HIV TAT GENE AND TRANSCRIPTIONAL ACTIVATION

人类免疫缺陷病毒(Hiv)的tat基因起着核心作用。 在艾滋病毒的激活和生命周期中。TAT在以下方面发挥作用 转录起始和延伸的水平。在这里，我们报道 TAT直接与基础转录因子TFIID结合。这个 HeLa提取物的转录活性在 在TAT亲和柱上进行层析，该亲和层析专门保留了 聚合酶II特异性转录因子TFIID。直接互动 TATA结合蛋白(TBP)和TBP组成的全息TFIID的TAT 观察相关因子(TAFs)变化。TAT，通过36-50个氨基酸， 直接与TFIID的TBP亚基结合。 用免疫共沉淀法检测TAT-TBP的相互作用 在感染HIV-1的细胞中。有趣的是，TAT-TBP复合体是 在病毒复制的早期阶段，而不是后期阶段，使用 被感染细胞的同步种群。TBP的领域 与TAT的相互作用已从163个氨基酸映射到196个氨基酸 TBP的缺失和定点突变。TBP的这个领域，它 包括H1和S2结构域，与H2结合位点不同 其他激活蛋白，如E1a。此站点上的TAT绑定到TFIID 在凝胶漂移实验中稳定TFIID与TFIIA的相互作用。在……里面 此外，TAT结合位点似乎与 负协因子dr1，因为TAT有效地竞争dr1 与TBP的互动。最后，我们证明了野生型、 或者人TBP的缺失突变体能够拯救HIV-1 在持续感染的ACH2细胞系中产生。这些结果 提示基础转录因子TBP/TFIID代表一种 HIV转录过程中的重要调控分子。

The Tat gene of human immunodeficiency virus (HIV) plays a central role in the activation and life cycle of HIV. Tat exerts its effects at the level of transcriptional initiation and elongation. Here we report that Tat binds directly to the basal transcription factor TFIID. The transcriptional activity of HeLa extracts was depleted after chromatography on a Tat affinity column, which specifically retained the polymerase II-specific transcription factor TFIID. Direct interaction of Tat with holo-TFIID composed of TATA-binding protein (TBP) and associated factors (TAFs) was observed. Tat, through amino acids 36-50, binds directly to the TBP subunit of TFIID. Using a coimmunoprecipitation assay, the Tat-TBP interaction was detected in HIV-1-infected cells. Interestingly, the Tat-TBP complex is demonstrated at early, but not later stages of viral replication using a synchronized population of infected cells. The domain of TBP interacting with Tat has been mapped from amino acids 163 to 196 using deletion and site-specific mutants of TBP. This domain of TBP, which includes the H1 and S2 domain, is distinct from the H2 binding site for other activator proteins such as E1A. Tat binding to TFIID at this site stabilizes the interaction of TFIID with TFIIA in a gel shift assay. In addition, the Tat binding site appears to overlap the binding site for the negative cofactor, Dr1, since Tat effectively competes for Dr1 interaction with TBP. Finally, we show that cotransfection of wild-type, or a deletion mutant, of human TBP is capable of rescuing HIV-1 production in the persistently infected ACH2 cell line. These results suggest that the basal transcription factor, TBP/TFIID, represents an important regulatory molecule in HIV transcription.