Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that establishes a latent infection in sensory ganglia and upon reactivation, can cause severe ocular disease. During lytic replication, viral proteins are expressed in a temporal cascade of immediate early (α), early (β) and late (γ) genes. The γ genes are further sub-classified into γ1 genes which are expressed prior to DNA replication and γ2 genes which are absolutely dependent on DNA replication for their expression. During a latent infection of the trigeminal ganglia (TG), no infectious virus is produced and latency is associated with a persistent virus-specific CD8+ T cell infiltrate that actively block reactivation. In C57BL/6 mice, approximately 50% of these CD8+ T cells are specific to a single epitope on a γ1 protein glycoprotein B (gB498-505; gB-CD8). In the TG, gB-CD8 are retained with an activated phenotype suggesting recent exposure to antigen during abortive reactivation events. While the kinetics of gene expression during lytic infection has been appreciated for some time, little is known about antigen expression during latency and reactivation, and its influence on antigen-specific CD8+ T cells in the TG. Using gB as our model antigen, in this work we show that during reactivation, gB expression occurs much sooner than gC expression, indicating that it expressed fairly early even during reactivation. Furthermore, by delaying gB expression to after DNA replication we observed a severe impairment in viral replication in the TG and a significant diminishment in the retention and activation phenotype of gB-CD8 in latently infected TG. However delaying gB did not prevent the ability of gB-CD8 to block reactivation indicating they can act very quickly even after DNA replication. Finally we demonstrate that by mutating the gB epitope such that it does not induce a gB-CD8 response, we abrogate the gB-CD8 infiltrate in the TG. These studies all demonstrate that during an HSV-1 infection, only antigen-specific CD8+ T cells infiltrate the TG and antigen exposure during latency is responsible for their retention in the TG. This work has great implications toward designing better immunogens for therapeutic vaccines to prevent HSV-1 reactivation.

Varicella-Zoster Virus (VZV) is the causative agent of chickenpox during primary infection and herpes-zoster or shingles following reactivation from neuronal latency. The VZV ORF66 protein kinase is a serine/threonine kinase and one of two VZV protein kinases. Its homologue in the alphaherpesviruses are termed the US3 kinases, and through phosphorylation of targets affect many events in infection, influencing processes such as survival of the infected cell to apoptosis, the state of permissivity to gene expression, avoidance of immunity, modulating cellular pathways affecting host actin dynamics, and influencing the nuclear structure and nuclear membrane to enable assembly of virus components. ORF66 is not essential in most cell culture but important for viral replication in T cells. In this work, we have found the ORF66 is critical for viral growth in primary corneal fibroblasts and thus established a model for further investigatation of cell-type dependent functions for ORF66. This finding may have important applications for viral pathogenesis as VZV reactivates and causes infection of the eye in herpes zoster ophthalmicus disease. Here we also describe a novel ORF66 cellular target, the nuclear matrix protein, matrin 3. Specific matrin 3 phosphorylation is conserved for herpes simplex virus - type 1 and pseudorabies virus US3 kinases. Thus, this finding may have important implications for the role of ORF66/US3 function in common alphaherpesvirus strategies to utilize host cell machinery in establishing a host cell environment conducive to viral replication. ORF66/US3-induced phosphorylation of matrin 3 was needed for matrin nuclear retainment late in viral infection, suggesting that ORF66/US3 may have a role in modulating matrin 3 nuclear functions needed for viral replication. The ORF66 kinase is clearly important for VZV growth in certain cell types relevant to human disease and our studies underscore the diverse roles of this protein in VZV infection.

Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin that is responsible for causing the symptoms of type A food poisoning, a leading cause of bacterial foodborne illness in the US. CPE-induced pore formation on intestinal epithelial cells results in ion permeability alterations leading to Ca2+ influx and activation of cell death pathways. Upon binding to its receptor, certain claudins, the interactions between CPE and the target membrane result in the formation of a series of toxin complexes (CH-1 and CH-2) that represent the formation of the functional CPE pore. Many bacterial toxins, particularly, pore-forming toxins, hijack cholesterol-rich lipid raft domains in the target cell membrane to aid in their virulence. Lipid rafts serve as platforms to cluster receptor proteins to allow for more efficient binding and oligomerization. Due to the pore-forming activity of CPE, we wished to determine if membrane rafts play a role in the mechanism of action of CPE. Interestingly, CPE was found to be a novel pore-forming toxin that does not require raft domains for its action in that CPE complexes do not form within lipid rafts and cholesterol depletion had no effect on CPE-induced cytotoxicity. These findings illustrate the unique interactions between CPE and target cells. Despite recent research findings indicating the presence of claudins in the various CPE complexes, these intricate interactions have not been fully elucidated, and the exact composition of the toxin complexes is unknown. Therefore, the research presented here describes the development of a two-step method of electroelution/immunoprecipitation that allows for the isolation and purification of the CPE complexes for compositional analysis by mass spectrometry. Finally, a mouse model has been developed and characterized to show that the molecular interactions that occur in cell culture models, such as complex formation and inflammatory cell death, also occur in vivo. Furthermore, the mouse model mimics the lethality that is occasionally seen in humans that suffer from type A food poisoning-related deaths.

