Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding
Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding
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DOI：
10.1128/jvi.73.9.7357-7367.1999
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发表时间：
1999-09-01
影响因子：
5.4
通讯作者：
Digard, P
中科院分区：
文献类型：
--
作者：
Elton, D;Medcalf, L;Digard, P
流感病毒核蛋白（NP）是与基因组和反基因组RNA相关的单链RNA结合蛋白，并且是病毒RNA转录和复制所必需的四种病毒蛋白之一。为了更好地表征NP结合RNA的机制，我们对多肽进行了物理和突变分析，首先确定与RNA直接接触的区域，然后确定所涉及的氨基酸类别，最后通过诱变确定关键残基。化学片段化和氨基酸测序的NP已UV交联的放射性标记的RNA显示，蛋白质-RNA接触发生在整个长度的多肽。化学修饰实验牵连精氨酸，但不是赖氨酸残基作为重要的RNA结合，而RNA依赖性的变化在NP的内在荧光光谱表明色氨酸残基的参与。支持这些观察，单密码子诱变确定了五个色氨酸，苯丙氨酸，和两个精氨酸残基作为必需的高亲和力RNA结合在生理温度下。此外，不能在体外结合RNA的突变体不能支持病毒基因在体内的表达。突变敏感残基不局限于NP的任何特定区域，而是分布在整个蛋白质中。总的来说，这些数据与先前的模型不一致，表明NP-RNA相互作用是由离散的N-末端结构域介导的。相反，我们建议，高亲和力的结合RNA的NP需要协同相互作用的蛋白质与RNA的多个区域，并介导的正电荷残基和磷酸骨架和平面之间的相互作用芳香族侧链和碱基之间的静电相互作用的组合，个别蛋白质-RNA的接触。
The influenza virus nucleoprotein (NP) is a single-strand-RNA-binding protein associated with genome and antigenome RNA and is one of the four virus proteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by which NP binds RNA, we undertook a physical and mutational analysis of the polypeptide, with the strategy of identifying first the regions in direct contact with RNA, then the classes of amino acids involved, and finally the crucial residues by mutagenesis. Chemical fragmentation and amino acid sequencing of NP that had been UV cross linked to radiolabelled RNA showed that protein-RNA contacts occur throughout the length of the polypeptide. Chemical modification experiments implicated arginine but not lysine residues as important for RNA binding, while RNA-dependent changes in the intrinsic fluorescence spectrum of NP suggested the involvement of tryptophan residues. Supporting these observations, single-codon mutagenesis identified five tryptophan, one phenylalanine, and two arginine residues as essential for high-affinity RNA binding at physiological temperature. In addition, mutants unable to bind RNA in vitro were unable to support virus gene expression in vivo. The mutationally sensitive residues are not localized to any particular region of NP but instead are distributed throughout the protein. Overall, these data are inconsistent with previous models suggesting that the NP-RNA interaction is mediated by a discrete N-terminal domain. Instead, we propose that high-affinity binding of RNA by NP requires the concerted interaction of multiple regions of the protein with RNA and that the individual protein-RNA contacts are mediated by a combination of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases.