The objectives of this study are to define how human cytomegalovirus (HCMV) infection upsets monocyte and endothelial cell functions and to elucidate the molecular mechanisms. The working hypothesis is that HCMV gene products, such as envelope glycoproteins, interact with cells of the monocyte/macrophage lineage and vascular endothelial cells and then subsequently disrupt the normal function of these cells. Monocytes and endothelial cells are two in vivo host cells for HCMV infection. Monocytes are responsible for protecting the body-from infections. Monocytes are responsible for protecting the body from infections and tumors through their release of cytokines, direct action towards invaders or aberrant cells, and interaction with T cells. Vascular endothelial cells are important angiogenic cells that physically form an integral vascular system and can secrete factors for vascularization or formation of extracellular matrix (ECM). To assess our hypothesis, we propose: (1) to continue to examine the effect of HCMV on the syntheses of monokines and cell adhesion molecules in monocytes. We shall determine the effect of HCMV and viral glycoprotein gB on monocyte immune mediator release by measuring the changes in cytokine and adhesion receptor expression; (2) to examine the direct or indirect effect of HCMV and ciral glycoprotein gB on endothelial cell function. We shall (i) measure the changes in cytokines, ECM, and adhesion molecules syntheses in infected or gB-treated human umbilical vein endothelial (HUVE) cells; these include the activation of VCAM-l, ICAM-1, and E-selectin expression in HUVEs treated with factors or monokines released from HCMV or gB-treated monocytes, (ii) determine endothelial cell function by measuring the endothelial leakage and IF staining of Plakoglobin, (iii) compare virus-infected HUVE cells with HUVE cells treated with culture fluid from HCMV-infected or gB- activated monocytes for their cytokine and ECM expression; (3) isolate and clone the ECMV gB receptor(s) on monocytes for studying its involvement in signaling. We will isolate the HCMV gB receptor by: (i) antibody-affinity chromatography; (ii) the use of a recombinant vaccinia virus-based screening system or (iii) using HCMV gB affinity chromatography; (4) to define the receptor-ligand mediated signal process by which viral binding affects cellular transcription factors and monokine expression. We will use gB M a mode ligand to: (i) examine the role of Ras and its related family members in the beginning stages of the signaling cascade; (ii) investigate the MAPK family pathways that are involved in gB-mediated signaling; and (iii) examine the role of the TRAFs, PKA, or other signaling molecules that result in NF-kappaB activation and subsequent monokine expression.
These aims are designed to characterize the effect of HCMV on monocyte and endothelial cell functions under conditions that mimic in vivo conditions. These results will provide a comprehensive understanding of HCMV infection on cell types believed to be crucial for inflammatory responses and virus dissemination in humans.
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