Thesis (M.S.)--Wisconsin. / Includes bibliographical references (leaves 55-60).

Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 98-107).

The replication of cytomegalovirus (CMV), Towne strain, in permissively infected cells is characterized by a long eclipse phase and lengthy growth cycle which may reflect the activity of mechanisms which regulate viral gene expression at the transcriptional level. To correlate the study of transcription with other events occurring during CMV replication, experiments were first conducted to determine the time post-infection for the onset of virus maturation and virus DNA synthesis under defined conditions. The onset of virus maturation was between 2 and 3 days post-infection. The hybridization kinetics of labeled CMV DNA with DNA extracted at different times post-infection indicated that the onset of viral DNA synthesis was between 24 and 36 hours post-infection in human foreskin fibroblast cells permissively infected with CMV. The results of hybridizations of stable viral RNA accumulating at various times post-infection, and at early times in the absence of protein synthesis, with labeled CMV DNA, showed that there is temporal regulation of transcription. The transcription of the genome is restricted in the presence of an inhibitor of protein synthesis to 5-6% of the genome (immediate early RNA). There is a rapid switch of immediate early to the early phase of transcription, which extends to at least 24 hours post-infection, and consists of transcripts homologous to approximately 30% of the viral genome throughout the entire phase. After the onset of viral DNA synthesis, the transcription extends into the late phase during which transcripts homologous to approximately 42% of the genome are synthesized. Early and late RNA was analyzed for the presence of symmetric transcripts. Transcription was found to be asymmetric at early times post-infection, with only 5% symmetric transcription during the late phase. The extent to which immediate early, early, and late stable RNA is transcribed from the repeat regions of the CMV genome was determined by hybridizations of stable RNA to labeled XbaI restriction fragments Q and M, which comprise the long repeat regions of the CMV genome. The hybridization of the probes indicated the presence of RNA transcripts at immediate early times that were homologous to 15% of the repeat sequences. Early RNA contained transcripts homologous to 18.5% of the repeat regions, and late RNA was homologous to 34% of the long repeat sequences.

Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Swiss white mice and C3H/HeJ mice were used occasionally. It was shown that in vitro infection resulted in the formation of infectious centers in only a small percentage of the spleen cell population even at high multiplicities of infection, when a proportionately higher number of virus particles were being taken up by the cells. Virus could be rescued from some of these infected cells by co-cultivation with susceptible mouse embryo fibroblasts, and the emerging infectious virus particles were detectable 40-50 hours after the start of co-cultivation. It appeared that allogeneic stimulation was not essential to virus rescue in our in vitro system, for both syngeneic and allogeneic mouse embryo fibroblasts were equally efficient in effecting virus reactivation from infected spleen cells. A fraction of the infected spleen cells was capable of supporting spontaneous MCMV replication. Such replication was not affected by incubation with the mitogens Con A or LPS prior to or after infection with the virus. However, the presence of an increased number of activated T cells due to Con A stimulation possibly inhibited virus replication to a certain extent. Virus replication was also reduced in the absence of some heat-labile factor in the fetal calf serum. Cell separation techniques such as nylon wool column adherence, plastic adherence, anti-serum treatment and y-xay irradiation, showed that macrophage-like cells were probably involved in harboring MCMV in a latent state although spontaneous replication did take place to a limited extent in such cells. Another cell fraction, with B-cell-like properties, was capable of supporting spontaneous MCMV replication. Another effect of in vitro MCMV infection on spleen cells was their suppressive effect on the mitogenic responses of such cells. Preliminary evidence suggested that the defective con A response might be mediated by macrophages exposed to MCMV. Moreover, the immunosuppressive effect could only be observed after exposure to infectious virus particles, and the degree of suppression of mitogen responses could be increased by using higher multiplicities of infection. In certain cases, a slight stimulatory effect on the spleen cells was observed about 30 hours after infection. In summary, the spleen cell population contained cell fractions that were capable of harboring MCMV in a latent state and supporting spontaneous viral replication. In addition, the mitogenic responses of infected cells were impaired. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells. Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development. Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--Murdoch University, 2002. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 210-245.

