Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and a serious problem for immunosuppressed individuals. It also may be a risk factor for the development of vascular disease. The multiple pathogenic effects of HCMV are likely manifested through a complex interplay of viral gene products and induced and repressed cellular functions. HCMV immediate early (IE) and early gene expression clearly play a pivotal role in this scheme. A major part of our research has been directed towards determining the cis-acting regulatory elements and the cellular and viral factors involved in the regulation of HCMV early gene expression, with primary focus on the functional properties of the major viral transactivator of these genes, IE2 86. We have developed a way to rapidly generate viruses with mutations in IE2 86, both viable and nonviable, and have gained significant insight into the multiple functions of IE2 86. We also are obtaining further understanding of the cell's molecular machinery from the perspective of how the virus utilizes the machinery for its own needs. In this renewal application, we propose to continue our studies on IE2 86 function and on the cellular and viral factors governing the expression of HCMV and cellular genes. We are now in an excellent position to elucidate the role of IE2 86 in the infection and determine the molecular mechanisms governing IE2 86 functions and its role in pathogenesis. The approach is to couple functional in vivo genetic analyses with biochemical and molecular assays to achieve the following aims: 1) identification of the viral and cellular proteins that interact with IE2 86;2) determination of the molecular mechanisms that are the basis of IE2 86 functions in the context of the viral genome;and 3) analysis of the functions of IE2 86 in endothelial cells, with focus on selected cellular processes that likely play a role in creating a procoagulant and proinflammatory environment. The long range goals of this research are to define at the molecular level the mechanisms which operate to control HCMV gene expression, thus providing an important foundation for understanding how the complex interactions of viral and host functions relate to the pathogenesis of the virus.
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