Virus cell entry is a multi-step process that often requires independent receptors for attachment and internalization. Reovirus infection is initiated by attachment to cell-surface carbohydrate and junctional adhesion molecule-A. Findings reported in this thesis indicate that following attachment to JAM-A, Beta1 integrin facilitates reovirus internalization into target cells. Importantly, tyrosine-based signaling motifs in the Beta1 integrin cytoplasmic tail are required for transport of reovirus to the appropriate endocytic organelle for viral disassembly. Together, these data provide evidence that Beta1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface. These results give new insights into mechanisms of reovirus internalization and functions of Beta1 integrin in endocytosis.

To efficiently initiate infection in target cells, viruses must become tethered to the plasma membrane by engaging cellular receptors. As the first step in the viral replication cycle, attachment is a key determinant of viral tropism and pathogenesis outcomes. The adhesion process is often mediated by a viral attachment protein and can involve binding to multiple receptors at the cell surface. Mammalian orthoreoviruses (reoviruses) are non-enveloped, double-stranded RNA viruses that serve as tractable models for studies of virus-receptor interactions and viral pathogenesis. Reoviruses also show potential as vectors for vaccine delivery and oncolytic therapeutics. The reovirus attachment protein, ó1, is a filamentous, trimeric molecule that extends from the icosahedral vertices of reovirus virions. Reovirus ó1 of some serotypes binds á-linked sialic acid, a carbohydrate, and ó1 of all serotypes binds junctional adhesion molecule-A (JAM-A), a dimeric protein involved in cell-cell adhesion and tight junction formation. Goals of my dissertation research have been to elucidate details of the mechanism of ó1-JAM-A engagement and understand features of ó1 that confer functionality. I characterized a novel trimerization motif, the aspartic acid sandwich, consisting of an unusual cluster of six aspartic acid residues sandwiched between residues with aromatic and hydrophobic side chains at the subunit interface of the ó1. This motif is likely to be important in mediating structural rearrangements in ó1. I systematically analyzed the contributions of individual residues in the JAM-A dimer interface to reovirus binding and JAM-A homodimer stability. Residues with charged side chains play critical roles in stabilizing both interactions. In addition, I report the structure of a complex between ó1 and JAM-A and identify sequences in ó1 required for efficient JAM-A binding and reovirus infectivity. These studies enhance an understanding of the contributions of structural features of ó1 and JAM-A to reovirus pathogenesis outcomes in vivo and provide a basis for vector retargeting as we explore the potential of reovirus for vaccine delivery and oncolysis.

The á2â1 integrin is expressed on many cell types throughout the immune system. Expression of the á2â1 integrin on mast cells is required for the early innate immune response to Listeria monocytogenes. Interaction between the á2â1 integrin and Listeria occurs through C1q within a Listeria immune complex, but is not sufficient for activation suggesting an additional co-receptor is required for activation. We demonstrate that Listeria immune complex activation of mast cells occurs through crosstalk between the á2â1 integrin and c-met. The best described mechanism of mast cell activation is IgE-mediated degranulation. We examined the mechanism of mediator release by mast cells following activation by Listeria immune complex. Activation by Listeria immune complex results in á2â1 integrin-dependent release of IL-6 from a granule pool that is distinct from known mast cell granules, identifying a novel population of mast cell granules. The á2â1 integrin-dependent early innate immune response to Listeria suggested that the integrin modulate later steps in the innate immune response or adaptive immunity. We demonstrate that serum IL-6 and TNF-á, but not IFN-ã are á2â1 integrin-dependent. Additionally, there is a diminished antigen specific T cell response in mice lacking the á2â1 integrin, but clearance of a secondary Listeria infection is not affected in these animals. These studies define a role for the á2â1 integrin and c-met in a novel mechanism of mast cell activation. Our studies also identify a pool of granules that contains IL-6 and can be selectively and differentially released following specific stimulation. Several studies have suggested that the á2â1 integrin may be important in the adaptive immune response. We demonstrate that there is a role for the á2â1 integrin in pro-inflammatory cytokine production and peak levels of antigen specific T cells, however we did not observe a defect in bacterial clearance or response to secondary infection. These studies provide a foundation for studies of the biology of mast cell mediator release and its role in modulation immune response.

