BIOCHEMICAL MECHANISM OF TRANS-ACTIVATION IN HTLV-I

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Human T-lymphotropic retrovirus type I (HTLV-I) is the prototype of a group of retroviruses (HTLV-I, HTLV-II, STLV, BLV) that encode regulatory proteins to modulate viral mRNA synthesis and utilization. The 40 kDa nuclear protein, Tax, encoded by the 3' region of HTLV-I genome is a transcriptional activator. The ability of tax to alter gene expression in HTLV-I infected cells appears to be causally related to HTLV-I pathogenesis. Tax activates transcription from three 21 bp repeats in the U3 region of the viral long terminal repeat (LTR). It also activates transcription from enhancer/promoters containing the NF-kB binding sites. Saturation mutagenesis of the 21 bp repeat indicates that the key element mediating tax response lies in a TGACGT motif homologous to the cAMP responsive element (CRE) found in the transcriptional regulatory region of many viral and cellular genes. Research efforts over the past two years have culminated in the identification of cAMP responsive element binding protein, CREB, as one of the primary targets of tax. Via protein-protein interaction, Tax forms a complex with CREB and enhances its binding to the HTLV-I 21 bp repeats. Furthermore, CREB exists both as a homodimer and a heterodimer in complex with a distinct CRE binding protein tentatively called CAF (CREB associated factor). Both CREB homodimer and CREB/CAF heterodimer interact with Tax. These findings provide the foundation for studying the mechanism of tax action. This proposal seeks to elucidate the protein-protein and protein-DNA interactions that mediate transcriptional activation by HTLV-I tax. Special emphasis will be directed towards using purified components including Tax, CREB, CAF, and the basal transcriptional factors to dissect the mechanism of Tax action. The reagents and experimental approaches developed for HTLV-I Tax will then be extended to the study of bovine leukemia virus Tax protein. Finally, the mechanism of tax activation from NF-kB binding sites will also be examined.