Chimeric proteins composed of Jun and CREB define domains required for interaction with the human T-cell leukemia virus type 1 Tax protein.

The regulation of human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat gene expression is dependent on three cis-acting elements known as 21-bp repeats and the transactivator protein Tax. Mutagenesis has demonstrated that sequences in each of the 21-bp repeats can be divided into three domains designated A, B, and C. Tax stimulates the binding of CREB to the B domain, which is essential for Tax activation of HTLV-1 gene expression. In this study, we demonstrate that Tax will stimulate the binding of CREB to the HTLV-1 21-bp repeats but does not stimulate CREB binding to the consensus cyclic AMP response element (CRE) element found in the somatostatin promoter. However, Tax stimulates CREB binding to a consensus CRE in the context of the 21-bp repeats, indicating the importance of these sequences in stimulating CREB binding. To determine the mechanism by which Tax stimulates CREB binding and determine potential interactions between Tax and CREB, we used the mammalian two-hybrid system in conjunction with in vitro binding and gel retardation assays. Two-hybrid analysis indicated that mutations in either the basic or leucine zipper region of CREB prevented interactions with Tax. Since several studies have demonstrated that Tax will also stimulate the binding of a variety of different basic region-leucine zipper proteins to their cognate binding sites, we assayed whether chimeric proteins composed of portions of CREB and another basic region-leucine zipper protein, Jun, could be used to map domains required for interactions with Tax. These studies were possible because we did not detect in vivo or in vitro interactions between Tax and Jun. The amino acid sequence of the CREB basic region and a portion of its leucine zipper were required for both in vivo and in vitro interactions with Tax and increased binding of CREB to the 21-bp repeats in response to Tax. These studies define the domains in CREB required for both in vivo and in vitro interactions by the HTLV-1 Tax protein.     

Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I

We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor (CREB)-like factor(s). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element.