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 ter minal repeat gene expression is dependent on three cis-acting elements known as 21-bp repeats and the transactivator protein Tax. Mutagenesi s 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 wil l stimulate the binding of CREB to the HTLV-1 21-bp repeats but does n ot stimulate CREB binding to the consensus cyclic AMP response element (CRE) element found in the somatostatin promoter. However, Tax stimul ates CREB binding to a consensus CRE in the context of the 21-bp repea ts, indicating the importance of these sequences in stimulating CREB b inding. To determine the mechanism by which Tax stimulates CREB bindin g and determine potential interactions between Tax and CREB, we used t he mammalian two-hybrid system in conjunction with in vitro binding an d gel retardation assays. Two-hybrid analysis indicated that mutations in either the basic or leucine zipper region of CREB prevented intera ctions with Tax. Since several studies have demonstrated that Tax will also stimulate the binding of a variety of different basic region-leu cine zipper proteins to their cognate binding sites, we assayed whethe r chimeric proteins composed of portions of CREB and another basic reg ion-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 leuc ine zipper were required for both in vivo and in vitro interactions wi th 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.