Abstract
The human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive T-cell malignancy in humans. While the virus appears to maintain a state of latency in most infected cells, high level virion production is an essential step in the HTLV-I life cycle. The virally-encoded Tax protein, a potent activator of gene expression, is believed to control the switch from latency to replication. Tax stimulation of HTLV-I transcription is mediated through cellular activating transcription factor/cAMP response element binding proteins, which bind the three 21-base pair (bp) repeat viral enhancer elements. In this report, we show that viral latency may result from a highly unstable interaction between CREB and the HTLV-I 21-bp repeats, resulting in rapid dissociation of CREB from the viral promoter. In the presence Tax, the dissociation rate of CREB from a 21-bp repeat element is decreased. This stabilization is highly specific, requiring the amino-terminal region of CREB and appropriate 21-bp repeat sequences. We suggest that Tax stabilization of CREB binding to the viral promoter leads to an increase in gene expression, possibly providing the switch from latency to high level replication of the virus.
Highlights
We show that viral latency may result from a highly unstable interaction between CREB and the human T-cell leukemia virus type I (HTLV-I) 21-bp repeats, resulting in rapid dissociation of CREB from the viral promoter
For th e ini ti al ex perime nt, we perform ed a n equilibrium binding assay to exa mine CREB binding affini ty to a conse ns us cAMP response element (CRE) sequence from t he human chorioni c gonadotropin gene promoter (Fig. lA ). In this expe rime nt, the a mount of lab eled CRE DNA was kep t consta nt, a nd purified recomb ina nt CREB protein was varied over a IOO-fold concentration ra nge
Th ese da t a essentia lly exclu de the possi bili ty that Tax transactivati on occurs th roug h direct interaction of Ta x with t he bound ATF/CREB prot eins at the promot er, as t he hig h a ffinity consens us CRE sh ould bin d mor e CREB and t ether more Tax, r esulting in h igher levels of transcriptional a cti va t ion. These da t a suggest that enh ancemen t in DNA binding activity only pa rti all y expla ins Tax transactivation, as we observed a significant increase in the affinity of both CREB and ATF-2 for the consensus CRE sequence, yet the consensus CRE was ineffective in mediating Tax transactivation
Summary
Several recent studies provided evidence suggesting that Tax stimulates viral transcription through enhancement in the DNA binding of cellular activator proteins, including CREB and ATF-2, to the 21-bp repeats [22,23,24, 26, 29, 30]. To further characterize the mechanism of Tax transactivation, we used equilibrium binding and dissociation kinetics to study the interaction ofCREB and ATF-2 with the 21-bp repeat sequences both in the presence and absence of Tax. We demonstrate in this report that the three 21-bp repeats represent lower affinity CRE sequences, consistent with a possible role for these elements in maintaining viral latency. This intera ction results in significa nt stabiliza tion of CREB binding to th e 21-bp repeat seque nces followed by t ra nscriptiona l activa tion of t he HTLV-I genome
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