Yin M J, Paulssen E, Seeler J, Gaynor R B
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA.
J Virol. 1995 Oct;69(10):6209-18. doi: 10.1128/JVI.69.10.6209-6218.1995.
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.
人类嗜T淋巴细胞病毒1型（HTLV-1）长末端重复序列基因表达的调控依赖于三个顺式作用元件，即21bp重复序列以及反式激活蛋白Tax。诱变实验表明，每个21bp重复序列中的序列可分为三个结构域，分别命名为A、B和C。Tax可刺激CREB与B结构域结合，这对于Tax激活HTLV-1基因表达至关重要。在本研究中，我们证明Tax可刺激CREB与HTLV-1的21bp重复序列结合，但不会刺激CREB与生长抑素启动子中发现的共有环磷酸腺苷反应元件（CRE）结合。然而，Tax可在21bp重复序列的背景下刺激CREB与共有CRE结合，这表明这些序列在刺激CREB结合方面的重要性。为了确定Tax刺激CREB结合的机制，并确定Tax与CREB之间潜在的相互作用，我们将哺乳动物双杂交系统与体外结合和凝胶阻滞分析相结合。双杂交分析表明，CREB的碱性或亮氨酸拉链区域发生突变会阻止其与Tax相互作用。由于多项研究表明Tax还可刺激多种不同的碱性区域-亮氨酸拉链蛋白与其同源结合位点结合，我们检测了由CREB部分序列和另一种碱性区域-亮氨酸拉链蛋白Jun组成的嵌合蛋白是否可用于定位与Tax相互作用所需的结构域。这些研究之所以可行，是因为我们未检测到Tax与Jun在体内或体外的相互作用。CREB碱性区域的氨基酸序列及其亮氨酸拉链的一部分对于与Tax的体内和体外相互作用以及Tax诱导的CREB与21bp重复序列结合增加都是必需的。这些研究确定了HTLV-1 Tax蛋白在体内和体外相互作用所需的CREB结构域。
