Ramazzotti E, Vignoli M, Re M C, Furlini G, La Placa M
Institute of Microbiology, University of Bologna, St Orsola General Hospital, Italy.
AIDS. 1996 May;10(5):455-61. doi: 10.1097/00002030-199605000-00002.
An enhanced nuclear factor (NF)-kappa B activation in response to tumour necrosis factor (TNF)-alpha has been observed in stably tat-transfected cells. Recent experimental evidence suggests that Tat may autocrinously influence both cellular physiology and HIV-1 long terminal repeat-directed gene expression in Tat-producing cells. Therefore, the possible association of a Tat autocrinous loop with the enhanced NF-kappa B-binding activity induced by TNF-alpha in Tat-producing cells was studied by anti-Tat antibody blocking experiments.
Permanently tat-transfected Jurkat cells, maintained either in the presence or absence of anti-Tat antibody, were studied for the presence of TNF-alpha-induced NF-kappa B-binding activity (quantified by electrophoretic mobility shift assays) and the presence of cell-surface-bound Tat (determined by flow cytometry and confocal microscopy of anti-Tat immunofluorescence-stained cell preparations.
The enhanced production of TNF-alpha-induced NF-kappa B binding activity exhibited by tat-transfected Jurkat cells was completely abolished in cell cultures maintained in the presence of anti-Tat antibody, thus indicating that the increased TNF-alpha-induced NF-kappa B binding activity observed in Jurkat-tat cells was dependent on the presence of Tat protein in an antibody-accessible location. In accordance with these findings, immunofluorescence-stained preparations of unfixed tat-transfected Jurkat cells showed the presence of cell-surface-bound Tat protein which was completely absent in cells incubated in the presence of anti-Tat antibodies.
This study demonstrates that the enhanced NF-kappa B activation exhibited by stably tat-transfected cells in response to TNF-alpha, is associated with cell surface interaction of extracellularly released Tat protein. These data add further evidence to the possible relevance of a Tat autocrinous loop in the physiology of Tat-producing cells and suggest that in HIV-1-infected cells Tat is likely to behave as a bifunctional molecule which not only acts from within facilitating NF-kappa B recruitment in the viral transcription complex, but may also act from without increasing the availability of activated NF-kappa B.
在稳定转染tat的细胞中观察到，对肿瘤坏死因子（TNF）-α的反应中核因子（NF）-κB激活增强。最近的实验证据表明，Tat可能自分泌地影响产生Tat的细胞中的细胞生理和HIV-1长末端重复序列指导的基因表达。因此，通过抗Tat抗体阻断实验研究了Tat自分泌环与TNF-α在产生Tat的细胞中诱导的增强的NF-κB结合活性之间的可能关联。
对在有或没有抗Tat抗体的情况下维持的永久转染tat的Jurkat细胞，研究其TNF-α诱导的NF-κB结合活性（通过电泳迁移率变动分析定量）的存在以及细胞表面结合的Tat的存在（通过抗Tat免疫荧光染色细胞制剂的流式细胞术和共聚焦显微镜确定）。
在有抗Tat抗体的细胞培养物中，tat转染的Jurkat细胞表现出的TNF-α诱导的NF-κB结合活性的增强产生被完全消除，因此表明在Jurkat-tat细胞中观察到的TNF-α诱导的NF-κB结合活性增加依赖于抗体可及位置中Tat蛋白的存在。与这些发现一致，未固定的tat转染的Jurkat细胞的免疫荧光染色制剂显示存在细胞表面结合的Tat蛋白，而在有抗Tat抗体的情况下孵育的细胞中则完全不存在。
本研究表明，稳定转染tat的细胞对TNF-α表现出的增强的NF-κB激活与细胞外释放的Tat蛋白的细胞表面相互作用有关。这些数据进一步证明了Tat自分泌环在产生Tat的细胞生理中的可能相关性，并表明在HIV-1感染的细胞中Tat可能表现为双功能分子，它不仅在病毒转录复合物中从内部促进NF-κB募集，而且还可能从外部作用增加活化的NF-κB的可用性。
