Turci Marco, Romanelli Maria Grazia, Lorenzi Pamela, Righi Paola, Bertazzoni Umberto
Department of Mother and Child, Biology and Genetics, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.
Gene. 2006 Jan 3;365:119-24. doi: 10.1016/j.gene.2005.09.043. Epub 2005 Dec 6.
Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.
人类嗜T细胞病毒（HTLV）I型和II型是密切相关的致癌逆转录病毒，与淋巴增殖性疾病和神经系统疾病有关。前病毒基因组编码一种反式调节Tax蛋白，该蛋白可激活病毒基因并上调参与细胞生长和转化的各种细胞基因。HTLV-I（Tax-I）和HTLV-II（Tax-II）的Tax蛋白具有超过77%的氨基酸同源性，Tax-I或Tax-II的表达足以使培养的T淋巴细胞永生化。Tax-I穿梭于细胞核和细胞质之间并在细胞核内积累，而Tax-II主要存在于细胞质中。在本研究中，我们使用重组载体分析了Tax-II蛋白序列中核定位域的大小和结构。Tax-II蛋白在HeLa细胞中表达为完整蛋白或其各个区域，这些区域分别与绿色荧光蛋白（GFP）融合。对融合的Tax-II产物进行免疫印迹分析证实了它们的表达和大小。荧光显微镜研究表明，完整的Tax-II以及N端截短形式呈现出点状细胞质分布，并且核定位决定簇局限于Tax-II的前60个氨基酸内。因此，对前60个氨基酸内特定序列进行定点诱变和缺失表明，核决定簇位于Tax-II的前41个残基内。这些结果表明Tax-II蛋白的氨基末端残基直接参与决定其核功能。
