T lymphocytes and mononuclear phagocytes are the primary host cells of HIV infection. Replication of HIV in these cells can be profoundly influenced by a variety of factors, including cytokines and viral regulatory proteins. A critical regulatory point in the virus life cycle is activation of HIV-transcription by the viral transcriptional activator Tat. The ability to inhibit a cellular factor which mediates Tat function is therefore an attractive target for antiviral strategies. A cellular protein kinase, TAK, has been identified in their laboratory that specifically associates with the activation domain of Tat and is likely to mediate its transcriptional stimulatory function. TAK phosphorylates the largest subunit of RNA polymerase II in a manner that is believed to provide critical regulatory control of transcription. Recent results indicate that TAK activity is induced in cells relevant to HIV infection - peripheral blood mononuclear cells and monocytic cell lines - by agents which are known to stimulate HIV gene expression. This application will investigate the effects of factors that activate or induce cellular differentiation of T lymphocytes and mononuclear macrophages on the activity of TAK. The hypothesis to be tested here is that TAK activity is regulated by factors that stimulate immune function and/or cellular differentiation and that TAK is a mediator of the transcriptional response of HIV to these factors. Activation of TAK may prime T lymphocytes and macrophages for efficient viral replication following initial infection of cells and/or it may play a role in the reactivation of viral gene expression from latency.
The specific aims of this proposal are: (1) the identification of cytokines, mitogens or other agents that regulate TAK activity and determination of cell type(s) in which regulation occurs, (2) an investigator of signal transduction pathways involved in regulation of TAK activity and (3) an analysis of molecular mechanisms involved in regulation of TAK activity. This analysis will be conducted both in primary monocytes and T lymphocytes as well as established cell lines.
These aims will be accomplished by the use of kinase assays, molecular biology techniques (transfections, CAT assays, RNA analysis, and nuclear run-on transcription assays), immunological techniques (isolation and activation of primary blood cells, immunoblotting, immunoprecipitation, and ELISA), and virological techniques (HIV infection). Long term goals of this project include an understanding of the normal function of TAK in uninfected cells,the regulation of TAK during disease progression of HIV-infected individuals and the role of TAK in AIDS pathogeneis, and the development of specific inhibitors of TAK activity. If the proposed hypothesis is confirmed, it will indicate that TAK plays a critical role in the life cycle of HIV and is therefore an attractive target for therapeutic intervention.
