Human cytomegalovirus (HCMV) infection is one of the most important opportunistic infections in patients with AIDS, and has been implicated as a cofactor in the progression of HIV-1 disease. Epidemiological evidence has identified HCMV as a possible factor or cofactor in diseases involving abnormal cell growth like restenosis and cervical carcinoma. Despite the recent advance in antiretroviral therapy, the incidence of HIV-associated malignancies has not decreased, such as invasive cervical cancer. Because of the oncogenic potential of HCMV as demonstrated by many in vitro studies and its ubiquity in human malignant tissues and patients with AIDS, this study will explore further and delineate the interactions between cellular and viral factors that may lead to the activation of host DNA replication machinery and, potentially, uncontrolled cellular proliferation, particularly in semi-permissive or nonproductive HCMV infection. Specific attention will be devoted to elucidating how the HCMV immediate-early (IE) protein, IE1-72, interacts with the cellular p107 and p53 proteins to activate host genes involved in DNA replication, while preventing the activation of the cellular apoptotic response. Our working hypotheses are: (a) that upon infection IE1-72 interacts with P107 and its associated E2F and cyclin/cdk complexes to activate E2F responsive promoters and cyclin/cdk kinase, respectively, to induce expression of genes associated with cellular proliferation; and (b) that IE1-72 also acts to prevent p53 activation and subsequent p53-mediated apoptosis via its interaction with p53. To support these hypotheses, we propose the following specific aims: (l) To further characterize the interactions between IE1-72 and P107. Deletion and point mutants of both proteins and viruses with point or deletion mutation in IE1- 72 will be constructed and used to access the domains necessary for interaction. (2) To describe the mechanism by which IE1-72 binding to the p107-E2F complex results in the alleviation of p107-mediated transcriptional repression function. The dissociation and/or activation of the p107-cyclin/cdk complexes following IE1-72 binding will be examined to determine if this leads to the alleviation of the p107-mediated growth suppression. (3) To understand the consequences of the interaction between p53 and IE1-72, and to study the effect of IE1-72 on the TGF-beta1 and p53 signal transduction pathways through the use of cell lines constitutively expressing IE1-72 and cell lines lacking p53. The domains on each protein necessary for interaction and the role this interaction plays in the preventing cellular apoptosis will be studied. The functional significance of the IE1-72 and p53 interaction on p53-responsive genes like p21 and mdm2 will be investigated to determine whether IE1-72 expression also has a transcriptionally repressive effect. These studies should provide us with a clearer picture of HCMV's role in cell cycle aberrations and, potentially, oncogenesis.
