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：
10.1101/024349
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发表时间：
2015-08
期刊：
影响因子：
6.7
通讯作者：
Eoin Leen;Frédéric Sorgeloos;S. Correia;Y. Chaudhry;F. Cannac;Chiara Pastore;Yingqi Xu;S. C. Graham;S. Matthews;I. Goodfellow;S. Curry
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文献类型：
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作者：
Eoin Leen;Frédéric Sorgeloos;S. Correia;Y. Chaudhry;F. Cannac;Chiara Pastore;Yingqi Xu;S. C. Graham;S. Matthews;I. Goodfellow;S. Curry
翻译起始是诺如病毒正向单链RNA基因组复制周期的关键早期步骤，诺如病毒是人类胃肠炎的主要原因。诺如病毒RNA既没有5‘端m7G帽子，也没有内部核糖体进入位点(IRES)，它采用一种不寻常的机制来启动蛋白质合成，这种机制依赖于病毒RNA 5’端共价连接的VPG蛋白与宿主细胞中的真核细胞起始因子(EIF)之间的相互作用。对于小鼠诺如病毒(MNV)，我们以前发现VPG与eIF4G的中间片段(4gm；残基652-1132)结合。在这里，我们使用下拉分析、荧光各向异性和等温滴定热法(ITC)证明了MNV VPG C末端的一段∼20氨基酸介导了与eIF4G 4gm片段中的热-1结构域的直接和特异性结合。我们的分析进一步表明，MNV的C末端通过在所有已知的诺如病毒中保守的基序与eIF4G HEAT-1结合。精细突变图谱表明，MNV VPG C末端可能以螺旋构象与eIF4G相互作用。用核磁共振波谱确定了eIF4G HEAT-1上的VPG结合位点，并通过诱变和结合实验证实了这一点。通过证明诺如病毒VPG可以形成VPG-eIF4G-eIF4A三元复合体，我们发现该位点与eIF4G HEAT-1上eIF4A的结合位点不重叠。VPG-eIF4G相互作用的功能意义在于，含有MNV VPG C末端肽的融合蛋白能够抑制诺如病毒RNA的翻译，但不能抑制CAP或IRES依赖的翻译。这些观察定义了诺如病毒VPG和eIF4G之间功能相互作用的重要结构细节，并揭示了可能被用作抗病毒治疗靶点的结合界面。
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 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.