Duvall J F, Kashanchi F, Cvekl A, Radonovich M F, Piras G, Brady J N
Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20892, USA.
J Virol. 1995 Aug;69(8):5077-86. doi: 10.1128/JVI.69.8.5077-5086.1995.
The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia and tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1 Tax1 gene product has been shown to transactivate transcription of viral and cellular promoters. To examine the biochemical mechanism of Tax1 transactivation, we have developed an in vitro transactivation assay in which wild-type Tax1 is able to specifically transactivate a polymerase II promoter through upstream Tax1-responsive elements. The in vitro system utilizes the HTLV-1 21-bp repeats cloned upstream of the ovalbumin promoter and G-free cassette. Purified Tax1 specifically transactivates this template 5- to 10-fold in a concentration-dependent manner. No transactivation of the ovalbumin promoter (pLovTATA) template control was observed. Tax1 transactivation was inhibited by low concentrations of alpha-amanitin and was effectively neutralized by anti-Tax1 but not control sera. Consistent with in vivo transactivating activity, Tax1 NF-kappa B mutant M22, but not cyclic AMP-responsive element-binding protein mutant M47, transactivated the template containing the tandem 21-bp repeat. In a reconstituted in vitro transcription assay, Tax1 transactivation was dependent upon basal transcription factors TFIIB, TFIIF, Pol II, TFIID, and TFIIA. TATA-binding protein did not functionally substitute for TFIID in the transactivation assay by Tax1 but was sufficient for basal transcription. Finally, we have used anti-TFIIA antibody (p55) to ask if Tax1 transactivation required TFIIA activity. Addition of TFIIA antibody to in vitro transcription reactions, as well as depletion of TFIIA by preclearing with antibody, showed that TFIIA was required for Tax1 transactivation. Only a slight (twofold) drop of basal transcription was observed. Tax1 transactivation was restored when purified HeLa TFIIA was added back into the reconstituted system. We propose that Tax1 utilizes a transactivation pathway involving the activator regulated basal transcription factors TFIID and TFIIA.
人类嗜T细胞病毒1型（HTLV-1）是成人T细胞白血病和热带痉挛性截瘫/HTLV-1相关脊髓病的病原体。HTLV-1 Tax1基因产物已被证明可反式激活病毒和细胞启动子的转录。为了研究Tax1反式激活的生化机制，我们开发了一种体外反式激活试验，其中野生型Tax1能够通过上游Tax1反应元件特异性地反式激活聚合酶II启动子。该体外系统利用克隆在卵清蛋白启动子和无G盒上游的HTLV-1 21碱基重复序列。纯化的Tax1以浓度依赖的方式特异性地将该模板反式激活5至10倍。未观察到卵清蛋白启动子（pLovTATA）模板对照的反式激活。低浓度的α-鹅膏蕈碱可抑制Tax1反式激活，抗Tax1血清可有效中和Tax1反式激活，但对照血清则不能。与体内反式激活活性一致，Tax1 NF-κB突变体M22可反式激活含有串联21碱基重复序列的模板，而环磷酸腺苷反应元件结合蛋白突变体M47则不能。在重组的体外转录试验中，Tax1反式激活依赖于基础转录因子TFIIB、TFIIF、Pol II、TFIID和TFIIA。TATA结合蛋白在Tax1的反式激活试验中不能在功能上替代TFIID，但足以进行基础转录。最后，我们使用抗TFIIA抗体（p55）来研究Tax1反式激活是否需要TFIIA活性。将TFIIA抗体添加到体外转录反应中，以及用抗体预先清除来耗尽TFIIA，结果表明TFIIA是Tax1反式激活所必需的。仅观察到基础转录略有（两倍）下降。当将纯化的HeLa TFIIA重新添加到重组系统中时，Tax1反式激活得以恢复。我们提出，Tax1利用了一条涉及激活剂调节的基础转录因子TFIID和TFIIA的反式激活途径。
