Importin α3/Qip1 is involved in multiplication of mutant influenza virus with alanine mutation at amino acid 9 independently of nuclear transport function.
Importin α3/Qip1 is involved in multiplication of mutant influenza virus with alanine mutation at amino acid 9 independently of nuclear transport function.
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DOI：
10.1371/journal.pone.0055765
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Aida Y
中科院分区：
文献类型：
--
作者：
Sasaki Y;Hagiwara K;Kakisaka M;Yamada K;Murakami T;Aida Y
甲型流感病毒核蛋白（NP）通过经典的输入蛋白α/β途径进入细胞核，并通过输入蛋白α分子识别的核定位信号（NLSs）进入细胞核。尽管NP与输入α亚型Rch1、Qip1和NPI-1结合，但每种亚型在NP核转运和流感病毒复制过程中的作用尚不清楚。在这项研究中，我们使用一组NP突变体，在NP的非常规NLS中包含一系列丙氨酸替代，研究了输入蛋白α亚型对NP核定位和病毒生长的贡献。氨基酸8 （R8A）的丙氨酸突变导致三种输入蛋白异构体的核定位和结合显著减少。R8A NP突变病毒不是通过反向遗传方法产生的。这表明8号位置是通过与Rch1、Qip1和NPI-1的相互作用介导核定位以及随后的病毒产生的主要位点。人类和禽流感病毒NP中氨基酸8的保存对病毒的传播是必要的，这一发现证实了这一点。相比之下，另一个突变体S9A NP定位于细胞核，导致病毒生长和vRNA转录减少，这表明NP内的非常规NLS可能通过独立的途径与核转运、vRNA转录和病毒复制有关。有趣的是，含有S9A突变的非常规NLS的n端110个氨基酸区域主要与Qip1结合。此外，沉默Qip1在不改变核定位的情况下降低了S9A NP突变体的vRNA转录和复制活性，表明Qip1独立于核转运功能参与了S9A突变体病毒的增殖。总之，我们的研究结果表明，NP中的非常规NLS可能具有通过与Qip1相互作用而独立于核定位活性的调节病毒复制的额外能力。
The nucleoprotein (NP) of influenza A virus is transported into the nucleus via the classical importin α/β pathway, and proceeds via nuclear localization signals (NLSs) recognized by importin α molecules. Although NP binds to importin α isoforms Rch1, Qip1 and NPI-1, the role of each individual isoform during the nuclear transport of NP and replication of the influenza virus remains unknown. In this study, we examined the contribution of importin α isoforms for nuclear localization of NP and viral growth using a panel of NP mutants containing serial alanine replacements within an unconventional NLS of NP. Alanine mutation at amino acid 8 (R8A) caused a significant reduction in the nuclear localization and binding to the three importin isoforms. The R8A NP mutant virus did not generate by reverse-genetics approach. This indicates that position 8 is the main site that mediates nuclear localization via interactions with Rch1, Qip1 and NPI-1, and subsequent viral production. This was confirmed by the finding that the conservation of amino acid 8 in human- and avian-origin influenza virus NP was necessary for virus propagation. By contrast, another mutant, S9A NP, which localized in the nucleus, caused a reduction in viral growth and vRNA transcription, suggesting that the unconventional NLS within NP may be associated with nuclear transport, vRNA transcription and viral replication through independent pathways. Interestingly, the N-terminal 110-amino acid region, which contained the unconventional NLS with S9A mutation, mainly bound to Qip1. Furthermore, activities of vRNA transcription and replication of S9A NP mutants were decreased by silencing Qip1 in without changing nuclear localization, indicating that Qip1 involves in multiplication of S9A mutant virus independently of nuclear transport function. Collectively, our results demonstrate the unconventional NLS within NP might have the additional ability to regulate the viral replication that is independent of nuclear localization activity via interactions with Qip1.
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