HUMAN FOAMY VIRUS BELL TRANSACTIVATOR CONTAINS A BIPARTITE NUCLEAR-LOCALIZATION DETERMINANT WHICH IS SENSITIVE TO PROTEIN CONTEXT AND TRIPLE MULTIMERIZATION DOMAINS
HUMAN FOAMY VIRUS BELL TRANSACTIVATOR CONTAINS A BIPARTITE NUCLEAR-LOCALIZATION DETERMINANT WHICH IS SENSITIVE TO PROTEIN CONTEXT AND TRIPLE MULTIMERIZATION DOMAINS
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DOI：
10.1128/jvi.69.2.801-808.1995
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发表时间：
1995-02-01
影响因子：
5.4
通讯作者：
SUNG, YC
中科院分区：
文献类型：
--
作者：
CHANG, J;LEE, KJ;SUNG, YC
人泡沫病毒的Bel 1蛋白是一个300个氨基酸的核调节蛋白，其反式激活由同源长末端重复序列和人免疫缺陷病毒1型长末端重复序列指导的基因表达。虽然以前的报道认为Bell的211 - 222和/或209 - 226位残基的单一碱性结构域对于有效的核定位是必要的和足够的（L. K.文卡特什角杨，P. A. Theodorakis和G. Chinnandurai，J. Virol. 67：161-169，1993; P. He，J. D.孙，E. D.加勒特和B。R. Cullen，J. Virol. 67：1896-1904，1993），我们最近的数据显示，Bell的核定位也需要另一个碱性结构域，从氨基酸残基199到200（C. W.李角，澳-地Jun，K. J. Lee和Y. C. Sung，J. Virol. 68：2708-2719，1994）。为了澄清这种差异，我们构建了各种bell-lacZ嵌合构建体和几种接头插入突变体，并确定了它们的亚细胞定位。当含有碱性结构域的Bell区域被置于lacZ基因的内部位点时，Bell的核定位信号（NLS）由两个不连续的碱性区域组成，所述两个不连续的碱性区域被间插序列分开。此外，在两个碱性区域之间插入特定的氨基酸破坏了Bell NLS的活性。另一方面，当Bell NLS附加到β-半乳糖苷酶的氨基末端时，Bell残基199和200不需要将Bell-β-半乳糖苷酶嵌合蛋白导向细胞核。这些结果表明Bell NLS的功能对该序列存在的蛋白质环境敏感。此外，我们证明，贝尔蛋白形成一个多聚体复合物在哺乳动物细胞的细胞核中，通过使用一个敏感的体内蛋白质-蛋白质相互作用测定。突变分析显示，介导多聚体形成的区域映射到Bell的三个结构域，即，残基1至31、42至82和82至111。此外，我们的研究结果表明，贝尔从残基202至226区域阻止贝尔形成多聚体复合物。
The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bell from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; P. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bell (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bell-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bell containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bell consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bell NLS. On the other hand, Bell residues 199 and 200 were not required to direct the Bell-beta-galactosidase chimeric protein to the nucleus when the Bell NLS was appended to the amino terminus of beta-galactosidase. These, results indicate that the function of the Bell NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bell protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bell, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bell from residues 202 to 226 prevents Bell from forming a multimeric complex.