The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin β1 and importin α/β-recognized nuclear localization signals
The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin β1 and importin α/β-recognized nuclear localization signals
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DOI：
10.1074/jbc.m204977200
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发表时间：
2002-09-27
影响因子：
4.8
通讯作者：
Vasudevan, SG
中科院分区：
文献类型：
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作者：
Brooks, AJ;Johansson, M;Vasudevan, SG
登革病毒NS5蛋白是一种多功能RNA依赖RNA聚合酶，对病毒复制至关重要。我们已经证明Dengue2 NS5的37个氨基酸域间间隔序列（残基(X2KKX14KKKX11RKX3405)-X-369）包含一个功能性核定位信号（NLS）。本研究分析了β -半乳糖苷酶融合蛋白携带带正电残基或结构域间连接区域截断点突变（残基369-389或残基386-405）的核输入和输入蛋白结合活性，结果表明，连接区域的n端部分(残基369-389，a/bNLS)对核定位至关重要，并被传统的nls结合的α / β异二聚体核输入受体以高亲和力识别。我们还发现，先前通过酵母双杂交分析发现的a/bNLS附近的输入蛋白β结合位点（残基320-368，bNLS）具有NLS的功能，被输入蛋白β高亲和力识别，并能够将β -半乳糖苷酶靶向到细胞核。有趣的是，bNLS在登革热和相关黄病毒中高度保守，这意味着该区域在感染周期中具有普遍作用并输入β。
Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37-amino acid interdomain spacer sequence (residues (X2KKX14KKKX11RKX3405)-X-369) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.