The purpose of the studies described in this proposal is to gain a fuller understanding of the mechanisms involved in regulation of human T cell leukemia virus (HTLV-I) gene expression. In addition to structural proteins, HTLV-I encodes several proteins which regulate viral gene expression at the level of transcription. One of these proteins, Tax1 is a 40 kD positive transactivator of viral gene expression. Taxi is unique from many other transcriptional regulators in that it appears to be incapable of binding directly to DNA. However, Tax1 can associate indirectly with DNA via a cellular transcription factor. This mechanism of indirect association with DNA is reminiscent of several other transcription factors including adenovirus ElA and herpesvirus VP16. These properties, as well as the absence of a classic acidic activating domain, suggest that Taxi may interact with a transcriptional coactivator/adaptor(s). In the proposed studies, the molecular mechanisms of transcription activation by Tax1 will be investigated with particular interest in possible interactions with cellular transcription factors. (i) The activation domain of Tax1, will be defined by constructing chimeric proteins containing a known DNA binding domain (GAl4, aa 1-147) fused to the Tax1, protein or various Tax1, mutants. These fusion proteins will be tested for their ability to transactivate LTR-CAT reporter constructs in which specific Taxi responsive elements have been replaced with GAL4 binding sites. (ii) Cellular proteins capable of interacting with Tax1 will be isolated and studied to determine if they play a specific role in Tax1 transactivation. (iii) An in vitro transcription system will be developed with which to study the mechanism of Tax1 transactivation. (iv) The crystal structure of Tax1 will be determinedin order to associate functional domains with specific structural features of the protein. Since Tax1 does not possess any strong homologies with known transcription activating domains, it is anideal candidate for revealing new structures which mediate these functions. In general, the insights generated by these studies will provide a more complete understanding of the mechanisms of eukaryotic gene regulation. Specifically, the results will strengthen our knowledge of HTLV-I latency and transformation, possibly resulting in therapeutic advances in treatment of HTLV-I associated diseases including adult T-cell leukemia and tropical spastic paraparesis-HTLV-l associated myelopathy.
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