Monomeric nucleoprotein of influenza A virus.
Monomeric nucleoprotein of influenza A virus.
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DOI：
10.1371/journal.ppat.1003275
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发表时间：
2013-03
期刊：
影响因子：
6.7
通讯作者：
Ruigrok RW
中科院分区：
文献类型：
--
作者：
Chenavas S;Estrozi LF;Slama-Schwok A;Delmas B;Di Primo C;Baudin F;Li X;Crépin T;Ruigrok RW
分离的甲型流感病毒核蛋白存在于单体和三聚体之间的平衡状态。只含单体或只含三聚体的样品可分别用低盐和高盐稳定。三聚体以高亲和力结合RNA，但仍保持修剪体，而单体聚合到RNA上，形成核蛋白-RNA复合体。当野生型(Wt)核蛋白结晶时，它形成三聚体，无论是以单体还是三聚体开始。因此，我们结晶了专有单体R416A突变核蛋白，并观察了通向三聚体结构中相邻原型的结构域交换环是如何与突变单体表面的等效位点相互作用的，从而避免了聚合。单体的C末端结合在RNA结合面的一侧，降低了它的正电荷。对突变型和野生型单体蛋白的生物物理鉴定结果相同，表明野生型蛋白的交换结构域以相同的方式折叠。在探索单体wt核蛋白在感染细胞中如何稳定的过程中，我们确定了从病毒颗粒中分离的核蛋白上的磷酸化位点。我们发现丝氨酸165在所有的甲型和乙型流感病毒中都是磷酸化和保守的。模拟磷酸化的S165D突变体是单体，与wt单体NP相比，对RNA的亲和力降低。这表明，磷酸化可能调节感染细胞中核蛋白的聚合状态和RNA结合。这种单体结构可以用于寻找新的抗流感药物，因为稳定单体的化合物可能会减缓病毒感染。负链RNA病毒的RNA被其特定的病毒核蛋白包裹，形成螺旋核蛋白-RNA结构，作为转录和复制的模板。所有这些核蛋白都有两个共同的活性：RNA结合和自我聚合，而且这些活性很可能是偶联的。所有这些病毒都必须在病毒RNA结合之前防止其核蛋白与细胞RNA结合和聚合。非片段病毒通过编码一种与核蛋白结合的磷蛋白来解决这一问题，从而阻止这两种活动。分段病毒，如流感病毒和本雅氏病毒，不编码磷酸蛋白，需要以不同的方式解决这个问题。在这里，我们介绍了单体流感病毒核蛋白的原子结构。虽然流感病毒和裂谷热病毒(Bunya病毒)核蛋白的结构不同，但当比较单体和聚合物结构时，功能上有相似之处。这两种核蛋白在单体和聚合物中都具有相同的核心结构。它们含有一条柔性臂，可移动到聚合物结构中的相邻原型上，但折叠到单体结构中的核心上，隐藏裂谷热病病毒核蛋白中的RNA结合槽，并改变流感病毒蛋白的RNA结合平台的静电势。
Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection. The RNAs of negative strand RNA viruses are encapsidated by their specific viral nucleoproteins, forming helical nucleoprotein-RNA structures that are the template for transcription and replication. All these nucleoproteins have two activities in common: RNA binding and self-polymerisation, and it is likely that these activities are coupled. All these viruses have to keep their nucleoprotein from binding to cellular RNA and from polymerisation before viral RNA binding. The non-segmented viruses solve this by coding for a phosphoprotein that binds to the nucleoprotein, blocking both activities. The segmented viruses, such as influenza and Bunyaviruses, do not code for a phosphoprotein and need to solve this problem differently. Here we present the atomic structure of monomeric influenza virus nucleoprotein. Although the structures of the influenza virus and the Rift Valley Fever Virus (Bunya virus) nucleoproteins are different, there are functional similarities when the monomer and polymer structures are compared. Both nucleoproteins have a core structure that is identical in the monomer and the polymer. They contain a flexible arm that moves over to a neighbouring protomer in the polymer structure but that folds onto the core in the monomer structure, hiding the RNA binding groove in the Rift valley Fever Virus nucleoprotein and modifying the electrostatic potential of the RNA binding platform of the influenza virus protein.
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影响因子：
3.8
作者：
KISTNER, O;MULLER, K;SCHOLTISSEK, C
通讯作者：
SCHOLTISSEK, C
影响因子：
6.7
作者：
Jorba N;Coloma R;Ortín J
通讯作者：
Ortín J
影响因子：
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作者：
COMPANS, RW;CONTENT, J;DUESBERG, PH
通讯作者：
DUESBERG, PH
影响因子：
3.7
作者：
Boulo, Sebastien;Akarsu, Hatice;Baudin, Florence
通讯作者：
Baudin, Florence
DOI：
10.1073/pnas.1108515108
发表时间：
2011-11-29
影响因子：
11.1
作者：
Hastie, Kathryn M.;Liu, Tong;Saphire, Erica Ollmann
通讯作者：
Saphire, Erica Ollmann