Valvatne H, Szilvay A M, Helland D E
Laboratory for Biotechnology, University of Bergen, Norway.
AIDS Res Hum Retroviruses. 1996 May 1;12(7):611-9. doi: 10.1089/aid.1996.12.611.
In the present study, a CAT assay, a beta-galactosidase assay, and immunofluorescence analysis have been used to study the cellular uptake of the HIV-1 Tat protein. An anti-Tat MAb binding to an epitope comprising both the basic domain and the RGD sequence inhibits trans-activation by exogenous Tat. Two different full-length recombinant Tat proteins were used in these studies. The inhibitory MAb, however, recognized only one of the recombinant Tat proteins. Immunofluorescence analysis demonstrated that only the Tat protein recognized by the inhibitory anti-Tat MAb was taken up by COS and HeLa cells. This indicates that there are conformational differences between the two Tat proteins and that a correct folding of the epitope recognized by the anti-Tat MAb is required for cellular uptake. The recombinant Tat taken up by the cells was distributed between the nucleoli, the nucleoplasm, and along the nuclear membrane. Interactions between Tat and serum components were shown in vitro and also inhibition of trans-cellular trans-activation by fetal calf serum in tissue culture was demonstrated. The specific inhibition of the cellular uptake of Tat by an anti-Tat monoclonal antibody and the blocking of uptake by serum components implies specific binding of Tat to the cell membrane.
在本研究中，采用了CAT检测、β-半乳糖苷酶检测和免疫荧光分析来研究HIV-1 Tat蛋白的细胞摄取情况。一种与包含碱性结构域和RGD序列的表位结合的抗Tat单克隆抗体可抑制外源性Tat的反式激活。在这些研究中使用了两种不同的全长重组Tat蛋白。然而，这种抑制性单克隆抗体仅识别其中一种重组Tat蛋白。免疫荧光分析表明，只有被抑制性抗Tat单克隆抗体识别的Tat蛋白能被COS细胞和HeLa细胞摄取。这表明两种Tat蛋白之间存在构象差异，并且抗Tat单克隆抗体识别的表位的正确折叠是细胞摄取所必需的。被细胞摄取的重组Tat分布在核仁、核质以及核膜周围。体外实验显示了Tat与血清成分之间的相互作用，并且在组织培养中也证明了胎牛血清对跨细胞反式激活的抑制作用。抗Tat单克隆抗体对Tat细胞摄取的特异性抑制以及血清成分对摄取的阻断意味着Tat与细胞膜的特异性结合。
