Schibeci S D, Clegg A O, Biti R A, Sagawa K, Stewart G J, Williamson P
Department of Clinical Immunology, Westmead Hospital, NSW, Australia.
AIDS. 2000 Aug 18;14(12):1701-7. doi: 10.1097/00002030-200008180-00003.
The Nef protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examine the effects of Nef on T lymphocyte activation and associated signalling events.
A recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, and either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates signal transduction pathways and results in IL-2 production and IL-2 receptor alpha-chain (CD25) expression. Cellular responses were examined in cells expressing either Nef or an irrelevant control protein.
Activation of signalling was assessed by immunoblot analysis, or by in-vitro phosphatidylinositol 3-kinase (PI3K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expression was examined using flow cytometry.
Infection of cells with recombinant vaccinia expressing HIV-nef resulted in a marked increase in the production of IL-2 when cells were activated. The enhanced IL-2 response was accompanied by an increase in the level of PI3K activity. IL-2 production remained sensitive to inhibition with the PI3K competitive inhibitor Ly294002, and to the fungal macrolide, rapamycin. In contrast, CD25 expression was not affected, and there were no measurable changes to nuclear factor kappaB (NFkappaB) activation pathways.
Enhanced IL-2 production in stimulated T cells expressing HIV-Nef is associated with increased activation of PI3K-dependent signalling pathways. The results support a model in which Nef affects HIV disease progression by distorting T cell responses.
Nef蛋白对HIV感染个体的疾病发病机制有重大影响。本研究的目的是检测Nef对T淋巴细胞活化及相关信号事件的影响。
使用重组痘苗表达系统在人T细胞系中表达Nef。用抗CD28抗体以及佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）或抗CD3刺激这些细胞，可激活信号转导通路并导致白细胞介素 - 2（IL - 2）产生和IL - 2受体α链（CD25）表达。在表达Nef或无关对照蛋白的细胞中检测细胞反应。
通过免疫印迹分析或体外磷脂酰肌醇3 - 激酶（PI3K）测定评估信号激活。通过酶联免疫吸附测定法测量IL - 2产生，并使用流式细胞术检测CD25细胞表面表达。
用表达HIV - nef的重组痘苗感染细胞后，细胞被激活时IL - 2的产生显著增加。IL - 2反应增强伴随着PI3K活性水平的增加。IL - 2的产生对PI3K竞争性抑制剂Ly294002和真菌大环内酯类药物雷帕霉素的抑制仍然敏感。相比之下，CD25的表达不受影响，核因子κB（NFκB）激活途径也没有可测量的变化。
在表达HIV - Nef的受刺激T细胞中IL - 2产生增强与PI3K依赖性信号通路的激活增加有关。这些结果支持了一种模型，即Nef通过扭曲T细胞反应影响HIV疾病进展。
