Niikura M, Dornadula G, Zhang H, Mukhtar M, Lingxun D, Khalili K, Bagasra O, Pomerantz R J
Dorrance H Hamilton Laboratories, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Oncogene. 1996 Jul 18;13(2):313-22.
Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.
先前在包括U87MG在内的中枢神经系统（CNS）来源的星形胶质细胞系中已证明，人类免疫缺陷病毒1型（HIV-1）Tat蛋白在没有反式激活应答元件（TAR）的情况下，对长末端重复序列（LTR）驱动的RNA表达具有独特的转录反式激活作用。在本研究中，利用核糖核酸酶保护试验来揭示这些细胞中HIV-1-LTR反式激活的分子机制。在不存在或存在Tat的情况下，均未检测到代表HIV-1转录失败的短转录本，并且在实验中，与3'探针相比，使用5'探针未检测到转录本水平的差异。因此，在U87MG细胞中，Tat的反式激活作用可能基于转录起始的增加，无论是在TAR依赖性还是非依赖性状态下都是如此。此外，通过使用新建立的含有HIV-1-LTR报告基因构建体的稳定细胞转化体，已证明TAR非依赖性反式激活主要在瞬时转染的U87MG细胞中有效发挥作用。如先前在其他星形胶质细胞中所报道的那样，用完整的HIV-1前病毒基因组稳定转染的U87MG细胞在长期培养后产生的病毒水平非常低。这些细胞显示出HIV-1基因组的转录受到严重限制，通过Northern印迹法未检测到HIV-1特异性RNA的可检测水平，这表明这些细胞中病毒产生的限制主要是由于5'HIV-1-LTR的总体转录水平较低。该细胞中HIV-1 RNA的转录不能被各种刺激物如佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）、肿瘤坏死因子-α（TNF-α）和丁酸钠显著上调。这些数据表明，这些细胞中HIV-1转录的限制可能受与淋巴细胞或单核细胞不同的机制控制。
