Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2007. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed July 16, 2008; title from "catalog record" of the print edition. Includes bibliographical references (leaves 173-182). Online version of the print edition.

Thesis (M.S.)--Georgia State University, 2008. / Title from title page (Digital Archive@GSU, viewed July 29, 2010) Yu-Sheng Hsu, committee chair; Gary Hastings, Jun Han, committee members. Includes bibliographical references (p. 41).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2008 Nov 30

Universiẗat, Diss., 2002--Bonn.

Universiẗat, Diss., 2000--Bonn.

Herpes simplex virus-1 (HSV-1) has a large double-stranded DNA genome encased within an icosahedral capsid. The capsid is surrounded by a protein-rich layer, termed tegument, and a membranous envelope containing viral glycoproteins. HSV-1 genome replication and encapsidation occurs in the nucleus, after which, DNA-loaded capsids enter the cytoplasm where they undergo tegumentation and assembly. This thesis presents a structural and functional investigation of two HSV-1 tegument proteins, UL7 and UL51 that are conserved across all herpesviruses. Deletion of UL7 or UL51 results in impaired viral replication, a small plaque phenotype and an accumulation of unenveloped capsids in the cytoplasm, the latter of which is indicative of a defect in tegumentation and/or secondary envelopment. Similar phenotypes have been observed upon deletion of homologous proteins in pseudorabies virus and human cytomegalovirus, suggesting a conserved role for these proteins. This thesis presents evidence for the formation of a UL7-UL51 complex in transfected and infected cells. Pull-down experiments using recombinant UL7 and UL51 protein purified from E. coli demonstrated that the interaction is direct, and mapped the UL7-binding region within UL51. The interaction was shown to be conserved between UL7 and UL51 homologues from murid herpesvirus, ORF42 and ORF55, respectively. The UL7-UL51 complex was purified from E.coli and, after optimisation of the purification protocol and the UL51 construct, a pure protein sample was obtained that was suitable for crystallisation trials. Two conditions were identified that produced reproducible crystals. These crystals proved to be thin and fragile, preventing their analysis by X-ray diffraction. Optimisation of the crystallisation conditions to produce more robust crystals and/or in situ diffraction measurements may yet yield X-ray diffraction data for the complex. Host-cell binding partners for UL7 and UL51 were identified by yeast-two-hybrid screen and quantitative proteomics (SILAC). An interaction between UL51 and the G-Box domain of the centriole protein CPAP was identified by Y2H screen, and validated by immunoprecipitation from transfected cells and in pull-down experiments using recombinant proteins purified from E. coli. The CPAP-binding region within UL51 was mapped and shown to resemble a motif present in the cellular protein STIL that mediates an interaction between STIL and CPAP. Two UL51 point-mutations within the putative CPAP-binding motif blocked the interaction between UL51 and the CPAP G-box domain. A mutant HSV-1 virus carrying these UL51 mutations was generated, but no difference was evident between single-step growth curves of wild-type HSV-1 and the UL51 mutant. Host-cell proteins pontin and reptin were identified as putative UL7/UL51 interaction partners in two SILAC screens. Purified GST-tagged UL7-UL51 complex was able to interact with pontin and reptin. However, it is likely that the interaction is non-specific since pontin and reptin were also found to bind a misfolded protein in a similar manner.

The cytopathology of herpes simplex virus (HSV) in H.Ep.2 and BHK-21 cells was studied using the techniques of light microscopy, immunofluorescence, electron microscopy, autoradiography and cytogenetics. Both cell types supported rapid growth cycles of HSV resulting in the production of maximum titres after 22 - 24 hours of infection. Cultures treated with 10 µg/ml ara-C or 100 µg/ml IDU at the time of infection showed a 99% decrease in infectious virus production. HSV-infected H.Ep.2 and BHK-21 cells revealed typical virus-induced inclusion bodies and a generalized disorganization of the nucleus and cytoplasm. Syncytia formation was not observed but after 24 hours of infection, nearly 100% of the cells were rounded and often detached from the glass surface. Addition of 10 µg/ml ara-C or 100 µg/ml IDU failed to prevent virus cytopathology but did cause a characteristic cytoplasmic disruption and rounding of uninfected cells. Virus-infected cells also revealed at least four separate immuno-fluorescent elements after exposure to hyperimmune serum prepared in guinea pigs. These elements included small nuclear granules, amorphous nuclear masses, diffuse cytoplasmic antigens, and intense surface fluorescence. The nuclear antigens and cytoplasmic fluorescence appeared after treatment with ara-C or IDU but the surface fluorescence was not produced in the presence of the anti-viral agents. Herpes simplex virus developed in the nucleus of infected H.Ep.2 and BHK-21 cells. The virions were enveloped at the inner lamella of the nuclear membrane and after passing into the cytoplasm, were released from the cells by a process of reverse phagocytosis. Ara-C and IDU allowed the synthesis of certain viral antigens and the development of nuclear cytopathology but completely prevented the formation of infectious HSV particles. Both drugs caused a marked distortion of the mitochondria and endoplasmic reticulum in uninfected cells. DNA synthesis in HSV-infected cells, as measured by ³H-thymidine incorporation, was almost completely inhibited by 4 hours of infection. This early inhibition of cellular DNA synthesis was followed by an immediate increase in ³H-thymidine uptake corresponding to the synthesis of viral DNA. Both cell types showed a brief stimulation of mitosis prior to the complete inhibition observed after 20 hours of infection. Cellular and viral DNA synthesis and mitosis appeared to be inhibited in virus-infected and uninfected cells treated with ara-C or IDU. Infection with HSV resulted in severe chromosomal damage to H.Ep.2 and BHK-21 cells. Chromosomal abnormalities included chromatid gaps and breaks, enhanced secondary constrictions, fragmentation, erosion, and endoreduplication, and were dependent on virus dose and time of infection. The capacity of the virus to induce chromosomal aberrations in cultured cells was UV-inactivated approximately five times less rapidly than the infectious property. Ara-C acted synergistically with the virus to produce a large number of cells with multiple chromosome breaks and also caused a significant number of abnormalities in uninfected cells. In contrast, IDU treatment resulted in few aberrations over and above those produced by HSV and little damage in uninfected cells. It was concluded that HSV was capable of producing severe morphological and genetic alterations in cultured human and hamster cells. The antiviral agents ara-C and IDU were able to completely inhibit virus multiplication but were unable to prevent any of the virus-induced cytopathic effects in vitro. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

