Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.
Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.
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DOI：
10.1038/srep21662
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发表时间：
2016-02-26
影响因子：
4.6
通讯作者：
Hou MH
中科院分区：
文献类型：
--
作者：
Liu CL;Hung HC;Lo SC;Chiang CH;Chen IJ;Hsu JT;Hou MH
核蛋白 (NP) 是甲型流感病毒感染细胞中最丰富的 RNA 结合病毒蛋白，是病毒 RNA 转录和复制所必需的。最近的研究表明，流感 NP 是抗病毒药物开发的有效靶点。甲型流感 NP 头域和体域之间的凹槽表面覆盖有大量保守残基，在 RNA 结合中发挥着至关重要的作用。为了探索 NP 结合 RNA 的机制，我们在 RNA 结合沟中进行了一系列定点诱变，然后进行表面等离子共振 (SPR)，以表征 RNA 和 NP 之间的相互作用。此外，还评估了 Y148 在 NP 稳定性和 NP-RNA 结合中的作用。发现 Y148 的芳香族残基与核苷酸碱基堆叠。通过中断Y148和RNA碱基之间的堆积相互作用，我们鉴定了流感病毒NP抑制剂，(E,E)-1,7-双(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮；这种抑制剂降低了 NP 的 RNA 结合亲和力并阻碍了病毒复制。我们的研究结果将有助于开发破坏流感病毒中 RNA 和病毒 NP 之间相互作用的新药。
Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus–infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.