GENETIC-EVIDENCE FOR MULTIPLE NUCLEAR FUNCTIONS OF THE HERPES-SIMPLEX VIRUS ICP8 DNA-BINDING PROTEIN
GENETIC-EVIDENCE FOR MULTIPLE NUCLEAR FUNCTIONS OF THE HERPES-SIMPLEX VIRUS ICP8 DNA-BINDING PROTEIN
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DOI：
10.1128/jvi.63.12.5258-5267.1989
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发表时间：
1989-12-01
影响因子：
5.4
通讯作者：
KNIPE, DM
中科院分区：
文献类型：
--
作者：
GAO, M;KNIPE, DM
我们已经分离出几种突变型单纯疱疹病毒，特别是在感染细胞蛋白8（ICP 8）基因突变，以定义ICP 8，主要的病毒DNA结合蛋白的功能域。为了促进这些突变体的分离，我们首先分离了突变体病毒HD-2，其中lacZ基因在5-溴-4-氯-3-吲哚基-β-吲哚存在下与表达ICP 8的细胞系融合。D-吡喃半乳糖苷突变的ICP 8基因质粒与HD-2 DNA共转染产生重组病毒，其中突变的ICP 8基因并入病毒基因组中。这些重组通过形成白色斑块来鉴定。将突变体分为四类：（i）表达ICP 8的突变体能与DNA结合，但不能定位于细胞核;（ii）表达ICP 8的突变体不能与DNA结合，但定位于细胞核;和（iv）一种表达定位于细胞核并在体外与DNA结合的ICP 8，但突变病毒不复制其DNA。这些类型的突变体提供了遗传证据，DNA结合和核定位是ICP 8的独特功能，并且ICP 8具有与DNA结合以外的核功能。此外，不同于DNA结合的核功能所需的ICP 8部分是显示与细胞蛋白质细胞周期蛋白或增殖细胞核抗原的序列相似性的ICP 8部分。
We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protien 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside. Mutated ICP8 gene plasmids contransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinations were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed to the cell nucleus and bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen.