Human cytomegalovirus (HCMV) is a significant pathogen in immuno- compromised individuals. HCMV is relevant to the oral environment for a number of reasons. The salivary glands are an important site of latent HCMV infection; salivary transmission of virus is one of the major routes of viral spread. HCMV infections of the gingiva and oral mucosa are becoming more prevalent with the increase in HIV infections and organ transplantation. Intraoral Kaposi's sarcoma, a malignancy composed of endothelial cells, is frequently found in association with HCMV, but causal relationships have not been established. An understanding of the basic processes in viral infection is the first step toward future therapeutic intervention. Upon infection, HCMV induces a cascade of gene expression changes in both viral and cellular genes, beginning with the immediate early (IE) viral genes. The ability of the IE proteins to activate a number of cellular genes is well documented. Activation of specific cellular genes in particular cell types may be involved in HCMV replication, latency, permissiveness, and transformation. The actual mechanisms of activation are not known. One of the ways HCMV IE proteins may activate cellular genes is by interacting with cellular transcription factors. Interaction with transcription factors may be directly, or indirectly through other cellular proteins. The experiments proposed in this study will investigate this mechanism through the following specific aims: 1. Identification of endothelial cell proteins that interact with the HCMV 72 kDa IE1 protein. Methods used to identify the cellular proteins include glutathione affinity and immunoprecipitation of 72 kDa lE1/cellular protein complexes, and the yeast two-hybrid system in which protein:protein interactions are detected by association of GAL4 DNA binding and activation domains in fusion proteins from an endothelial cell library and the 72 kDa IE1 protein. 2. Determination of critical regions of the HCMV 72 kDa IE1 protein necessary for interaction with cellular proteins. Once endothelial proteins have been identified that bind wild type 72 kDa IE1 protein, deletion mutations will be made in the IE1 coding region and mutant proteins will be tested in the yeast system for binding. Point mutations will be introduced to further determine critical amino acids.
