Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding.
Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding.
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巨细胞病毒激酶 pUL97 亚型的核输入是由 NLS1 和 NLS2 的差异活性介导的，两者均通过经典输入蛋白-α 结合起作用
DOI：
10.1099/vir.0.040592-0
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Marschall
中科院分区：
文献类型：
--
作者：
Solbak;S.M.Ø;Milbradt;Eichler;Wittenberg;Jardin;Sticht;Fossen;Marschall
人巨细胞病毒（human cytomegalovirus， HCMV）多功能蛋白激酶pUL97对病毒复制效率有重要影响。以前，有两种pUL97异构体的存在，它们由交替翻译起始产生，并显示出主要的核定位。在大异构体的N端鉴定出NLS1和NLS2两个二部核定位序列，而小异构体只含有NLS2。目前的研究发现：(i)在hcmv感染的原代成纤维细胞中，pUL97的核定位显示在病毒复制中心和其他核切片中有积累；（ii）在NLS活性的定量评价系统中，大异构体的核易位效率高于小异构体；（iii） NLS1定位于aa 6 ~ 35， NLS2定位于aa 190 ~ 213；（iv）利用表面等离子体共振光谱，证实了NLS1和NLS2与人类输入蛋白α的结合，强调了在二部共识基序中单个精氨酸残基的重要性；(v) pUL97肽的核磁共振波谱证实了先前关于NLS1嵌入完整α-螺旋结构的功能要求；（vi）对溶剂可及表面的生物信息学研究表明，NLS1具有较高的可及性，而NLS2在与输入蛋白α相互作用时具有同工型特异性的可变可及性。因此，这两种异构体的核胞质转运机制似乎受到了不同的调控，这可能对病毒复制过程中pUL97的异构体依赖功能产生影响。
The multifunctional protein kinase pUL97 of human cytomegalovirus (HCMV) strongly determines the efficiency of virus replication. Previously, the existence of two pUL97 isoforms that arise from alternative translational initiation and show a predominant nuclear localization was described. Two bipartite nuclear localization sequences, NLS1 and NLS2, were identified in the N terminus of the large isoform, whilst the small isoform exclusively contained NLS2. The current study found the following: (i) pUL97 nuclear localization in HCMV-infected primary fibroblasts showed accumulations in virus replication centres and other nuclear sections; (ii) in a quantitative evaluation system for NLS activity, the large isoform showed higher efficiency of nuclear translocation than the small isoform; (iii) NLS1 was mapped to aa 6–35 and NLS2 to aa 190–213; (iv) using surface plasmon resonance spectroscopy, the binding of both NLS1 and NLS2 to human importin-α was demonstrated, stressing the importance of individual arginine residues in the bipartite consensus motifs; (v) nuclear magnetic resonance spectroscopy of pUL97 peptides confirmed an earlier statement about the functional requirement of NLS1 embedding into an intact α-helical structure; and (vi) a bioinformatics investigation of the solvent-accessible surface suggested a high accessibility of NLS1 and an isoform-specific, variable accessibility of NLS2 for interaction with importin-α. Thus, the nucleocytoplasmic transport mechanism of the isoforms appeared to be differentially regulated, and this may have consequences for isoform-dependent functions of pUL97 during virus replication.
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