK cell response to HMPV infection, as well as the CD8+ T cell (TCD8) response to virus-like particle (VLP) vaccination. I show that while activated lung NK cell numbers increase after human metapneumovirus (HMPV) infection, and these cells degranulate and produce IFNγ, NK cell depletion does not alter weight loss, peak HMPV titer, or time required for viral clearance. In addition, NK cell depletion does not affect the adaptive T cell response to HMPV infection. Thus, despite the importance of NK cells during the immune response against infections by many other viruses, these cells are expendable during HMPV infection, at least in a mouse model. Additionally, I show that a non-replicating VLP vaccine fully protects wild-type C57BL/6 mice from HMPV viral challenge in the lungs for at least six months post-vaccination. Vaccination alone elicited TCD8 that recognized specific epitopes in each of the two HMPV proteins incorporated into the VLPs. Lung TCD8 responding to non-replicating VLPs expressed lower levels of the inhibitory receptors PD-1 and TIM-3 than those responding to replicating virus; however, inhibitory receptor expression on spleen TCD8 was much lower than that of lung TCD8 in all groups of mice. Viral challenge after vaccination abrogated the low-inhibitory receptor expression phenotype of TCD8 in vaccinated mice prior to challenge. These results have important implications for design of novel therapeutics and vaccine strategies against respiratory viruses.

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (iNKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of iNKT cells in respiratory infectious diseases remains poorly understood. Hence, their function was assessed in a murine model of pulmonary tularemiabecause tularemia is a sepsis-like proinflammatory disease and iNKT cells are known to control the cellular and humoral responses underlying sepsis. These studies show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of iNKT cells within the lung interstitium. Activated iNKT cells produced interferon-gamma upon recognition of a Francisella-derived glycosphingolipid, and promoted both local and systemic proinflammatory responses. Consistent with these results, iNKT cell-deficient mice showed a reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, iNKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, iNKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice.

Thousands of genes encoding long noncoding RNAs were identified throughout the genome; however, less than 1% of known long noncoding RNAs have been assigned a biologic function. At present, long noncoding RNAs mediate positive or negative regulation target gene transcription within nearly all developmental systems establishing an additional layer of noncoding regulation of protein-coding genes. Within the immune system we identified TMEVPG1, an enhancer lncRNA that contributes to the Th1 differentiation program of key adaptive immune cells resulting in the expression of the pleiotropic cytokine interferon gamma. As with other developmental programs, our results demonstrate that a lncRNA plays an essential role in Th1 cell biology.

Coronaviruses (CoV) are positive-strand RNA viruses that encode large replicase polyproteins that are processed by two or three viral proteases to yield intermediate and mature nonstructural proteins (nsps). Nsp5 (3CLpro, Mpro) is a cysteine protease that is essential for virus replication and mediates processing at 11 cleavage sites, yielding nsps 4 through 16. Due to its critical role in replication, nsp5 is a key target for development of antivirals against coronaviruses. However, the intramolecular and intermolecular mechanisms that govern nsp5 protease structure and function remain unclear. These issues and the limited ability to culture many human coronaviruses make testing of inhibitors against nsp5 challenging during infection in culture. This dissertation discusses the role of newly identified intramolecular residue interactions on protease function, and the conservation of these interactions across coronaviruses. Using mutagenesis, I identified temperature-sensitive and second-site suppressor mutations in murine hepatitis virus (MHV) nsp5 that are distant from known functional determinants and have a profound impact on viral replication and nsp5-mediated polyprotein processing. To evaluate the role of these mutations and their associations in other viruses, I have engineered nsp5-substitution chimeras by introducing the nsp5 proteases of closely and distantly related coronaviruses into the background of MHV. These data show that coronavirus nsp5 protease activity is governed by complex long-distance residue interactions that span the protease structure and have tightly co-evolved within the context of the greater polyprotein and viral background. Collectively, these data define a new approach for the study of human coronavirus nsp5 proteases in an efficiently replicating non-human coronavirus, provide a platform for testing antivirals against the proteases from virus that are difficult to cultivate, and provide key new insights into the regulatory mechanisms directing nsp5 structure and function.

Bloodstream spread is an essential step in the pathogenesis of many viruses. However, mechanisms that promote viremia are not well understood. Reoviruses are neurotropic viruses that disseminate systemically through the circulatory system. Junctional adhesion molecule-A (JAM-A) serves as a receptor for reovirus and is expressed in tight junctions and on hematopoietic cells. In infected mice, JAM-A is required for reovirus spread to sites of secondary replication. To examine functions of endothelial and hematopoietic JAM-A in reovirus bloodstream dissemination, I generated mice with altered JAM-A expression in these cell types and assessed systemic reovirus spread. Endothelial but not hematopoietic JAM-A facilitates reovirus bloodstream entry and egress. To determine how reovirus traverses endothelia, I examined reovirus infection of polarized endothelial cells (ECs). Reovirus infection of polarized ECs is JAM-A-dependent but does not alter tight junction integrity. Viral release occurs exclusively from the luminal surface in the absence of cell lysis. These data implicate the endothelium as an important mediator of viral pathogenesis. Understanding hematogenous viral spread may aid in development of therapeutics that inhibit this key step in viral pathogenesis.