Kamine J, Elangovan B, Subramanian T, Coleman D, Chinnadurai G
Institute for Molecular Virology, St. Louis University School of Medicine, Missouri 63110, USA.
Virology. 1996 Feb 15;216(2):357-66. doi: 10.1006/viro.1996.0071.
The Tat protein of the human immunodeficiency virus (HIV) is a powerful activator of HIV gene expression. Genetic and biochemical evidence suggests that one or more cellular cofactors may be important for Tat activity. We have used two-hybrid interactive cloning in yeast to identify a partial cDNA clone (clone 10) from a human B-lymphoblastoid library that specifically interacts with the N-terminal 31 amino acids of HIV-1 Tat which contains the essential cysteine-rich portion of the Tat activation domain. The encoded protein also binds to purified Tat in vitro. Mutation of single essential cysteine residues in Tat abolishes interaction between Tat and clone 10, suggesting that interaction with the encoded protein is important for Tat activity. We have identified the full-length cDNA for the Tat binding protein and shown that overexpression of the encoded protein, Tip60 (Tat interactive protein, 60 kDa), results in a fourfold augmentation of Tat transactivation of the HIV-1 promoter in transient expression assays without increasing the basal activity of the HIV promoter or activating the heterologous RSV promoter. These data together with the genetic and in vitro binding data support the notion that Tip60 might be a cofactor of Tat involved in the regulation of HIV gene expression.
人类免疫缺陷病毒（HIV）的反式激活因子（Tat）蛋白是HIV基因表达的强力激活剂。遗传学和生物化学证据表明，一种或多种细胞辅助因子可能对Tat活性至关重要。我们利用酵母双杂交相互作用克隆技术，从人B淋巴母细胞样文库中鉴定出一个部分cDNA克隆（克隆10），它能与人免疫缺陷病毒1型（HIV-1）Tat蛋白的N端31个氨基酸特异性相互作用，这31个氨基酸包含Tat激活域中富含半胱氨酸的关键部分。该编码蛋白在体外也能与纯化的Tat蛋白结合。Tat蛋白中单个关键半胱氨酸残基的突变消除了Tat与克隆10之间的相互作用，这表明与该编码蛋白的相互作用对Tat活性很重要。我们已鉴定出Tat结合蛋白的全长cDNA，并表明在瞬时表达实验中过表达该编码蛋白Tip60（Tat相互作用蛋白，60 kDa）可使HIV-1启动子的Tat反式激活增加四倍，而不会增加HIV启动子的基础活性或激活异源劳斯肉瘤病毒（RSV）启动子。这些数据连同遗传学和体外结合数据支持这样一种观点，即Tip60可能是参与HIV基因表达调控过程的Tat辅助因子。
