Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells

Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells

Bibliographic Details
Main Author: Maxwell Stropes, Melissa Page
Language:English
Published: University of Cincinnati / OhioLINK 2009
Subjects:
Biochemistry
Biomedical Research
Molecular Biology
Virology
US28
RANTES
Fractalkine
Cytomegalovirus
HCMV
GPCR
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=ucin1236016578
id ndltd-OhioLink-oai-etd.ohiolink.edu-ucin1236016578
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Biochemistry
Biomedical Research
Molecular Biology
Virology
US28
RANTES
Fractalkine
Cytomegalovirus
HCMV
GPCR
spellingShingle Biochemistry
Biomedical Research
Molecular Biology
Virology
US28
RANTES
Fractalkine
Cytomegalovirus
HCMV
GPCR
Maxwell Stropes, Melissa Page
Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells
author Maxwell Stropes, Melissa Page
author_facet Maxwell Stropes, Melissa Page
author_sort Maxwell Stropes, Melissa Page
title Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells
title_short Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells
title_full Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells
title_fullStr Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells
title_full_unstemmed Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells
title_sort signaling and regulation of the human cytomegalovirus g-protein coupled receptor us28 in hcmv infected cells
publisher University of Cincinnati / OhioLINK
publishDate 2009
url http://rave.ohiolink.edu/etdc/view?acc_num=ucin1236016578
work_keys_str_mv AT maxwellstropesmelissapage signalingandregulationofthehumancytomegalovirusgproteincoupledreceptorus28inhcmvinfectedcells
_version_ 1719432927970852864
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-ucin12360165782021-08-03T06:13:08Z Signaling and Regulation of the Human Cytomegalovirus G-Protein Coupled Receptor US28 in HCMV Infected Cells Maxwell Stropes, Melissa Page Biochemistry Biomedical Research Molecular Biology Virology US28 RANTES Fractalkine Cytomegalovirus HCMV GPCR The human Cytomegalovirus (HCMV) is a species specific herpes virus which encodes several G-protein coupled receptors (GPCRs) that likely serve important roles in immunomodulation and viral dissemination during infection. One of these receptors, called US28, has been shown to act through multiple G-protein dependent and G-protein independent signaling pathways, which contribute to diverse US28-mediated effects following HCMV infection such as cell migration and tumorigenesis. One of these signaling pathways occurring in HCMV infected cells, the Gα<sub>q/11</sub> mediated PLC-β pathway, has previously been shown to be dependent on US28, but does not necessarily require ligand activation by chemokines. Additionally, these signaling events are likely attenuated or “desensitized” in a sequence of events including receptor phosphorylation and β-arrestin recruitment to the carboxy terminus of the receptor. The full repertoire of signaling events activated by US28, their dependence on chemokine binding, and attenuation by cellular regulatory factors is still unknown. Most studies regarding US28 activity have been performed by over-expressing the receptor or various mutants in cell culture in the absence of the other 200 gene products encoded by HCMV, and with expression levels of US28 which may not be physiologically relevant. To address this, recombinant HCMVs knocked-out for US28 or encoding FLAG-tagged versions of either wild type US28, a signaling deficient DRY box mutant US28(R129A), a chemokine binding mutant US28ΔΝ a carboxy terminal mutant US28(1-314) were engineered into the HCMV clinical isolate VR1814 via BAC recombineering. These viruses were first used to verify that US28 primarily exhibits early kinetics, with expression peaking at 48-72 hours post infection, although very low levels can be detected as early as 6 hpi. We then verified that the FLAG-tagged US28 wild type has the same ability to induce PLC-beta signaling potential as the parental HCMV strain, indicating that the addition of the FLAG epitope did not alter US28 signaling ability. Infection with FLAG-US28(R129A) virus resulted in deficient signaling similar to the US28 knockout, formally demonstrating that G-protein coupling to US28 is required for PLC-beta signaling during an HCMV infection. The chemokine binding mutant US28ΔΝ signals to 80% of wild type, providing further evidence that chemokine binding is not critical for US28 signaling during infection but may be necessary for trafficking of US28 to the plasma membrane. Additionally, US28/1-314 exhibits a more potent signal than US28/WT in HCMV infected cells, indicating that the carboxy terminus is involved in US28 desensitization and therefore likely to be critical in maintaining an appropriate threshold of signaling activity. Using our recombinant viruses, we also demonstrated that while RANTES and Fractalkine function as neutral or inverse agonists in PLC-beta assays, respectively, these chemokines function as agonists in acute signaling assays such as the release of intracellular calcium. Since PLC-beta activity is measured over a two hour period, these data suggest that both RANTES and Fractalkine induce the stabilization of an active conformation of US28 and that the apparent “inverse” agonist effects of Fractalkine is due to receptor internalization or down regulation and not due to the stabilization of an inactive conformation of the receptor. The data presented in this dissertation are the first to utilize US28 functional mutants in HCMV infected cells, which is important in establishing a relationship between overexpression studies in transfected cells and events occurring during infection. Elucidating the mechanisms by which US28 signals during infection will provide important insight into HCMV pathogenesis and possibly its effects on immune system modulation and immune evasion. 2009-07-20 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1236016578 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1236016578 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.

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