Yu L, Zhang Z, Loewenstein P M, Desai K, Tang Q, Mao D, Symington J S, Green M
Institute for Molecular Virology, Saint Louis University School of Medicine, Missouri 63110, USA.
J Virol. 1995 May;69(5):3007-16. doi: 10.1128/JVI.69.5.3007-3016.1995.
The mechanism by which human immunodeficiency virus type 1 Tat transactivates the long terminal repeat promoter is not understood. It is generally believed that Tat has one or more transcription factors as its cellular target. One might expect a cellular target for Tat to possess several properties, including (i) the ability to bind to the Tat activation region, (ii) the possession of a transcriptional activation domain, and (iii) the ability to contact the cellular transcription machinery. Here we describe the cloning, expression, and characterization of a human protein, termed TAP (Tat-associated protein), which possesses some of these properties. TAP is highly conserved in eukaryotes and is expressed in a variety of human tissues. The major intracellular species of TAP is a highly acidic 209-amino-acid protein that likely is formed by removal of a highly basic 70-amino-acid N-terminal segment from a primary translation product. By deletion analysis, we have identified a TAP C-terminal region rich in acidic amino acids and leucine residues which acts as a strong transcriptional activator when bound through GAL4 sites upstream of the core long terminal repeat promoter, as well as flanking sequences that mask the activation function. Amino acid substitution of two leucine residues within the core activation region results in loss of the TAP activation function. Two lines of evidence suggest that Tat interacts with TAP in vivo. First, promoter-bound Tat can recruit a TAP/VP16 fusion protein to the promoter. Second, transiently expressed Tat is found associated with endogenous TAP, as demonstrated by coimmuno-precipitation analysis. As shown in an accompanying report, the TAP activation region binds the Tat core activation region and general transcription factor TFIIB (L. Yu, P.M. Loewenstein, Z. Zhang, and M. Green, J. Virol. 69:3017-3023, 1995). These combined results suggest the hypothesis that TAP may function as a coactivator that bridges Tat to the general transcription machinery of the cell via TFIIB.
1型人类免疫缺陷病毒（HIV-1）的反式激活因子Tat激活长末端重复序列启动子的机制尚不清楚。一般认为，Tat以一种或多种转录因子作为其细胞靶点。人们可能期望Tat的细胞靶点具有多种特性，包括：（i）与Tat激活区域结合的能力；（ii）拥有转录激活结构域；（iii）与细胞转录机制接触的能力。在此，我们描述了一种被称为TAP（Tat相关蛋白）的人类蛋白的克隆、表达及特性，该蛋白具有其中一些特性。TAP在真核生物中高度保守，且在多种人类组织中表达。TAP的主要细胞内形式是一种高度酸性的209个氨基酸的蛋白，它可能是由初级翻译产物中一个高度碱性的70个氨基酸的N端片段去除后形成的。通过缺失分析，我们确定了一个富含酸性氨基酸和亮氨酸残基的TAP C端区域，当通过核心长末端重复序列启动子上游的GAL4位点结合时，该区域可作为一个强大的转录激活因子，同时还有掩盖激活功能的侧翼序列。核心激活区域内两个亮氨酸残基的氨基酸替代导致TAP激活功能丧失。有两条证据表明Tat在体内与TAP相互作用。首先，与启动子结合的Tat可将TAP/VP16融合蛋白招募至启动子。其次，共免疫沉淀分析表明，瞬时表达的Tat与内源性TAP相关。如一篇附带报告所示，TAP激活区域与Tat核心激活区域及通用转录因子TFIIB结合（L. Yu、P.M. Loewenstein、Z. Zhang和M. Green，《病毒学杂志》69:3017 - 3023，1995）。这些综合结果提示了一个假说，即TAP可能作为一种辅激活因子，通过TFIIB将Tat与细胞的通用转录机制连接起来。
