Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.
Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.
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DOI：
10.1371/journal.ppat.1001244
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发表时间：
2011-01-06
期刊：
影响因子：
6.7
通讯作者：
Golovanov AP
中科院分区：
文献类型：
--
作者：
Tunnicliffe RB;Hautbergue GM;Kalra P;Jackson BR;Whitehouse A;Wilson SA;Golovanov AP
疱疹病毒蛋白HSV-1、ICP27和HVS ORF57通过利用细胞内的mRNA输出机制促进病毒的mRNA输出。这一功能是由与转录-输出(TREX)复合体的蛋白质结合触发的，特别是与REF/Aly结合，REF/Aly将病毒mRNA导向TAP/NFX1途径，随后进入核孔，输出到细胞质。在这里，我们确定了REF-ICP27相互作用界面的原子分辨结构，并用溶液状态核磁共振详细比较了ICP27、ORF57和REF之间的结合界面。尽管没有任何明显的序列相似性，但这两种病毒蛋白通过简短但特异的识别位点结合在REF的折叠RRM结构域的同一位置。参与REF结合的ICP27和ORF57区域分别被定位为残基104-112和103-120。我们已经确定了对REF/Aly识别至关重要的残基模式，这对ICP27和ORF57都是共同的。通过定点突变证实了这些结合位点中关键氨基酸残基的重要性。ORF57-REF/Aly相互作用的功能意义也通过体外细胞质病毒mRNA积累实验进行了探讨，这表明降低蛋白质-蛋白质相互作用的突变体显著降低了ORF57介导无内含子病毒mRNA核输出的能力。总之，这些数据精确地定位了负责病毒适配器和细胞ref/Aly之间直接相互作用的氨基酸残基，并提供了疱疹病毒如何进入细胞mRNA输出途径的第一个分子细节。当侵入宿主细胞时，疱疹病毒会劫持细胞成分，使其能够复制。长期以来，人们已经认识到，每种疱疹病毒都有一个特定的标志性接头蛋白，除了其他功能外，它还将病毒mRNA插入到细胞mRNA核输出途径中，使宿主细胞能够产生病毒蛋白。这一过程在体内和体外都得到了广泛的研究，但尽管做了很多努力，但病毒接头与细胞mRNA输出因子之间关键相互作用的分子和结构机制尚未被描述。在这里，我们展示了原型病毒接头ICP27(来自单纯疱疹病毒1)和细胞mRNA输出因子REF之间的关键复合体的第一个原子分辨结构，REF负责将病毒mRNA引入细胞核输出途径。我们证明，尽管没有明显的序列相似性，但来自不同疱疹病毒(疱疹病毒SAIMIRI)的适配蛋白ORF57以相似的方式在相同的位置结合REF。我们已经鉴定并研究了与REF识别有关的氨基酸残基。这些数据一起提供了关于疱疹病毒签名蛋白如何识别细胞蛋白的第一个分子洞察力，从而获得了进入细胞mRNA输出机制的途径。
The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway. When invading host cells, herpes viruses highjack cellular components to allow them to replicate. It has been long recognized that each herpes virus has a specific signature adaptor protein which, among other functions, inserts viral mRNA into the cellular mRNA nuclear export pathway, enabling production of viral proteins by the host cell. This process has been extensively studied in vivo and in vitro, but despite many efforts, the molecular and structural mechanisms of key interactions between viral adaptors and cellular mRNA export factors have not been described. Here we present the first atomic-resolution structure of the key complex between the archetypal viral adaptor ICP27 (from Herpes simplex virus 1) and the cellular mRNA export factor REF, responsible for introducing viral mRNA into the cellular nuclear export pathway. We demonstrate that despite the absence of obvious sequence similarity, the adaptor protein ORF57 from a different herpes virus (Herpesvirus saimiri) binds REF in the same site and in a similar way. We have identified and studied amino acid residues responsible for REF recognition. Together the data provide the first molecular insight into how herpesviral signature proteins recognize cellular proteins, obtaining access to the cellular mRNA export machinery.
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