Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction
Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction
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DOI：
10.1016/j.jmb.2004.05.015
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发表时间：
2004-07-02
影响因子：
5.6
通讯作者：
Yeo, RP
中科院分区：
文献类型：
--
作者：
Bhella, D;Ralph, A;Yeo, RP
麻疹病毒是一种高度传染性的病毒，尽管存在有效的疫苗，但它仍然是全世界疾病和死亡的主要原因。该病毒具有负义单链 RNA 基因组，由核衣壳蛋白 (N) 包裹，形成螺旋核糖核蛋白复合物，称为核衣壳。该结构充当转录和复制的模板。副粘病毒核衣壳是柔性结构，这一特征迄今为止阻碍了结构分析，即使在低分辨率下也是如此。我们研究了这种结构可塑性的程度，使用实空间方法从低温负染色透射电子显微照片中计算重组核衣壳的三维重建。根据螺距（每圈的轴向上升）和扭曲（每圈的亚基数量）对螺旋短截面的图像进行分类。我们的分析表明，这些结构具有广泛的构象灵活性，螺距范围从 50 埃到 66 埃，而扭曲度从至少 13.04 到 13.44 不等，每圈包含约 13.1 个亚基的更多螺旋。我们还研究了 N 的 C 末端对螺旋构象的影响，通过胰蛋白酶消化去除该结构域后分析核衣壳。我们发现这会导致螺距和扭曲发生显着变化，螺距变短，范围从 46 埃到 52 埃，而更多的螺旋每圈扭曲约 13.3 个子单元。我们的发现使我们提出了一种机制，即受病毒或宿主蛋白与 N 的 C 末端之间相互作用影响的构象变化可能在病毒感染期间调节转录和复制的平衡中发挥作用。 (C) 2004 Elsevier Ltd. 保留所有权利。
Measles virus is a highly contagious virus that, despite the existence of an effective vaccine, is a major cause of illness and mortality worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form a helical ribonucleoprotein complex known as the nucleocapsid. This structure serves as the template for both transcription and replication. Paramyxovirus nucleocapsids are flexible structures, a trait that has hitherto hampered structural analysis even at low resolution. We have investigated the extent of this structural plasticity, using real-space methods to calculate three-dimensional reconstructions of recombinant nucleocapsids from cryo-negative stain transmission electron micrographs. Images of short sections of helix were sorted according to both pitch (the axial rise per turn) and twist (the number of subunits per turn). Our analysis indicates that there is extensive conformational flexibility within these structures, ranging in pitch from 50 Angstrom to 66 Angstrom, while twist varies from at least 13.04 to 13.44 with a greater number of helices comprising around 13.1 subunits per turn. We have also investigated the influence of the C terminus of N on helix conformation, analysing nucleocapsids after having removed this domain by trypsin digestion. We have found that this causes a marked change in both pitch and twist, such that the pitch becomes shorter, ranging from 46 Angstrom to 52 Angstrom, while more helices have a twist of approximately 13.3 subunits per turn. Our findings lead us to propose a mechanism whereby changes in conformation, influenced by interactions between viral or host proteins and the C terminus of N, might have a role in regulating the balance of transcription and replication during virus infection. (C) 2004 Elsevier Ltd. All rights reserved.