Anderson M G, Scoggin K E, Simbulan-Rosenthal C M, Steadman J A
Institute of Molecular Medicine and Genetics, Program in Gene Regulation, Medical College of Georgia, Augusta, Georgia 30912, USA.
J Virol. 2000 Mar;74(5):2169-77. doi: 10.1128/jvi.74.5.2169-2177.2000.
Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax.
人类1型T细胞白血病病毒(HTLV-1)编码一种转录激活因子Tax,其活性被认为对细胞转化有显著贡献。Tax刺激前病毒启动子以及多种细胞基因启动子的转录。Tax与通用转录因子和RNA聚合酶II沟通的机制尚未完全确定。我们研究了Tax是否能直接通过通用转录因子和RNA聚合酶II发挥作用,或者是否需要其他中间因子或共激活因子。我们的结果表明,由纯化的重组TFIIA、TFIIB、TFIIE、TFIIF、CREB和Tax组成的系统,连同高度纯化的RNA聚合酶II、亲和纯化的表位标记TFIID和半纯化的TFIIH,支持HTLV-1启动子的基础转录,但对Tax无反应。Tax刺激转录还需要另外两种活性。我们证明其中一种活性是聚(ADP-核糖)聚合酶(PARP),一种先前已被鉴定为转录共激活因子PC1的分子。在我们的实验中,PARP作为共激活因子发挥作用,其摩尔浓度大约与DNA相等,且等于或低于实验中转录因子的浓度。我们进一步证明PARP在体内刺激Tax激活的转录,表明这种生化方法在功能上鉴定出了逆转录病毒转录激活因子Tax的一个新靶点。