The human T-cell leukemia virus type I (HTLV-I) is a retrovirus that is the causative agent of a variety of clinical disorders, including an aggressive and fatal T-cell leukemia. Malignant transformation associated with HTLV-I infection is strongly linked to the synthesis of the vitally encoded transcription factor Tax. The Tax protein is critical to the life cycle of the virus, as it plays a major role in the transition from viral latency to high-level virion production. Tax mediates this transition via strong activation of HTLV-I transcription, which leads to efficient replication of the virus. Viral gene expression is stimulated following the formation of a stable promoter-bound complex containing Tax, the cellular transcription factor CREB and the cellular coactivators CBP/p300. Much of our current understanding of Tax- activated HTLV-l transcription has come from in vitro binding and transient transfection assays, neither of which directly assess physiological relevance of the interactions under study. In this application, we propose to explore the transcriptional regulation of the natural, chromosomally integrated proviruses in HTLV-I infected T-cells. These studies will investigate the dynamic interaction between Tax, cellular transcription factors, coactivators and corepressors at the HTLV-I promoter in living cells. We will also characterize the kinetics of transcription complex assembly, transcriptional activation and RNA synthesis, following Tax induction in human T-cells. Similar studies will also be carried out on chromatin templates in vitro. These experimental approaches are designed to mimic to further our understanding of transcriptional regulation of HTLV-I. We also propose to characterize the effects of rationally designed polyamides on HTLV-I transcription and Tax function both in vivo and in vitro. We have a comprehensive set of polyamides that should enable inhibition of Tax binding specifically at each viral CRE. This will allow dissection of the role of each viral CRE in Tax-transactivation. The long-term goal of this study will be the synthesis and characterization of specific, high affinity polyamides that will block Tax function in the cell. Together these studies provide a diverse, integrated approach to the study of Tax function, both in vitro and in vivo. Our studies should yield a greater understanding of the multiple mechanisms of Tax transcriptional activation in the HTLV-I-infected T-cell.
