Date

August 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Human Cytomegalovirus (HCMV) is a ubiquitous human pathogen that is associated with the development of numerous inflammatory diseases including vascular disease in solid allografts and certain forms of cancer. HCMV establishes life-long persistent/latent infections via nuanced manipulation of the host immune response. As such. HCMV encodes both chemokines and chemokine receptor homologs and is able to subvert the host chemokine-signaling network in infected cells and tissues. The pathological consequences of CMV chemokine mimicry are only beginning to be understood. In this dissertation, we investigate signaling from the HCMV-encoded chemokine receptor US28 in multiple HCMV susceptible cell types and identify a novel CMV-encoded chemokine. In Chapter 2, we demonstrate that US28 is a functionally selective chemokine receptor. Binding of CC-chemokines is pro-migratory when US28 is expressed in SMC and Fractalkine is an anti-migratory stimulus to SMC. Conversely, Fractaline stimulus is chemotactic to US28-expressing macrophages but CC-chemokines block US28-mediated migration of macrophages. We determine that US28 functional selectivity occurs at the level of G-protein coupling to the ligand-bound receptor. In Chapter 3, we identify the non-receptor protein tyrosine kinase Pyk2 as the critical cellular mediator of US28 singaling in SMC. We demonstrate that Pyk2 autophosphorylation is required for US28 pro-migratory singaling via activation of the GTPase RhoA. Moreover, we identify US28 singaling to Pyk2 as a potential mechanistic link between HCMV biology and the pathogenesis of malignant glioblastoma. We determine US28-specific Pyk2 binding partners in both SMC and U373 glioma cells. Importantly, Pyk2 binding partners are cell type-specific, providing further evidence that the functional consequences of US28 signaling are highly context-specific. The results presented herein significantly refine and extend our understanding of US28 as a functionally selective chenokine receptor and provide new mechanisms for the participation of HCMV in the pathogenesis of inflammatory diseases. In Chapter 4, I identify and characterize the RCMV gene product r129 as a functional CC-chemokine. I map the functional domains of r129 with respect to its chemotactic activity and determine that r129 targets immature CD4+ T cells in vitro. Moreover, I determine that r129 is expressed with late kinetics in RCMV infection and that r129 is incorporated into progeny virions. I construct and perform initial characterization of two recombinant RCMV strains containing critical mutations of the r129 ORF. These mutants will be important for future studies of RCMV r129 in the pathogenesis of inflammatory diseases and transplant vascular sclerosis.

Identifier

doi:10.6083/M4TQ5ZHQ

School

School of Medicine

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