Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17
Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17
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DOI：
10.1128/jvi.00851-19
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发表时间：
2019-10-01
影响因子：
5.4
通讯作者：
Liu, Hung-Jen
中科院分区：
文献类型：
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作者：
Chiu, Hung-Chuan;Huang, Wei-Ru;Liu, Hung-Jen
禽呼肠孤病毒(ARV)p17蛋白通过转录依赖和染色体区域维持1(CRM1)非依赖机制在细胞核和细胞质之间持续穿梭。然而，细胞蛋白是否调节p17的核质穿梭尚不清楚。这是首次报道异质性核核糖核蛋白A1作为载体蛋白调节p17的核质穿梭。体外和体内研究表明，p17与hnRNP A1的直接相互作用位于p17的氨基末端(氨基酸[AA]19~40)和hnRNP A1的C末端富含甘氨酸的区域。此外，我们的结果表明，p17-hnRNP A1-Transportin 1载体-货物复合体的形成是调节p17核输入所必需的。利用序列分析和突变分析，我们已经确定了p17的核输出信号(NES)(19)LSLRELAI(26)()。这些残基的突变导致p17的核滞留。在这项工作中，我们发现组成NES的p17的N端21个氨基酸(AA 19到40)可以调节p17和hnRNP A1的相互作用和p17的核质穿梭。在这项工作中，p17与lamin A/C的相互作用部位被定位在p17和p17的氨基末端(aa41~60)，并与核膜上的lamin A/C共定位。HnRNP A1或lamin A/C的敲除导致p17核质穿梭的抑制和病毒产量的降低。总之，这项研究的结果为hnRNP A1和lamin A/C调节ARV p17蛋白的核质穿梭提供了机械性的见解。ARV p17蛋白在细胞核和细胞质之间持续穿梭，调节多种细胞信号通路，并与多种细胞蛋白相互作用，导致翻译关闭、细胞周期停滞和自噬小体形成，所有这些都促进了病毒的复制。到目前为止，p17核质穿梭的机制在很大程度上仍然未知。在这里，我们报告了hnRNP A1和lamin A/C作为载体和中介蛋白来调节p17的核质穿梭。P17-hnRNP A1-Transportin 1载体-货物复合体的形成是调节p17核进口的必要条件。此外，我们还发现了p17的一个含有NES的核质穿梭结构域(AA 19-40)，它对与hnRNP A1结合和p17的核质穿梭至关重要。这项研究为hnRNP A1和lamin A/C如何调节ARV p17蛋白的核质穿梭提供了新的见解。
Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) (19)LSLRELAI(26)( )of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.