Suñé C, García-Blanco M A
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Virol. 1995 May;69(5):3098-107. doi: 10.1128/JVI.69.5.3098-3107.1995.
Expression of human immunodeficiency virus type 1 (HIV-1) genes is regulated by the trans activator Tat. Tat exerts its effects by increasing the rate of transcription, but the mechanism by which it does so is still unknown. To study the cellular factors required for Tat trans activation, we have expressed functional Gst-Tat fusion protein and used it to construct affinity columns. Our findings are as follows. (i) A Gst-Tat affinity matrix depleted HeLa nuclear extracts of a factor(s) required for Tat function. A Tat mutant bearing the missense mutation lysine to alanine at position 41 was incapable of this depletion. (ii) Tat trans activation was recovered by addition of unfractionated nuclear extract, the 0.5 M KCl elution fraction from the Tat affinity column, or sedimentation gradient fractions of HeLa extracts. The activity from the gradients sedimented with an apparent molecular mass of 200 kDa. (iii) Tat trans activation could not be recovered by use of recombinant human TATA-binding protein or partially purified TFIID. (iv) trans activation by Tat was blocked by heating of the nuclear extract under conditions in which basal transcription was not decreased. Our data demonstrate for the first time the existence of unique Tat coactivators distinct from factors required for general basal transcription.
1型人类免疫缺陷病毒（HIV-1）基因的表达受反式激活因子Tat调控。Tat通过提高转录速率发挥作用，但其作用机制仍不清楚。为了研究Tat反式激活所需的细胞因子，我们表达了功能性Gst-Tat融合蛋白并用其构建亲和柱。我们的研究结果如下：（i）Gst-Tat亲和基质耗尽了HeLa细胞核提取物中Tat功能所需的一种或多种因子。在第41位带有赖氨酸到丙氨酸错义突变的Tat突变体无法实现这种耗尽。（ii）通过添加未分级的核提取物、来自Tat亲和柱的0.5M KCl洗脱级分或HeLa提取物的沉降梯度级分，可恢复Tat反式激活。梯度中的活性以表观分子量200 kDa沉降。（iii）使用重组人TATA结合蛋白或部分纯化的TFIID无法恢复Tat反式激活。（iv）在基础转录未降低的条件下，加热核提取物可阻断Tat的反式激活。我们的数据首次证明存在不同于一般基础转录所需因子的独特Tat共激活因子。