Thesis (Ph.D.) - University of Glasgow, 2007. / Ph.D. thesis submitted to the Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

The purpose of this investigation was to examine the structure of the murine cytomegalovirus (MCMV) genome and to study its expression during productive and non-productive infections caused by the virus. The kinetic complexity of MCMV DNA was not less than its molecular weight implying the absence of major reiterations. The restriction endonuclease EcoR^ cleaved this molecule into twenty-five fragments, which were present in the digest in equimolar amounts and ranged in molecular weights from 20 to 1 million. The sum of the molecular weights of the fragments was 136 million. The genomes of the 'K 181' and 'Smith' strains of MCMV appeared to share more than 99 percent of their sequences, although the DNAs exhibited slightly different fragmentation patterns when treated with EcoR^ and Hind III endonucleases. Control was exerted on the transcription of the MCMV genome at temporal, quantitative, and processing levels. During productive infections, approximately 25 percent of the genome was represented as stable transcripts in the cell at 6 hours post infection, i.e., before the onset of viral DNA synthesis, whereas RNA transcribed from 35 to 40 percent of the DNA was present in the cells in the later stages of infection. RNA sequences corresponding to 6 h (early) transcripts would be detected in the cell throughout the infectious cycle. Both 'early' and 'late' RNA comprised two RNA classes differing about 7 to 10 fold in concentration. Viral DNA synthesis in the host cell was required for the expression of 'late' genes since in the presence of inhibitors of protein and DNA synthesis only 'early' transcription occurred. Control was also exerted on the transport of transcripts from the nucleus to the cytoplasm of infected cells. Although RNA extracted from the nuclei of infected cells arose from 25 (early) and 35 (late) percent of the viral genome, transcripts from only 11 (early) and 15 (late) percent of the DNA were detected in the cytoplasm. Cells of mouse origin (3T3.cells), arrested in the G^ phase of the cell cycle, retained the viral genome in a non-replicating state, but could be induced to enter the lytic cycle by serum activation. Transcripts from 19 percent of the genome were observed in G^ arrested, MCMV-infected cells. Viral RNA in these cells comprised only one abundance class, which was similar to the scarce class in 'early' RNA from infected exponentially growing cells. Some evidence was also obtained for the transmission of latent MCMV genomes from mother to progeny. Cells cultured from embryos of infected mice did not normally produce infectious virus. However, the presence of the virus, at least in some of these cells, could be demonstrated by immunofluorescence, and by in-situ hybridization, using iodinated MCMV DNA as probe. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The interaction between Murine Cytomegalovirus (MCMV) and the cell cycle has been investigated in synchronized murine cells. Based on the following evidence it was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of viral growth in exponentially growing 3T3 cells (12 h). was protracted until the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support viral replication; (3) entry of the virus was equally efficient in G-l, S and exponential cells; (4) in exponential 3T3 cells viral DNA synthesis began at 10 h post infection, and in synchronized cells it began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of viral DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in its ability to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the ability of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a viral thymidine kinase, and thymidine kinase was not essential for normal viral replication. When G-l arrested 3T3 cells were infected with MCMV, viral DNA synthesis did not initiate and the lytic cycle was reversibly blocked. The non-replicating viral genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the cell cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was first studied in G-l, S phase, and exponential 3T3 cells. Under the appropriate conditions, nuclei from infected cells synthesized viral DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized cell DNA only and not viral DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative viral DNA polymerase was inhibited by antiserum prepared against infected cell proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a viral gene product. The second approach involved a comparison of viral transcription in permissive and non-permissive 3T3 cells. The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of viral RNA classes and the fraction of the viral genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before viral DNA synthesis), two classes of viral transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the viral DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the viral DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the viral DNA. (4) in non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the viral DNA. Summation hybridization experiments indicated that in non-permissive (G-l) cells, only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe cell cycle dependent replication of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the initiation of viral DNA synthesis directly, or for the transcription of viral DNA sequences. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate