ABSOLUTE DEPENDENCE ON KAPPA-B RESPONSIVE ELEMENTS FOR INITIATION AND TAT-MEDIATED AMPLIFICATION OF HIV TRANSCRIPTION IN BLOOD CD4 T-LYMPHOCYTES
ABSOLUTE DEPENDENCE ON KAPPA-B RESPONSIVE ELEMENTS FOR INITIATION AND TAT-MEDIATED AMPLIFICATION OF HIV TRANSCRIPTION IN BLOOD CD4 T-LYMPHOCYTES
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DOI：
10.1002/j.1460-2075.1995.tb07141.x
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发表时间：
1995-04-03
期刊：
影响因子：
11.4
通讯作者：
ARENZANASEISDEDOS, F
中科院分区：
文献类型：
--
作者：
ALCAMI, J;DELERA, TL;ARENZANASEISDEDOS, F
通过系统比较从人外周血中提纯的人CD4T细胞和转化的淋巴母细胞T细胞株中HIV调节区的活性，分析了依赖于NF-kappa B的信号在激活HIV调节区(LTR)转录活性中的作用。在正常的CD4T细胞中，我们还分析了病毒kappa B反应元件在HIV复制中所起的作用。对静息的正常T淋巴细胞的核抽提物的分析表明，存在p50，但不存在p65，NF-kappa B亚单位，以及佛波酯诱导真正的(p50-p65)NF-kappa B复合体的存在。同时，我们观察到明显的增强子依赖的HIV LTR反式激活，其强度与淋巴母细胞类似。我们发现，未经刺激的CD4T淋巴细胞提供了一种对HIV LTR功能具有极低通透性的细胞环境。这与在淋巴母细胞类T细胞中观察到的高自发LTR活性形成鲜明对比，在淋巴母细胞T细胞中，LTR活性基本上不依赖于kappa B反应元件。由于静息T淋巴细胞的基础LTR活性较低，依赖于NF-kappa B的反式激活是诱导HIV LTR的必要条件。令人惊讶的是，甚至HIV Tat在静止的CD4T淋巴细胞中的功能也被发现完全依赖于LTRkappa B反应元件。在瞬时转染中获得的这些观察结果的相关性得到了证实，即感染了携带kappa B反应元件临界点突变的HIV感染前病毒的血液CD4T淋巴细胞在细胞激活和体外长期培养时无法显示任何可检测到的转录活性。我们的观察强调了分析正常CD4T淋巴细胞为HIV感染提供的自然环境中HIV调节域功能的重要性，并证明了血液CD4T淋巴细胞中TAT依赖和TAT非依赖的HIV转录绝对需要NF-kappa B反应元件。
The role of NF-kappa B-dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF-kappa B subunit and the induction by phorbol esters of bona fide (p50-p65) NF-kappa B complexes. In parallel, we observed clear enhancer-dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF-kappa B-dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro. Our observations emphasize the importance of analyzing the functioning of HIV regulatory domains in the natural environment provided by normal CD4 T lymphocytes for HIV infection, and demonstrate an absolute requirement for NF-kappa B responsive elements for Tat-dependent and Tat-independent HIV transcription in blood CD4 T lymphocytes.