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Characterization of Cytomegalovirus US28 vGPCR Signaling within the ARPE cell line

Campbell, Emily Lo
http://orcid.org/0000-0003-3188-3574
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1530267484318891

Abstract Details

2018, MS, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
Human cytomegalovirus (HCMV) is a member of the β-herpesviridae subfamily. It contains a 230kb genome of linear dsDNA and is known to establish persistent and latent lifelong infection that is generally asymptomatic in immunocompetent individuals. Approximately 50-75% of adults in developed countries are seropositive for HCMV but will not develop symptoms unless the virus is reactivated. For immunocompromised individuals, HCMV is an important pathogen and it is known to cause vascular disease, birth defects, and retinitis. Current knowledge of this virus lacks understanding of how the latent cycle is established and maintained. US28, a viral G protein-coupled receptor (vGPCR) encoded by HCMV, is thought to be important during latent replication and is known to influence cellular signaling. Recently, the crystal structure of US28 was published which highlighted differences in amino acid sequence between US28 and human homologs. To better understand the molecular mechanisms of US28 in HCMV latent replication, we asked how these differences may affect US28 constitutive signaling within a cell model. US28 mutants with specific amino acid switches were constructed and cloned into a pSLIK-Venus lentivirus. Retinal Pigmented Epithelium (ARPE) cells were infected, creating a stably transduced Tet-ON regulatable cell system. Two mutants were made: a single K223E and a Triple Mutant which combined the K223E mutation with a lengthening of the intracellular loop 2 and the mutation of E124L (which is thought to be important for stabilizing the active conformation of the vGPCR). Protein expression was induced by doxycycline (dox) and measured by flow cytometry and immunoprecipitation/western blotting while signaling was measured by inositol triphosphate (IP3) and calcium (Ca2+) assays. Parental (non-transduced) ARPEs, WT, and R129A were used as experimental controls. Flow cytometry and immunoprecipitation/western blot demonstrated that US28 was dox inducible within the stably transduced ARPEs. In IP3 and Ca2+ signaling assays K223E showed markedly decreased constitutive signaling but did have ligand-induced signaling activity similar to WT. Triple Mutant had no constitutive or ligand-induced signaling activity when compared to basal parental ARPEs. In human GPCRs, there is a glutamic acid residue in transmembrane 6 that creates an “ionic lock” with the arginine residue in the DRY motif located in transmembrane 3. The decreased constitutive activity of K223E while maintaining ligand-induced signaling activity demonstrates the importance of this glutamic acid in stabilizing the inactive conformation of the GPCR. Due to the role of US28 in HCMV latent infection, this provides a target for modifying the course of HCMV.
William Miller, Ph.D. (Committee Chair)
Edmund Choi, Ph.D. (Committee Member)
Rhett Kovall, Ph.D. (Committee Member)
56 p.
Molecular Biology
HCMV; US28; Epithelial; Structure Function; Signaling

Recommended Citations

Citations

  • Campbell, E. L. (2018). Characterization of Cytomegalovirus US28 vGPCR Signaling within the ARPE cell line [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1530267484318891

    APA Style (7th edition)

  • Campbell, Emily. Characterization of Cytomegalovirus US28 vGPCR Signaling within the ARPE cell line. 2018. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1530267484318891.

    MLA Style (8th edition)

  • Campbell, Emily. "Characterization of Cytomegalovirus US28 vGPCR Signaling within the ARPE cell line." Master's thesis, University of Cincinnati, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1530267484318891

    Chicago Manual of Style (17th edition)

ucin1530267484318891
194
© 2018, some rights reserved.Creative Commons License Characterization of Cytomegalovirus US28 vGPCR Signaling within the ARPE cell line by Emily Lo Campbell is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.