The development of vascular diseases such as atherosclerosis, arterial restenosis following angioplasty, and solid organ transplant vascular sclerosis are chronic inflammatory disease processes that involve multiple factors. Infectious agents such as human cytomegalovirus (HCMV), which chronically persists in individuals following primary infection, have been implicated in the acceleration of these vascular diseases although the mechanism has remained elusive. Recently, we have reported that HCMV infection of arterial smooth muscle cells (SMC) results in cellular migration due to the expression of a virally encoded G protein coupled receptor (US28). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemo kinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. Therefore, the primary focus of this project is to elucidate the mechanism(s) involved in US28 induced SMC migration and develop an animal model to test the contribution of vitally encoded chemokines in this process. We plan to accomplish these goals in the following specific aims. First, we will identify the structural domains of US28 that mediate SMC migration by generating chimeras between the viral GPCR and CCR5, which cannot induce cell movement. We will also identify the protein tyrosine kinases (PTKs), which are stimulated during the US28 signaling process as a marker for SMC activation. Lastly, in this specific aim we will determine whether all chemokines, which bind US28 induce SMC migration and whether some of these ligands can act as antagonists of GPCR activity. In the second specific aim, we will characterize another Viral GPCR (m33), which is encoded within murine cytomegalovirus (MCMV) and induces mouse SMC movement. We will utilize an infectious MCMV BAC clone and shuffle mutagenesis system to identify m33 structural domains that are key to SMC migration. In addition we will identify the PTKs stimulated during m33 stimulation. In the last specific aim, we will examine the contribution of m33 induced SMC migration in the generation of atherosclerosis in ApoE and ApoE/CCR2 knockout mice. The wild type and mutant MCMV BAC clones derived above will be used to infect mice in this part of the project. These experiments will determine the role of m33 in the development of vascular disease and the structural domains of m33, which mediate this process. Completion of these experiments will provide a molecular basis for the role of HCMV in the acceleration of vascular disease as well as provide an animal model to test future intervention therapies.
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