Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal
Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal
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DOI：
10.1128/jvi.00736-07
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发表时间：
2008-01-01
影响因子：
5.4
通讯作者：
Sugimoto, Nakaba
中科院分区：
文献类型：
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作者：
Isegawa, Yuji;Miyamoto, Yoichi;Sugimoto, Nakaba
为了阐明人类疱疹病毒6型（HHV-6）U69蛋白激酶在体内的功能，我们首先利用抗U69蛋白的多克隆抗体分析了其在感染HHV-6的Molt 3细胞中的亚细胞定位。免疫荧光研究表明，在HHV-6感染和HHV-6转染的细胞中，U69信号以网状模式定位于细胞核。一个计算机程序预测两个重叠的经典核定位信号（NLS）在N-末端区域的蛋白质，这NLS基序是高度保守的N-末端区域的大多数疱疹病毒蛋白激酶检查到目前为止。蛋白质的N-末端缺失突变形式未能进入细胞核，而绿色荧光蛋白（GFP）和/或谷胱甘肽S-转移酶（GST）与U69 N-末端区域的融合蛋白被转运到细胞核中，表明预测的蛋白质的N-末端NLS实际上起NLS的作用。含有U69的N-末端NLS的GST-GFP融合蛋白的核转运被麦胚凝集素和Q69 L Ran-GTP突变体抑制，表明U69蛋白通过经典的核转运机制从细胞质转运到细胞核中。无细胞输入试验表明，U69蛋白的核转运是由输入素α/β与小GTdR Ran共同介导的。当使用低浓度的每种输入素-α亚型进行输入测定时，NPI 2/输入素-α 7引起比Rch 1/输入素-α 1或Qip 1/输入素-α 3更有效的转运活性。这些结果表明NPI 2/importin-α 7的定位与HHV-6的细胞嗜性之间的关系。
To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha 7 elicited more efficient transport activity than did Rch1/importin-alpha 1 or Qip1/importin-alpha 3. These results suggest a relationship between the localization of NPI2/importin-alpha 7 and the cell tropism of HHV-6.