Adachi Y, Copeland T D, Hatanaka M, Oroszlan S
Laboratory of Molecular Virology and Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201.
J Biol Chem. 1993 Jul 5;268(19):13930-4.
Rex protein, the post-transcriptional regulator of human T-cell leukemia virus type I, is located predominantly in the cell nucleolus and is associated with the cytoplasmic accumulation of unspliced and singly spliced viral mRNAs. The N-terminal 19-amino acid segment of Rex has been identified as the nucleolar targeting signal (NOS) and shown to be important for Rex function. To study the molecular interaction between the NOS region of Rex and its binding host protein(s) in the nucleolus, we chemically synthesized a functional NOS peptide (wild type) and mutant NOS peptides. Fluorescein isothiocyanate-conjugated functional NOS peptide was rapidly taken up by human cells and was transported to the nucleolus. Using affinity chromatography, we identified nucleolar protein B-23 as the major protein that binds to NOS. We also identified two highly acidic regions of B-23 (amino acids 120-132 and 161-188) as acceptor regions for NOS. Previous experiments have suggested that B-23 functions as a shuttle protein for the nucleolar transport of ribosomal components. Our results suggest that B-23 may also serve as a shuttle for the import of Rex from the cytoplasm to the nucleolus coupled to the export of viral mRNAs containing the Rex-responsive element.
雷克斯蛋白是I型人类T细胞白血病病毒的转录后调节因子，主要位于细胞核仁中，并与未剪接和单剪接的病毒mRNA在细胞质中的积累有关。雷克斯蛋白的N端19个氨基酸片段已被确定为核仁靶向信号（NOS），并显示对雷克斯蛋白的功能很重要。为了研究雷克斯蛋白的NOS区域与其在核仁中结合的宿主蛋白之间的分子相互作用，我们化学合成了一种功能性NOS肽（野生型）和突变型NOS肽。异硫氰酸荧光素偶联的功能性NOS肽被人类细胞迅速摄取并转运到核仁。通过亲和层析，我们确定核仁蛋白B-23是与NOS结合的主要蛋白。我们还确定了B-23的两个高酸性区域（氨基酸120-132和161-188）作为NOS的受体区域。先前的实验表明，B-23作为核糖体成分核仁运输的穿梭蛋白发挥作用。我们的结果表明，B-23也可能作为一种穿梭蛋白，将雷克斯蛋白从细胞质导入核仁，同时将含有雷克斯反应元件的病毒mRNA输出。
