Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: Implication of cytoplasmic function of EBNA-LP
Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: Implication of cytoplasmic function of EBNA-LP
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DOI：
10.1128/jvi.74.21.10104-10111.2000
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发表时间：
2000-11-01
影响因子：
5.4
通讯作者：
Hirai, K
中科院分区：
文献类型：
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作者：
Kawaguchi, Y;Nakajima, K;Hirai, K
EB病毒核抗原前导蛋白(EBNA-LP)由W1W2重复序列和一个独特的Y1Y2结构域组成，在EBV诱导的转化过程中起重要作用。为了确定与EBNA-LP相互作用的细胞因子，我们进行了酵母双杂交筛选，以含有4个W1W2重复序列的EBNA-LP cDNA为诱饵，以EBV转化的人外周血淋巴细胞cDNA文库为细胞基因来源。我们的研究结果如下。(I)在阳性酵母菌落中，所有三个cDNA都编码相同的细胞蛋白HS1相关蛋白X-1(HAX-1)，该蛋白主要定位于细胞质中，并被认为参与了B细胞信号转导和凋亡的调节。(Ii)EBNA-LP的突变分析表明，与HAX-1的关联是由W1W2重复结构域介导的。(Iii)纯化的谷胱甘肽S转移酶与EBNA-LP融合的嵌合蛋白，与HAX-1在COS-7细胞中瞬时表达形成特异性的复合物。(4)当EBNA-LP和HAX-1在COS-7细胞中共表达时，EBNA-LP与HAX-1特异性地共沉淀。(5)EBV感染的淋巴母细胞系的仔细细胞分裂实验表明，EBNA-LP定位于胞浆和胞核。(6)当含有4个W1W2重复序列的EBNA-LP在COS-7细胞中表达时，免疫荧光法检测到EBNA-LP主要在细胞核中表达。有趣的是，当含有单个W1W2重复序列的EBNA-LP在COS-7细胞中表达时，EBNA-LP主要定位于细胞质，并与HAX-1共定位。这些结果表明，EBNA-LP实际上是存在的，并且可能通过与HAX-1的相互作用和影响其功能而在细胞质中具有重要的功能。
Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1, (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1.