The long-term objectives of this study are to define how human cytomegalovirus(HCMV) infection upsets endothelial cell functions and to elucidate the molecular mechanisms involved. Vascular endothelial cells, one of in vivo host cells for HCMV, are a group of important angiogenic cells that form an integral vascular system, and secrete factors for vascularization and formation of the extracellular matrix. HCMV infection is associated with several cardiovascular diseases, such as atherosclerosis and coronary restenosis. In the past funding period we identified that HCMV uses the epidermal growth factor receptor (EGFR) as a primary cellular receptor for viral entry and signaling. Therefore, in this study we hypothesize that HCMV infection of human umbilical vein endothelial (HUVE) cells is EGFR dependent and that HCMV glycoproteins and immediate-early proteins play major roles in initiating viral pathogenesis by interacting with its surface receptors and cell cycle regulating proteins, respectively. To assess our hypothesis, we propose: (1) To investigate whether HCMV infection of HUVE cells is EGFR dependent. The status of EGFR on cell surfaces of HUVE, HEL, and breast cancer cells, and the consequences of HCMV infection will be studied to determine the role of EGFR in cell susceptibility to HCMV infection, (2) To define the interacting domains of HCMV gB that bind to EGFR. The truncated, site-directed point mutant, and tryptic-digested gB fragments will be expressed and purified for interaction studies in CHO cells transfected with erbB1 and erbB3. We will use also the phage display approach to identify the domains of gB that bind EGFRs. The gB mutant virus will be developed by using the bacterial artificial chromosome (BAG) technique to study the receptor-ligand interaction in the context of viruses, (3) To determine the interaction domains on EGFR that serve as receptor domains for gB binding. Truncated and site-directed mutants of erbB1 molecules will be expressed on EBFR(-) CHO or MB453 cells and the phage display approach will be employed to identify the active interaction domain(s) on EGFR, and (4) To search for anti-HCMV agents targeting viral binding and entry. Compounds targeting EGFR kinase activity, EGFR degradation, or mimicking the active domain(s) of cellular receptor will be studied and developed. Studies proposed in this project would provide a comprehensive understanding of how HCMV infects cell types crucial for angiogenesis. Thus, potential anti-HCMV agents that target viral entry and signaling can be developed.
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