REGULATION OF VIRAL AND CELLULAR GENE EXPRESSON

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Human T-cell lymphotropic virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). The Tax protein of HTLV-I is a 40 kDa transcriptional activator which is critical for HTLV-I gene regulation and viral-induced cellular transformation. Through interaction with cellular transcription factors, Tax1 regulates the level of viral and cellular gene expression. Tax is localized to the DNA through its interaction with the site-specific activators CREB, NF-kappaB and SRF. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. Based on several independent assays, we have demonstrated a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35 kD (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized point mutants of Tax, M32 and M41, shown to be defective in Tax-activated transcription, were unable to interact with TFIIA in this assay. Second, using a GST affinity binding assay, the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, in HTLV-I-infected human T-lymphocytes, Tax and TFIIA were found to associate in a coimmunoprecipitation assay. Finally, TFIIA was observed to influence the ability of Tax to activate transcription both in vivo and in vitro. In vitro transcription studies showed significantly reduced levels of Tax transactivation when using TFIIA-depleted cell extracts. In addition, transfection of human T-lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-I-LTR CAT reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation. In a separate study, a Jurkat T-cell cDNA library was screened for proteins which interact with the HTLV-I Tax protein. These studies have identified and characterized a lymphocyte specific Tax1 binding protein, TRX. TRX interacts with the cyclin B/cdc2 complex in a cell cycle dependent manner, suggesting a role in cell cycle regulation.