Tange T O, Jensen T H, Kjems J
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, DK-8000 Aarhus C, Denmark.
J Biol Chem. 1996 Apr 26;271(17):10066-72. doi: 10.1074/jbc.271.17.10066.
Continuous replication of human immunodeficiency virus type 1 requires the expression of the regulatory protein Rev, which binds to the Rev response element (RRE) and up-regulates the cytoplasmic appearance of singly spliced and unspliced mRNA species. It has been demonstrated that the murine protein YL2 interacts with Rev in vivo and modulates the activity of Rev (Luo, Y., Yu, H., and Peterlin, B. M. (1994) J. Virol. 68, 3850-3856). Here we show that the YL2 human homologue, the p32 protein, which co-purifies with alternative splicing factor ASF/SF2, interacts directly with the basic domain of Rev in vitro and that the Rev-p32 complex is resistant to high concentrations of salt or nonionic detergent. Protein footprinting data suggest that Rev interacts specifically with amino acids within the 196-208 region of p32. An analysis of the ternary complex, formed among p32, Rev, and RRE RNA, shows that Rev can bridge the association of p32 and RRE. Furthermore, we demonstrate that exogenously added p32 specifically relieves the inhibition of splicing in vitro exerted by the basic domain of Rev. Our data are consistent with a model in which p32 functions as a link between Rev and the cellular splicing apparatus.
1型人类免疫缺陷病毒的持续复制需要调节蛋白Rev的表达，Rev可与Rev反应元件（RRE）结合，并上调单剪接和未剪接mRNA种类在细胞质中的出现。已证明鼠蛋白YL2在体内与Rev相互作用并调节Rev的活性（Luo，Y.，Yu，H.，和Peterlin，B.M.（1994）J.Virol.68，3850 - 3856）。在此我们表明，YL2的人类同源物，即与可变剪接因子ASF/SF2共纯化的p32蛋白，在体外直接与Rev的碱性结构域相互作用，并且Rev - p32复合物对高浓度盐或非离子去污剂具有抗性。蛋白质足迹数据表明Rev与p32的196 - 208区域内的氨基酸特异性相互作用。对由p32、Rev和RRE RNA形成的三元复合物的分析表明，Rev可介导p32与RRE的结合。此外，我们证明外源添加的p32可特异性缓解Rev碱性结构域在体外对剪接的抑制作用。我们的数据与一种模型一致，即p32作为Rev与细胞剪接装置之间的连接物发挥作用。
