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Title Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo
Publication Date
Degree PhD
Discipline/Department Medicine: Molecular Genetics, Biochemistry, and Microbiology
Degree Level doctoral
University/Publisher University of Cincinnati
Abstract The betaherpesvirus Human Cytomegalovirus (HCMV) is estimated to be present in 40-80% of world population. HCMV infection in a healthy person causes mild symptoms, although the virus persists in the host in the latent form. Immunocompromised patients such as HIV and organ transplant patients as well as fetuses and neonatal babies with underdeveloped immune system are most affected by HCMV infection. CMVs are characterized by their strict species specifity; therefore humans are the only host for HCMV. The mouse cytomegalovirus (MCMV) has been the most useful animal model to explore HCMV spread and disease. CMVs encode G Protein-Coupled Receptors (GPCR) and these viral GPCRs are increasingly recognized as important regulators of pathogenesis and disease. HCMV for example encodes four of these GPCRs, whereas MCMV expresses two: M33 and M78. M33 shows constitutive signaling properties and activates various signaling pathways. M33 stimulates the PLC-&beta/PKC pathway via G&alphaq/11, however activation of NF-&kappaB and p38 MAP-kinase is independent of G-proteins. Importantly, M33 is essential for dissemination to or growth in salivary glands of immunocompetent mice and the G-protein signaling ability of M33 is necessary for viral growth at this site. Here we used the non-obese diabetic (NOD) and the immunocompromised NOD scid gamma (NSG) mouse models to assess a potential role for M33 as an immunomodulator and to further investigate the mechanism(s) by which M33 promotes viral growth in vivo. We are able to detect replication of &DeltaM33 viruses in the salivary glands of immunocompromised NSG mice; however it exhibited a 400x defect compared to wildtype MCMV. Since the growth phenotype is not completely reverted in the NSG mice, we conclude that M33 is not solely functioning to modulate the immune system. We also showed that M33 is dispensable for hematogenous dissemination within the host and that activation of G&alphaq signaling pathways alone is not sufficient to promote salivary gland growth as neither HCMV US28 nor a constitutively active form of G&alphaq are able to complement the unique functions of M33. We also describe here the generation and characterization of an in vitro cell culture model with cells extracted from mouse salivary glands. Specialized growth conditions resulted in cells that express markers of salivary gland epithelium, but none resulted in maintenance of epithelial markers similar to those expressed by the organ in vivo. Despite growth conditions that enable sustained expression of at least low levels of the salivary epithelial markers, M33 was not required for viral growth in these primary cells. Taken together, these studies suggest that M33 is necessary to allow viral growth within the three-dimensional structure of the gland in vivo but that this activity is not required once the structure of the gland is disrupted. An understanding of CMV replication in the salivary gland and how M33 modulates salivary gland function to promote viral replication will provide insights into mechanisms that…
Subjects/Keywords Virology; Murine Cytomegalovirus; Viral GPCR; M33
Contributors Miller, William (Committee Chair)
Language en
Rights unrestricted ; This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
Country of Publication us
Format application/pdf
Record ID oai:etd.ohiolink.edu:ucin1397234044
Repository ohiolink
Date Indexed 2016-12-22
Grantor University of Cincinnati

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…disease. Among them, murine cytomegalovirus (MCMV) has been well characterized as a pathogen with similar genome structure and pathogenesis to that of its counterpart, HCMV (4-6). Infection of immunocompetent individuals with HCMV…

…Gammaherpesvirus ORF74 EBV Gammaherpesvirus BILF1 Table 1: GPCRs encoded by herpesviruses. MCMV: murine cytomegalovirus; RCMV: rat cytomegalovirus; HCMV: human cytomegalovirus; HHV6: human herpesvirus 6; HHV7: human herpesvirus 7; MHV68: murine

…to immunocompromised individuals and developing fetuses. Due to the species specificity of CMVs, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. MCMV like other beta- and gamma- herpesviruses…

…x29;. CMVs show strict species specificity, which has severely restricted in vivo studies of HCMV pathogenesis as the virus is unable to replicate and disseminate in model organisms like the mouse. For this reason, murine cytomegalovirus (MCMV…

…List of Abbreviations: CMV: Cytomegalovirus CRE: Cyclization Recombinase CREB: cAMP response element binding DAG: Diacylglycerol EBV: Epstein-Barr virus EGFR: Epidermal growth factor receptor ERK: Extracellular signal-regulated kinase FACS: Fluorescence…

…activated cell sorting HCMV: Human cytomegalovirus IFN-: Interferon gamma IP3: Inositol 1,4,5-trisphosphate GPCR: G protein-coupled receptor KSHV: Kaposi’s sarcoma-associated herpesvirus MAPK: Mitogen activated protein kinase MCMV: Mouse cytomegalovirus MEF…

…x28;non-obese diabetic) Scid Gamma xi ORF: Open reading frame PBL: Peripheral blood leukocyte PFU: Plaque forming units PKA: Protein Kinase A PKC: Protein kinase C PLC-: Phospholipase C-beta RCMV: Rat cytomegalovirus VEGF: Vascular endothelial…

…persistence. Alpha subfamily members include Herpes Simplex and Varicella Zoster viruses, which persist in neurons. Beta subfamily members include Cytomegalovirus and Roseolavirus, which persist in cells of myeloid lineage. Lastly, the Gamma subfamily…