The pathogen-host interaction between bacteria and invariant NKT (iNKT) cells is explored in this dissertation. iNKT cells are innate-like lymphocytes that recognize glycolipid antigens in the context of the MHC class Ilike antigen-presenting molecule CD1d. The impact of bacterial infection on the phenotype and function of these cells have been carefully dissected in this dissertation, and the results indicate that mouse iNKT cells activated in vivo by multiple bacterial microorganisms, or by bacterial LPS or flagellin, become unresponsive to subsequent activation with alpha-GalCer. This hyporesponsive phenotype of iNKT cells required IL-12 expression and was associated with changes in the surface phenotype of these cells, reduced severity of concanavalin Ainduced hepatitis, and alterations in the therapeutic activities of alpha-GalCer. These findings may have important implications for the development of iNKT cellbased therapies.

Mammalian reoviruses serve as important models for studies of viral replication and pathogenesis. These viruses have been isolated from many mammalian species, including humans, and cause disease primarily in the very young. Reoviruses induce apoptosis by engagement of cell-surface receptors, intracellular signal transduction, and activation of nuclear factor-kB (NF-kB). However, receptor binding alone is not sufficient to evoke these events. Internalization of reovirus by antibody-mediated uptake into Fc-receptor expressing cells indicates a critical function for the membrane penetration protein, mu1, in NF-kB activation and apoptosis. Reovirus activates NF-kB by stimulation of the multisubunit IkB kinase complex (IKK) resulting in phosphorylation and degradation of IkB alpha. Experiments using genetically deficient cell lines indicate a key role for the alpha and gamma IKK subunits in the mechanism used by reovirus to activate NF-kB. Mice lacking the NF-kB p50 subunit display diminished apoptosis in the brain following reovirus infection but enhanced apoptosis in the heart. IFN-alpha is upregulated following reovirus-induced NF-kB activation and protects cardiac myocytes against viral infection in cell culture and in vivo. Collectively, these findings identify both viral and cellular determinants of reovirus-induced apoptosis and define a novel mechanism employed by reovirus to cause disease in the infected host.

Diverse families of viruses bind immunoglobulin superfamily (IgSF) proteins located in tight junctions and adherens junctions of epithelium and endothelium. However, little is known about the roles of these receptors in the pathogenesis of viral disease. Junctional adhesion molecule-A (JAM-A) is an IgSF member that localizes to tight junctions and serves as a receptor for mammalian reovirus. Peroral inoculation of wild-type and isogenic JAM-A-/- mice demonstrated that JAM-A is dispensable for viral replication in the intestine but required for high-titer viremia and virus-induced encephalitis. Reovirus replication in the brain and tropism for discrete regions in neural tissues are equivalent in wild-type and JAM-A-/- mice following intracranial inoculation, suggesting a function for JAM-A in reovirus dissemination from the intestine. Hematogenous but not neural routes of reovirus dissemination are blunted in JAM-A-/- mice, and infection of neurons is JAM-A-independent, suggesting that JAM-A promotes dissemination of reovirus through the blood. JAM-A promotes reovirus infection of endothelial cells, providing a conduit for the virus into the bloodstream. These findings suggest a role for junction-associated viral receptors in hematogenous dissemination.

CD8+ T cells are a critical component of an HIV-specific immune response. Our studies aimed to define the T cell receptor (TCR) structural determinants as well as effector functions associated with CD8+ T cell suppression of HIV replication. We demonstrate biased gene usage in dominant HIV epitope-specific TCR repertoires during chronic HIV infection. Despite this evidence for convergent evolution to a highly conserved viral epitope, our results indicate TCR diversity can still provide the structural ability to recognize viral variants. To better understand the role of proliferative capacity in CD8+ T cell-mediated suppression of HIV replication, we directly assessed proliferation and suppression simultaneously in vitro. We found that low proliferating CD8+ T cells suppressed HIV replication in vitro similar to levels of suppression of high-proliferating CD8+ T cells. Our data also revealed low-proliferating cells to have higher frequencies of HIV-specific IFNã+TNFá+ T cells. These results suggest a critical role for remaining effector functions in the absence of proliferation. Together our findings have implications for improved assessment of candidate HIV vaccine elicited immune responses and further investigations into the correlates of control of HIV-1 viremia.

The bulk of the developing B cell repertoire consists of autoreactive specificities; accordingly, effective maintenance of immune tolerance at this stage is critical for the prevention of autoimmune disease, the third most prevalent disease category in the U.S. Using bone marrow culture, we provide the first evidence that functional silencing, manifested by reduced surface IgM, impaired Ca2+ mobilization, and impaired proliferation, occurs in developing anti-insulin B cells exposed to insulin, a key type 1 diabetes autoantigen. This form of anergy occurs following short-term insulin exposure, and removal of antigen restores normal immature B cell function, suggesting that a high degree of functional elasticity is maintained. Development of a novel Igê-targeted BCR transgenic model reveals that a second mechanism of tolerance, receptor editing, can additionally censor immature anti-insulin B cells by replacing the autoreactive BCR specificity. These data therefore indicate that low concentrations of small molecules trigger developing B cell tolerance and highlight a potential therapeutic window for insulin autoimmunity, as the involvement of B cells in type 1 diabetes is increasingly appreciated.

Apolipoprotein B mRNA-editing complex 3G and 3F (APOBEC3G, A3G, A3F) reduce endogenous retrotransposition and also limit the replication of HIV-1. These cytidine deaminases have anti-HIV activity when packaged into virions, if not antagonized by the HIV-1 protein vif, and also when present in an endogenous target cells cytoplasm. The aim of this study was to gain an understanding of how expression of A3G is regulated in primary cells and if physiological levels of expression of A3G could overcome the vif counter measure of HIV-1. These studies demonstrate that differentiation from naïve T cells to specific CD4+ T helper subtypes regulates the expression of A3G; with increases in T helper type 1 (Th1) subtype cells and relative decreases in T helper type 2 (Th2) subtype cells. Within the Th1 subtype cells, sufficient A3G is expressed to overcome vif in the producer cell. This allows for packaging of A3G in virions, reducing infectivity of Th1 cell-produced virions relative to virions produced from Th2 cells. These studies also reveal that Th1 cells maintain a greater cytoplasmic A3G-dependent restriction to incoming wild-type HIV-1 infection than do Th2 cells. These data support the conclusion that A3G expression in primary T helper cells is capable of partially restricting vif-positive HIV-1 by both its virion-packaged and target cell activities and suggest strategies for novel therapies.

The human pathogen Staphylococcus aureus is capable of acquiring heme during infection for use as a source of iron, a nutrient that is scarce in its free form in host tissues. However, heme acquisition is dangerous to bacteria due to the toxicity of the heme molecule. We have found that S. aureus solves the heme paradox by sensing heme toxicity through the novel HssRS two-component system, which induces the expression of a transporter essential for overcoming the lethal effects heme exerts on staphylococci. In vitro and in vivo studies of HssRS signaling as well as a small molecule library screen for HssRS activators have provided insights into the signaling events regulating heme resistance in S. aureus. Furthermore, we have found that heme sensing through HssRS also occurs in Bacillus anthracis, the causative agent of anthrax. Our findings indicate that heme sensing may also occur throughout the life cycles of a number of other related Gram-positive bacteria. Together, these studies reveal the functional details of a novel bacterial heme detoxification system conserved among multiple Gram-positive bacteria that associate with host tissues rich in heme.