A role has been hypothesized for human cytomegalovirus (HCMV) in the etiology and pathogenesis of many different diseases. In vivo, HCMV has been shown to infect leukocytes, especially monocyte/macrophages and these have been proposed as a reservoir for HCMV persistence. Viral gene expression in peripheral blood monocytes is limited to the immediate early (IE) genes, whereas infiltrating monocytes have been observed to have a more complete expression of HCMV genes in patients with disseminated HCMV disease. The activation of monocytes during infiltration apparently makes these cells amenable to HCMV replication. The initiating event of this process is monocyte adhesion ot extracellular matrix (ECM) or cell substrates. Recent work has demonstrated that the degree and type of monocytic activation response is dependent on the substrate to which the monocyte initially binds making adhesion the key regulatory step in directing monocyte function. We hypothesize that HCMV infection (or IE expression) of monocytes during this early stage (0 to 8 hr after adhesion) will greatly alter normal monocyte activation and function. Furthermore, adhesion to specific matrices could, alone or with secondary signals influence viral gene induction and replication. Therefore, it is necessary to understand the involvement HCMV infection on normal monocyte function and activation, and to define the effect(s) that cell adhesion has on HCMV gene expression. This proposal is designed to address these issues with the following specific aims; (1) Define the susceptibility, expression pattern of HCMV genes and the cellular consequences of infection on monocytes under the influence of ECM or activating stimuli. Monocytes will be activation by adhesion alone (to fibronectin, collagen or endothelial cells), or in combination with chemical stimulation (PMA, LPS, or sodium butyrate) to identify conditions amenable to virus replication. (2) Determine the extent of IE1 and IE2 function in monocytes under normal and activating conditions using transfection functional and peptide mapping (structural) assays.
This aim should provide insight into why the viral IE products do not stimulate later virus gene expression and replication under most conditions. (3) Examine the changes in monocyte-mediated immune responses following HCMV infection and cell activation. Immune functions to be assayed include cytokine production, alterations in surface markers an cytoskeleton and adhesion (in infections of unadhered monocytes) to cellular substrates (endothelial cells).
These aims are designed to fully characterize the effect of HCMV on monocytes under laboratory conditions which closely model in vivo activating conditions. This will provide a comprehensive understanding of HCMV infections on cell types associated with inflammation, including abnormal cytokine expression in monocytes, and virus dissemination in humans.
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