A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation.
A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation.
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DOI：
10.1371/journal.ppat.1005379
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发表时间：
2016-01
期刊：
影响因子：
6.7
通讯作者：
Curry S
中科院分区：
文献类型：
--
作者：
Leen EN;Sorgeloos F;Correia S;Chaudhry Y;Cannac F;Pastore C;Xu Y;Graham SC;Matthews SJ;Goodfellow IG;Curry S
翻译起始是诺如病毒（一种导致人类胃肠炎的主要病原体）正链单链RNA基因组复制周期中关键的早期步骤。诺如病毒RNA既无5´ m7G帽结构，也无内部核糖体进入位点（IRES），它采用一种独特的机制来启动蛋白质合成，这种机制依赖于与病毒RNA 5´端共价连接的VPg蛋白和宿主细胞中的真核起始因子（eIFs）之间的相互作用。对于鼠诺如病毒（MNV），我们之前已表明VPg与eIF4G的中间片段（4GM；残基652 - 1132）结合。在此，我们利用下拉实验、荧光各向异性和等温滴定量热法（ITC）证明，MNV VPg的C末端约20个氨基酸的一段序列介导与eIF4G的4GM片段内的HEAT - 1结构域直接且特异性的结合。我们的分析进一步揭示，MNV的C末端通过一个在所有已知诺如病毒中都保守的基序与eIF4G的HEAT - 1结合。精细的诱变图谱表明，MNV VPg的C末端可能以螺旋构象与eIF4G相互作用。利用核磁共振波谱确定了eIF4G的HEAT - 1上的VPg结合位点，这通过诱变和结合实验得到了证实。我们发现这个位点与eIF4G的HEAT - 1上的eIF4A结合位点不重叠，因为我们证明了诺如病毒VPg能够形成VPg - eIF4G - eIF4A三元复合物。含有MNV VPg的C末端肽的融合蛋白能够抑制诺如病毒RNA的体外翻译，但不抑制依赖帽结构或IRES的翻译，这表明了VPg - eIF4G相互作用的功能重要性。这些观察结果明确了诺如病毒VPg和eIF4G之间功能性相互作用的重要结构细节，并揭示了一个可能被用作抗病毒治疗靶点的结合界面。
诺如病毒感染导致急性胃肠炎，是全球人类健康中一个日益严重的问题。感染的一个关键早期步骤是病毒RNA基因组的翻译，以产生组装新病毒颗粒所需的蛋白质。在鼠诺如病毒（MNV）中，它为研究人类诺如病毒提供了一个有用的模型，与病毒RNA相连的VPg蛋白对这一过程至关重要，因为它与一种细胞蛋白eIF4G相互作用，而eIF4G通常参与从宿主基因的信使RNA启动蛋白质合成。我们利用多种生化和生物物理实验来测量MNV VPg与eIF4G结合的紧密程度，并确定参与这种相互作用的两种蛋白的部分。我们表明，MNV VPg一端（C末端）约20个氨基酸的序列使其能够与eIF4G内一个明确的结构域（称为HEAT - 1）结合，并且在结合时可能采取螺旋结构。我们的数据表明，这种相互作用在所有诺如病毒中都很常见，包括感染人类的类型。我们还表明，MNV VPg的C末端肽能够抑制诺如病毒的蛋白质合成，这增加了在抗病毒药物设计中靶向VPg - eIF4G相互作用的可能性。
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. Norovirus infections cause acute gastroenteritis and are a growing worldwide problem in human health. A critical early step in infection is translation of the viral RNA genome to produce the proteins needed to assemble new virus particles. In mouse noroviruses (MNV), which provide a useful model for studying human noroviruses, the VPg protein attached to the viral RNA is essential for this process because it interacts with a cellular protein, eIF4G, that is normally involved in initiating protein synthesis from the messenger RNA of host genes. We have used a variety of biochemical and biophysical experiments to measure how well MNV VPg binds to eIF4G and to identify the parts of both proteins that are involved in this interaction. We show that a sequence of about 20 amino acids at one end of MNV VPg–the C terminus– allows it to bind to a well-defined domain within eIF4G (called HEAT-1), and that it may adopt a helical structure when doing so. Our data suggest that this interaction is common to all noroviruses, including types that infect humans. We have also shown that the MNV VPg C-terminal peptide can inhibit norovirus protein synthesis, which raises the possibility that the VPg-eIF4G interaction could be targeted in the design of antiviral drugs.
影响因子：
0.9
作者：
Akabayov SR;Wagner G
通讯作者：
Wagner G