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Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo

Bittencourt, Fabiola M.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234044

Abstract Details

2014, PhD, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
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 cytomegaloviruses use to promote persistence and access to saliva, a main bodily fluid by which horizontal viral transmission occurs.
William Miller, Ph.D. (Committee Chair)
Nancy Sawtell, Ph.D. (Committee Member)
Kasper Hoebe, Ph.D. (Committee Member)
Richard Thompson, Ph.D. (Committee Member)
Alison Weiss, Ph.D. (Committee Member)
177 p.
Virology
Murine Cytomegalovirus; Viral GPCR; M33

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Citations

  • Bittencourt, F. M. (2014). Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234044

    APA Style (7th edition)

  • Bittencourt, Fabiola. Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo. 2014. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234044.

    MLA Style (8th edition)

  • Bittencourt, Fabiola. "Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo." Doctoral dissertation, University of Cincinnati, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234044

    Chicago Manual of Style (17th edition)

ucin1397234044
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This open access ETD is published by University of Cincinnati and OhioLINK.