Herpes simplex virus type 1 replicates its DNA and builds progeny nucleocapsids in the nucleus of the infected cell. Replicated viral DNA is a concatemer that is cleaved to unit-length genomes and packaged into capsid precursors, and successful DNA packaging is required for the mature capsid to exit the nucleus and become incorporated into virions. The DNA cleavage and packaging machinery is highly conserved across herpesvirus subfamilies; they represent novel targets for anti-herpesviral treatments. Seven genes have been identified that are essential for the packaging reaction. Of these, the UL25 gene product is unique because it is required for the completion rather than the initiation of DNA packaging. In the absence of functional UL25, the nuclei of infected cells accumulate empty capsids and free, close to genome-length viral DNA. This phenotype differs from null mutations of the other packaging genes, which block cleavage of the concatemer and expulsion of the capsid scaffold protein. While the functions of several members of the packaging machinery have been deduced by analogy with the dsDNA bacteriophages, the role of UL25 protein (pUL25) in this process is unclear. pUL25 is stably associated with the capsid vertices, where it forms a heterodimer with the packaging protein UL17. The goal of this project is to elucidate the pUL25 capsid-binding mechanism and its significance for DNA packaging and virion assembly. Mutational analysis of UL25 mapped the capsid-binding domain to the pUL25 N-terminus. pUL25 interactions with the capsid surface proteins were visualized with cryo-electron microscopy and 3D image reconstructions of capsids containing pUL25 fusion proteins. Finally, we demonstrated that UL25 mutants aberrantly cleave viral DNA before the second packaging signal. These data support a model in which pUL25 provides structural support to capsid vertices without interacting directly with DNA during the packaging reaction.

HSV-1 is a ubiquitous human pathogen with a biphasic lifecycle. During latency, gene expression is globally repressed, most likely due to epigenetic mechanisms. The HSV protein ICP0 is a promiscuous transactivator of gene expression, and is required for efficient viral reactivation from latency. Evidence indicates that ICP0 interacts with a number of cellular pathways that mediate chromatin structure. Using a cell culture model of latency, the effects of cell-type, ICP0 expression, and ICP0 functional domains on the chromatin structure of viral genomes was examined. During viral entry into quiescence, ICP0 expression increased gene expression and histone hyperacetylation, while limiting the association of histones and heterochromatin with the viral genome. In the absence of ICP0, expression from viral promoters was rapidly repressed, and heterochromatin formed on viral promoters in a cell-type specific manner. Once quiescence was fully established, HSV genomes were found in a highly heterochromatic state. Heterochromatin, measured by the presence of heterochromatin protein 1γ and trimethylation of histone H3 lysine 9, was rapidly removed upon provision of ICP0 in trans. The changes in epigenetic structure were global, and preceded reactivation of gene expression. Overexpression of ICP0 resulted in the removal of histones from the quiescent genome, an effect not seen when ICP0 was expressed at physiological levels. This indicated that ICP0 may be mediating its effects through multiple functions, potentially through the enzymatic function of its RING finger (RF) E3 ubiquitin ligase domain when expressed at low levels, and through physical protein-protein interaction when overexpressed. The effects on chromatin structure and gene activation of quiescent HSV were determined by superinfection with mutants of the RF domain and the C-terminus, which is important for the disruption of the CoREST/HDAC repressor complex. The RF domain was necessary and sufficient for reactivation of gene expression, but full transcriptional activation and removal of heterochromatin and histones required both the RF and C-terminus of ICP0. These results indicate that ICP0 relieves repression of gene expression through interactions with multiple cellular pathways. Expression of ICP0 removes epigenetic repression at multiple levels of chromatin, including heterochromatin, histone deposition, and the acetylation of histones.

Mycobacterium tuberculosis continues to be a leading cause of death by an infectious agent world wide with approximately two million deaths attributable to it annually. Once infected, the host mounts a robust Th1 type immune response that contains, but does not eliminate the bacteria. A small percentage (5-10%) fail to contain the infection, develop active disease, and are contagious. Most remain asymptomatic and are considered latently infected. BCG, while the gold standard for vaccination, ultimately appears to limit disease, but not prevent infection. In addition, chemotherapeutic treatment is long, requires multiple agents, and compliance is difficult to maintain. With one third the worlds population infected, inadequate vaccine efficacy, and difficult treatment regimen, it is imperative that a better understanding of which factors are responsible for containment of the infection be achieved. CD4 T cells are an essential component of the immune response that controls M. tuberculosis. CD4 T cells are able to both promote inflammation and dampen its effect. As a pro-inflammatory Th1 cell, these cells secrete pro-inflammatory cytokines, participate in macrophage and dendritic cell activation and help prime CD8 T cells. As regulatory T cells, CD4 T cells prevent autoimmunity and may act to protect surrounding tissue from immunopathologic damage. One main function of pro-inflammatory CD4 T cells is IFNγ production, however other cells can and do produce IFNγ. In addition, the role of regulatory T cells during infection and how they relate to disease progression has yet to be elucidated. The work presented in this dissertation provides a new model for addressing the role of IFNγ from sources other than CD4 T cells, and addresses what pro-inflammatory functions they may have in addition to IFNγ production. In addition, these studies address regulatory T cells and the anti-inflammatory effects of long term IL-12 treatment as well as Tregs relationship to disease outcome in non-human primates.

Human papillomaviruses (HPVs) are involved in the pathogenesis of different types of human cancers, especially cancer of the cervix (CaCx). MicroRNAs (miRNAs) are post-transcriptional gene regulators that have recently been associated with many types of cancers. We analyzed the expression of cellular miRNAs in HPV-16 positive cervical cell lines and tissues via microarray, Northern blotting, and quantitative RT-PCR. Three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. An HPV-negative CaCx cell line, C-33A, showed underexpression of four miRNAs compared to the normal cervix. Also, nine miRNAs were overexpressed and only one underexpressed in cell lines containing integrated HPV-16 DNA compared to C-33A. MicroRNA-218 (miR-218) was specifically underexpressed in cell lines, cervical lesions and cancer tissues containing integrated HPV-16 DNA compared to both C-33A and the normal cervix. Exogenous expression of the HPV-16 E6 oncogene reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16 positive cell line increased miR-218 expression. Furthermore, exogenous expression of miR-218 in HPV-16 positive cell lines decreased expression of the epithelial-specific gene LAMB3, which is involved in cell migration and tumorigenicity. We analyzed the expression of cellular miRNAs in additional clinical samples including six HPV-16 positive cervical cancers, three cervical dysplasias, and four normal cervical tissues using TaqMan® MicroRNA Arrays V2.0. Eighteen miRNAs were overexpressed and two underexpressed in cervical cancer tissues compared to normal cervical tissues. Nine miRNAs were consistently overexpressed, and two miRNAs were consistently underexpressed, including miR-218, in cervical dysplasias and cervical cancer tissues compared to normal tissues. We also found that exogenous expression of miR-218 in HPV-16 positive cell lines decreased expression of the extracellular matrix protein MMP3, which is involved in the epithelial-mesenchymal transition. We also demonstrated that HPV-16 E6 regulates miR-218 via the histone acetyltransferase p300 in cervical cells. The expression of miR-218 was found to be reduced in HPV-negative cells upon p300 knock-down or p300 inhibition with anacardic acid, and expression of miR-218 was reactivated in HPV-16 positive cells upon EZH2 knock-down or inhibition with adenosine dialdehyde. Reactivation of miR-218 was enhanced upon treatment with a combination of 5-azacytidine, trichostatin A, and adenosine dialdehyde. We also demonstrated that miR-218 reduces the migration and invasion of SiHa cervical cancer cells, indicating that miR-218 functions as a tumor suppressor in cervical cancer and that it may have therapeutic value.

CD8+ T cells can inhibit HIV-1 replication in infected CD4+ T cells in a non-cytolytic manner by suppressing transcription from the viral promoter. The factor mediating this response, referred to as CD8 Antiviral Factor (CAF), and its mechanism of action, are unknown. The overall aim of this project was to elucidate the mechanisms by which CAF mediates its HIV-1 transcription suppressing effects. Towards this, we first studied the interaction of CAF with CD4+ target cells; then, investigated the signaling molecules involved in the process of viral transcription suppression; and thirdly, identified a region on the HIV-1 Long Terminal Repeat (LTR) promoter necessary for CAF-mediated transcription suppression. We used exosomes secreted by CD8+ T cells as a source of CAF to examine the interactions of CAF with its CD4+ target cells. Exosomes interacted with the cells at their surface, within 10 minutes of addition. However, maximal suppression of transcription was seen in the cells only 12 to 16 hours after addition. These results suggest the involvement of an intracellular signaling cascade and, possibly, secondary gene expression. Previous reports, using HVS-transformed CD8+ T cell lines, have shown the requirement for the intracellular signal transducer, STAT1, in CAF-mediated viral transcription suppression. However, we found that the requirement for STAT1 in the suppressive process was seen only when CAF from transformed CD8+ T cells (CAFtransformed) was used. In contrast, CAF from primary CD8+ T cells (CAFprimary), from HIV-1 infected patients, suppressed HIV-1 transcription in a STAT1-independent manner. Our investigations on the region of the viral promoter necessary for suppression showed the importance of the minimal promoter region of HIV-1 LTR. This 90bp region, containing 3 SpI sites and TATA box, was sufficient for transcription suppression to occur. Hence, our studies implicate a multi-pronged mechanism of CAF action on the LTR minimal promoter, and indicate major differences between CAF from transformed and primary CD8+ T cells.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.

Thesis (Ph. D., Pathology)--Ohio State University, 2002. / Title from first page of PDF file. Document formatted into pages; contains xix, 167 p.: ill. (some col.). Includes abstract and vita. Advisor: Christoph Plass, Dept. of Medical Microbiology and Immunology. Includes bibliographical references (p. 145-167).