Weber F, Kochs G, Gruber S, Haller O
Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Freiburg, D-79008, Germany.
Virology. 1998 Oct 10;250(1):9-18. doi: 10.1006/viro.1998.9329.
We have previously shown that the nucleoprotein (NP) of Thogoto virus (THOV), a tick-borne member of the Orthomyxoviridae family, accumulates in the cell nucleus. Here we demonstrate that THOV NP contains a motif (KRxxxxxxxxxKTKK) at amino acid positions 179-193 that represents a classical bipartite nuclear localization signal (NLS). This sequence motif (named cNLS) was able to translocate a cytoplasmic 80-kDa reporter protein into the nucleus. Targeted mutations substituting lysines for alanines in the downstream cluster of the bipartite motif abolished the capacity of cNLS to mediate nuclear import. In contrast, identical mutations had no effect on nuclear localization when introduced into THOV NP, indicating that additional transport signals are present in NP. Amino-acid sequence comparisons revealed that THOV NP lacks the N-terminal nonconvential NLS (named here nNLS), which has been implicated in nuclear import of influenza A virus NP. Accordingly, THOV NP failed to interact in coprecipitation assays with the cellular NPI-1/3 transport factors of the karyopherin alpha family. A highly conserved motif identified in THOV NP was the so-called nuclear accumulation sequence (NAS). Mutating NAS alone, or in combination with cNLS, had no gross effect on the intracellular distribution of the protein, indicating that a functional NAS is not required for nuclear accumulation of THOV NP in mammalian cells. We also studied nuclear transport of influenza A/PR/8/34 virus NP. Interestingly, we found a cNLS motif at amino acid positions 198-216 in addition to the previously described nonconventional nNLS. To further assess the functional role of cNLS, nNLS, and NAS, we analyzed single, double, and triple mutants of influenza A virus NP. When nNLS was destroyed, the protein stayed in the cytoplasm as expected. When NAS was disrupted in addition to nNLS, the double mutant accumulated in the nucleus, suggesting that cNLS was active. Indeed, when cNLS was also inactivated, the triple mutant protein localized again predominantly to the cytoplasm. These findings suggest that NP of orthomyxoviruses have two independent NLSs, namely cNLS and nNLS. They further suggest that NAS and NLSs may assume opposing roles in nucleocytoplasmic transport of NP.
我们之前已经表明，正粘病毒科蜱传成员托戈托病毒（THOV）的核蛋白（NP）在细胞核中积累。在此我们证明，THOV NP在氨基酸位置179 - 193处含有一个基序（KRxxxxxxxxxKTKK），它代表一个典型的双分型核定位信号（NLS）。这个序列基序（命名为cNLS）能够将一种细胞质80 kDa的报告蛋白转运到细胞核中。在双分型基序下游簇中用丙氨酸取代赖氨酸的靶向突变消除了cNLS介导核输入的能力。相比之下，将相同的突变引入THOV NP时对核定位没有影响，这表明NP中存在其他转运信号。氨基酸序列比较显示，THOV NP缺乏N端非常规NLS（在此命名为nNLS），该NLS与甲型流感病毒NP的核输入有关。因此，在共沉淀试验中，THOV NP未能与核转运蛋白α家族的细胞NPI - 1/3转运因子相互作用。在THOV NP中鉴定出的一个高度保守的基序是所谓的核积累序列（NAS）。单独突变NAS或与cNLS一起突变，对该蛋白的细胞内分布没有明显影响，这表明功能性NAS对于THOV NP在哺乳动物细胞中的核积累不是必需的。我们还研究了甲型流感病毒A/PR/8/34 NP的核转运。有趣的是，除了先前描述的非常规nNLS外，我们在氨基酸位置198 - 216处发现了一个cNLS基序。为了进一步评估cNLS、nNLS和NAS的功能作用，我们分析了甲型流感病毒NP的单突变体、双突变体和三突变体。当nNLS被破坏时，该蛋白如预期那样留在细胞质中。当除了nNLS之外NAS也被破坏时，双突变体在细胞核中积累，这表明cNLS是有活性的。确实，当cNLS也失活时，三突变体蛋白再次主要定位于细胞质中。这些发现表明，正粘病毒的NP有两个独立 的NLS，即cNLS和nNLS。它们进一步表明，NAS和NLS在NP的核质转运中可能发挥相反的作用。