No description available.

2,3-Dipalmitoyl-S-Glycerylcystein (Pam2Cys), ein Toll-like-Rezeptor 2 (TLR2)-Ligand, aktiviert dendritische Zellen und erhöht die Expression von HLA-DR auf deren Zelloberfläche. Natürliche Killerzellen (NK-Zellen) können über die Ausschüttung von Zytokinen das erworbene Immunsystem stimulieren. Zudem weist die Entdeckung von HLA-DR auf NK-Zellen, besonders auf CD56bright-NK-Zellen, auf eine Verbindung zwischen angeborenem und erworbenem Immunsystem hin. Gekoppelt an ein immundominantes Peptidepitop aus dem Glykoprotein D des Herpes-Simplex-Virus (HSV) Typ 2, wurde Pam2Cys in dieser Arbeit als potentieller lipopeptidbasierter HSV-Impfstoff eingesetzt, um die Effekte dieser Vakzine auf NK-Zellen zu untersuchen. Die Einflüsse der Vakzine wurden anhand der CD69-, HLA-DR- und TLR2-Expression auf und in NK-Zellen nach Inkubation mit Pam2Cys untersucht. Die Analyse der Gesamt-NK-Zellpopulation ließ nur mit Hilfe der quantitativen Real-Time-PCR einen signifikanten Anstieg der TLR2-Expression erkennen, jedoch nicht durchflusszytometrisch. Inkubierte NK-Zellen zeigten deutlich mehr HLA-DR auf der Zelloberfläche, als unstimulierte NK-Zellen. Die Untersuchung der zwei großen NK-Zellsubpopulationen, CD56bright und CD56dim, ergab, dass stimulierte CD56bright-Zellen wesentlich mehr HLA-DR und TLR2 aufwiesen, als stimulierte CD56dim-Zellen. Die Expression des Aktivierungsmarkers CD69 stieg in der Gesamtpopulation der NK-Zellen nicht in signifikantem Maße an, zeigte jedoch eine Zunahme nach Inkubation mit den Pam2Cys-Molekülen. Die Aufnahme der Lipopeptide in NK-Zellen wurde mit der konfokalen Mikroskopie bestätigt. Diese Ergebnisse zeigen, dass die hier verwendete HSV-Vakzine von NK-Zellen aufgenommen wird und sie die Ausbildung von Oberflächenrezeptoren auf diesen Zellen moduliert. Insbesondere die Expressionssteigerung von HLA-DR auf CD56bright-NK-Zellen deutet auf die Fähigkeit dieser Zellen hin, Antigene zu präsentieren und stellt einen weiteren Mechanismus vor, der eine impfstoffbezogene Immunantwort unterstützen kann. Die vorliegende Arbeit bildet eine Basis für weiterführende Studien an NK-Zellen hinsichtlich der Antigenpräsentation im Allgemeinen und der Antigenpräsentation nach Stimulation durch eine Vakzine im Besonderen. / The Toll-like receptor 2 (TLR2) ligand 2,3-Dipalmitoyl-S-Glycerylcystein (Pam2Cys) enhances the surface expression of HLA-DR on dendritic cells and activates them. Natural killer cells (NK cells) may stimulate the adaptive immune system via the production of cytokines. Furthermore the detection of HLA-DR on NK cells, especially on CD56bright NK cells, hints at a connection between innate and adaptive immunity. In this study Pam2Cys was used as a potential lipopeptide-based HSV vaccine attached to an immunodominant epitope in herpes simplex virus (HSV) type 2 glycoprotein D. The effects of this vaccine on NK cells were analysed here. Examination of the influence of this vaccine was performed by measuring the expression of CD69, HLA-DR and TLR2 on and in NK cells after incubating them with Pam2Cys. Using quantitative real-time PCR, a significant increase of TLR2-mRNA in the entire NK cell population was detectable; however flow cytometry was not able to confirm these results. Incubated NK cells showed considerably more HLA-DR than unstimulated NK cells. Analysis of the two big NK cell subpopulations, CD56bright and CD56dim, revealed essentially more HLA-DR and TLR2 on stimulated CD56bright cells, compared to stimulated CD56dim cells. Expression of the activation marker CD69 after incubation was not triggered significantly on NK cells altogether; nevertheless an increase could be measured. Confocal microscopy confirmed an uptake of lipopeptides into NK cells. These data demonstrate the surface receptor modulation on NK cells by a potential HSV vaccine and the ability of NK cells to endocytose Pam2Cys. Particularly, the increase of HLA-DR on CD56bright NK cells points at the ability of these cells to present antigens, introducing a new mechanism able to support a potent immune response to a vaccination. This study provides a basis for further work on NK cells in terms of antigen presentation in general and antigen presentation after stimulation by a vaccine in particular.