LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells
LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells
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DOI：
10.1074/jbc.m303594200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Engelborghs, Y
中科院分区：
文献类型：
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作者：
Maertens, G;Cherepanov, P;Engelborghs, Y
我们报道了人类免疫缺陷病毒1型（HIV-1）整合酶（IN）与人晶状体上皮衍生生长因子/转录共激活因子p75 （LEDGF/p75）蛋白形成特异性核复合物。我们现在利用IN和LEDGF/p75与增强的绿色荧光蛋白和远红色荧光蛋白HcRed1融合，研究IN-LEDGF/p75在活细胞中的相互作用和IN的核输入。我们发现n端锌结合结构域和IN的中心核心结构域都参与了与LEDGF/p75的相互作用。这两个结构域对于IN的核定位以及有丝分裂过程中IN与浓缩染色体的关联都是必不可少的。然而，当LEDGF/p75过表达时，IN的核心结构域片段被招募到细胞核和有丝分裂染色体上，其分布模式具有全长蛋白的特征，表明它是与LEDGF/p75相互作用的主要决定因素。虽然IN的c端结构域在细胞核/染色体定位中是不可缺少的，但c端IN片段与增强的绿色荧光蛋白的融合只在细胞核中被发现，并且具有弥漫性的核/核仁分布，这表明c端结构域也可能在IN的核输入中发挥作用。与LEDGF/p75相反，它的另一种剪接变体p52在体外和活细胞中不与HIV-1 In相互作用。最后，RNA干扰介导的内源性LEDGF/p75表达的敲除消除了IN的核/染色体定位。因此，我们得出结论，与LEDGF/p75的相互作用解释了HIV-1 IN的亲核特性和染色体靶向性。
We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN.