Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus
Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus
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DOI：
10.1016/j.jmb.2012.01.017
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发表时间：
2012-03-16
影响因子：
5.6
通讯作者：
Stockley, Peter G.
中科院分区：
文献类型：
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作者：
Bakker, Saskia E.;Ford, Robert J.;Stockley, Peter G.
我们使用冷冻电子显微镜确定了萝卜皱病毒 (TCV) 的天然形式和扩展形式的三维结构，该显微镜可以直接观察到在病毒粒子的高分辨率 X 射线结构中看不到的衣壳单链 RNA 和外壳蛋白 (CP) N 端区域。扩展形式是感染过程中假定的分解中间体，由 CP 亚基衣壳形成域的分离产生。衣壳扩张导致孔的形成，从而允许病毒 RNA 排出。 CP N 端区域的一个子集以扩展形式变得可以蛋白水解，但核酸酶仍然无法接近 RNA。沉降速度测定表明膨胀状态是亚稳态的并且膨胀不是完全可逆的。蛋白水解裂解的 CP 亚基与衣壳分离，可能导致病毒 RNA 内的静电排斥力增加。与这一想法一致，电子显微镜图像显示蛋白水解将不对称性引入 TCV 衣壳中，并允许基因组从指定位点最初挤出。小麦胚芽提取物中多核糖体的明显形成表明随后的脱壳与翻译有关。这意味着病毒RNA及其衣壳在原发感染期间发挥多种作用，这与核糖体介导的基因组脱壳以避免宿主抗病毒活性一致。 (C) 2012 Elsevier Ltd. 保留所有权利。
We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity. (C) 2012 Elsevier Ltd. All rights reserved.