Human Cytomegalovirus (HCMV) infection is a leading cause of viral associated congenital birth defects and has proven to be a significant public health problem in organ transplant recipients as well as in individuals with AIDS. Accumulating evidence indicates that there may be a relationship between HCMV and the development of vascular disease. The interplay between HCMV and the immune system is a critical factor in the events leading to initiation of disease. Interestingly, HCMV encodes several genes that appear to modulate immune system function. One of these genes, termed US28, is homologous to the mammalian chemokine G-protein coupled receptors (GPCRs). While progress has recently been made in the identification of signaling pathways downstream of US28, very little is known regarding the molecular details proximal to US28 that contribute to the activation of signaling pathways. The objective of this proposal is to identify specific receptor properties and molecular events proximal to US28 that are critical for activation of known signal transduction pathways. We hypothesize that a detailed study of the molecular interactions of betaarrestin proteins with US28 will generate novel insights into the regulation of US28 signaling. The betaarrestins were originally identified in the context of termination of """"""""traditional"""""""" G-protein signaling to effectors such as adenylyl cyclases and phospholipases. This termination process, also known as desensitization, involves receptor phosphorylation by GRKs and binding of betarrestin proteins. It is now clear that the betaarrestins simultaneously function as adapter/scaffold molecules to recruit signaling proteins to receptors in order to activate additional """"""""non-traditional"""""""" signaling pathways. We have identified a US28 mutant that is unable to interact with betaarrestin and have generated a significant amount of preliminary data regarding signaling from this mutant. We propose to analyze the involvement of betaarrestin proteins in US28 signaling in order to gain a greater understanding of the proximal events leading to activation of US28 signaling pathways. To accomplish this goal, we propose three specific aims. First, we will characterize """"""""traditional"""""""" signaling from this US28 mutant in the context of HCMV infection and will analyze how this mutant affects the ability of US28 to stimulate smooth muscle cell migration. Second, we will analyze """"""""non-traditional"""""""" signaling pathways from US28 potentially mediated by betaarrestin recruitment to the US28 carboxy terminal regulatory domain. Third, we will analyze US28 signaling in cells deficient for betaarrestin expression in order to specifically define the involvement of betaarrestins. Our preliminary data indicate that US28 engages ?arrestin proteins suggesting that we will be able to make rapid progress toward our goal of defining the role of these important regulatory proteins in US28 signaling. These studies will identify mechanisms regulating US28 mediated signal transduction and will enhance our understanding of how US28 participates in pathogenesis and initiation of HCMV disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI058159-03
Application #
7188515
Study Section
Virology - B Study Section (VIRB)
Program Officer
Beisel, Christopher E
Project Start
2005-06-01
Project End
2010-02-28
Budget Start
2007-03-01
Budget End
2008-02-29
Support Year
3
Fiscal Year
2007
Total Cost
$254,706
Indirect Cost
Name
University of Cincinnati
Department
Genetics
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Wu, Shu-En; Miller, William E (2016) The HCMV US28 vGPCR induces potent G?q/PLC-? signaling in monocytes leading to increased adhesion to endothelial cells. Virology 497:233-243
Wu, Shu-En; Miller, William E (2015) The human cytomegalovirus lytic cycle is induced by 1,25-dihydroxyvitamin D3 in peripheral blood monocytes and in the THP-1 monocytic cell line. Virology 483:83-95
Bittencourt, Fabiola M; Wu, Shu-En; Bridges, James P et al. (2014) The M33 G protein-coupled receptor encoded by murine cytomegalovirus is dispensable for hematogenous dissemination but is required for growth within the salivary gland. J Virol 88:11811-24
Schneider, Olivia D; Millen, Scott H; Weiss, Alison A et al. (2012) Mechanistic insight into pertussis toxin and lectin signaling using T cells engineered to express a CD8?/CD3? chimeric receptor. Biochemistry 51:4126-37
Miller, William E; Zagorski, William A; Brenneman, Joanna D et al. (2012) US28 is a potent activator of phospholipase C during HCMV infection of clinically relevant target cells. PLoS One 7:e50524
Sherrill, Joseph D; Stropes, Melissa P; Schneider, Olivia D et al. (2009) Activation of intracellular signaling pathways by the murine cytomegalovirus G protein-coupled receptor M33 occurs via PLC-{beta}/PKC-dependent and -independent mechanisms. J Virol 83:8141-52
Stropes, Melissa P; Schneider, Olivia D; Zagorski, William A et al. (2009) The carboxy-terminal tail of human cytomegalovirus (HCMV) US28 regulates both chemokine-independent and chemokine-dependent signaling in HCMV-infected cells. J Virol 83:10016-27
Stropes, Melissa P M; Miller, William E (2008) Functional analysis of human cytomegalovirus pUS28 mutants in infected cells. J Gen Virol 89:97-105
Sherrill, Joseph D; Miller, William E (2008) Desensitization of herpesvirus-encoded G protein-coupled receptors. Life Sci 82:125-34
Sherrill, Joseph D; Miller, William E (2006) G protein-coupled receptor (GPCR) kinase 2 regulates agonist-independent Gq/11 signaling from the mouse cytomegalovirus GPCR M33. J Biol Chem 281:39796-805