Abstract
The human T-cell leukemia virus type I (HTLV-I)-encoded transcriptional activator protein Tax is strongly implicated in HTLV-I pathogenesis. Tax regulates HTLV-I gene expression through three 21-base pair (bp) repeat enhancer elements located in the transcriptional control region of the virus. Tax does not bind these elements directly, but mediates transactivation through the cellular transcription factors that recognize a cAMP response element (CRE)-like sequence centered within each of the 21-bp repeats. In this report, we identify activating transcription factor-2 (ATF-2) and CRE-binding protein (CREB) as the principal T-cell proteins that bind the three 21-bp repeats in vitro. Purified Tax protein augments the level of RNA synthesis induced by ATF-2 and CREB in a cell-free transcription assay, providing evidence that Tax cooperates with these cellular proteins to activate HTLV-I transcription. Furthermore, Tax dramatically increases the binding of both the T-cell-derived and recombinant forms of ATF-2 and CREB to each of the 21-bp repeats. The target sequences for this enhancement reside within the DNA binding/dimerization domains of these proteins. These data suggest that Tax transactivates HTLV-I gene expression by increasing the number of bound ATF-2 and CREB molecules at the viral promoter.
Highlights
The cellular transcription factors that recogniczAeMaP Altman et a l . , 1988)
We show that ATF-2 and CREB are present as significant components in protein preparations obtained from cultured human T-lymphocytes by DNA affinity chromatography using a 21-bp repeat
The evidence supporting thisconclusion includes the following. 1)The mobilities of the two major affinity-purified T-cell proteins on SDS-PAGE correlate with the mobilities ofATF-2 and CREB. 2) The66-kDa T-cell protein is identified by the anti-ATF-2 antibody, and the43-kDa T-cell protein is identified by the anti-CREB antibody
Summary
Lymphocyte Cell Culture, Nuclear Extract Prepamtion, a n d T-cell Protein Purification-The HTLV-I-negative CEM T-lymphocytecell line was grown in spinner culturaet 37 "C in Iscove's medium supplemented with 5% fetal calf serum. Purified by gel filtration chromatography (Matthews et al, 1992), ali- GTCTCCCCA-3'; HTLV-I 2nd repeat (site 2). Reactions contained Taxor a n equivalent volumeof Superdex buffer (withmM4 fresh P-mercaptoethanol) to givaefinal concentrationof 12.5 mM HEPES, pH proteins that bind the HTLV-I 21-bp repeats (Nyborg and Dynan, 1990). These proteins were obtained from the uninfeded human T-lymphocyte cell line CEM by threerounds of sequence-specific DNA affinity chromatography using thesecond.
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