Heisig V, Benter T, Josephs S F, Sadaie M R, Okamoto T, Gallo R C, Wong-Staal F
Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20892.
Haematol Blood Transfus. 1987;31:423-9. doi: 10.1007/978-3-642-72624-8_90.
The location of cis-acting regulatory sequences within the long terminal repeat (LTR) of the human T-cell lymphotropic virus type III was determined by eukaryotic cell transfection and chloramphenicol acetyltransferase (CAT) assay or in vitro cell-free transcription. A 160 base pair (bp) region of the LTR at position - 104 to 56 is required for trans-activation (cap site 1). A 24 bp enhancer element (EHE) capable of increasing the rate of transcription, irrespective of orientation, is located between nucleotides -105 to -80. It contains two 10 bp repeats. Three Sp1 binding sites (Sp1 III-I) are located between -78 and -45. A deletion of Sp1 III allowed for limited TATIII response while the presence of a functional enhancer restored the activity in HTLV-III infected cells. Complete loss of transcriptional activity and CAT gene expression could be attributed to the absence of EHE and Sp1 III-I at position -48. However, reinsertion of the enhancer restored accurate initiation but at a decreased level suggesting that the presence of a Sp1 binding site is not a prerequisite for the accurate initiation of transcription but is required for transcriptional activation independent of a promoter. The presence of a negative regulatory element (NRE) has been demonstrated by removal of the 5' part of U3 to position -117. Nucleotide sequences around the cap site and poly (A) site contain a trans-activator response element (TRE) and could be arranged into a unique secondary structure. A deletion of four nucleotides TCTGAGCCTGGGAGCTC causes a loss of three dimer linkage sequence binding. The CAT gene enzyme expression is completely abolished but transcriptional activity remains at reduced level.
通过真核细胞转染和氯霉素乙酰转移酶（CAT）测定或体外无细胞转录，确定了人类III型嗜T细胞淋巴细胞病毒长末端重复序列（LTR）内顺式作用调节序列的位置。LTR位于 -104至56位的160个碱基对（bp）区域是反式激活所必需的（帽位点1）。一个24 bp的增强子元件（EHE），无论其方向如何，都能够提高转录速率，它位于核苷酸 -105至 -80之间。它包含两个10 bp的重复序列。三个Sp1结合位点（Sp1 III-I）位于 -78和 -45之间。Sp1 III的缺失使得TATIII反应受限，而功能性增强子的存在则恢复了HTLV-III感染细胞中的活性。转录活性和CAT基因表达的完全丧失可归因于 -48位处EHE和Sp1 III-I的缺失。然而，增强子的重新插入恢复了准确起始，但水平降低，这表明Sp1结合位点的存在不是转录准确起始的先决条件，但对于独立于启动子的转录激活是必需的。通过去除U3的5'部分至 -117位，已证明存在负调节元件（NRE）。帽位点和聚（A）位点周围的核苷酸序列包含反式激活因子反应元件（TRE），并且可以排列成独特的二级结构。四个核苷酸TCTGAGCCTGGGAGCTC的缺失导致三个二聚体连接序列结合的丧失。CAT基因酶表达完全被消除，但转录活性仍处于降低水平。
