Competitive and cooperative interactions mediate RNA transfer from herpesvirus saimiri ORF57 to the mammalian export adaptor ALYREF.
Competitive and cooperative interactions mediate RNA transfer from herpesvirus saimiri ORF57 to the mammalian export adaptor ALYREF.
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DOI：
10.1371/journal.ppat.1003907
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发表时间：
2014-02
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;Wilson SA;Kalra P;Golovanov AP
在所有其他疱疹病毒中具有同源物的疱疹病毒必需衔接蛋白HVS ORF 57通过利用细胞mRNA输出机制促进病毒mRNA输出。ORF 57蛋白特异性识别病毒mRNA转录物，并结合细胞转录-输出（TREX）复合物的蛋白质，特别是TREREF。这种相互作用将病毒mRNA引入NXF 1途径，随后将其引导至核孔以输出至细胞质。在这里，我们使用了一系列的技术来揭示的网站之间的直接接触RNA和ORF 57的存在和不存在的情况下，ORF 57。ORF 57内的结合位点被表征为识别特定的病毒mRNA基序。当有α-REF存在时，该ORF 57 RNA结合位点的一部分（由α-螺旋组成）优先与α-REF结合。这竞争性地将病毒RNA从α-螺旋中置换出来，但与RNA的接触仍然由侧翼区域维持。与此同时，ERAREF的柔性N-末端结构域与病毒RNA接触，从而与ERAREF和ORF 57形成广泛的协同相互作用网络。通过UV交联和诱变证实了RNA转移到三元复合物中的ORF 57残基，以及单个ORF 57残基参与RNA识别。确定了ORF 57-BREF界面的原子分辨率结构，其明显不同于同源ICP 27-BREF结构。总之，这些数据提供了第一个位点特异性描述病毒mRNA如何被疱疹病毒接头蛋白与细胞内的CRISPR REF复合物锁定，使疱疹病毒能够进入细胞mRNA输出机制。所使用的NMR策略可能更普遍地适用于研究涉及柔性多肽区域的模糊蛋白质-蛋白质-RNA复合物。疱疹病毒侵入细胞，劫持细胞成分以维持其生命周期和复制。感染的一个关键步骤是将病毒mRNA从细胞核输出到细胞质，细胞质是产生蛋白质的分子机制所在。为了在它们的mRNA和mRNA输出途径的细胞组分之间提供联系，所有疱疹病毒都使用特殊的衔接蛋白。这些衔接蛋白特异性地从细胞核中存在的混合物中选择病毒mRNA，并将它们引入细胞mRNA输出因子，如BMPREF。这些病毒适配器是如何设法欺骗CRAREF接受外来遗传物质的，还没有在分子水平上被理解。在这项研究中，我们揭示了一个典型的病毒衔接蛋白ORF 57如何识别特定的病毒RNA基序，以及它如何与细胞蛋白ORF REF结合。我们揭示了ORF 57如何将病毒RNA转移到CRFREF，将其锁定在合作的三元复合物中的细节。我们还描述了ORF 57-BREF相互作用界面的原子分辨率结构。这些数据一起提供了关于病毒mRNA如何在病毒和细胞蛋白之间转移的第一个分子见解，从而帮助病毒劫持细胞。
The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions. Herpes viruses invade cells, hijacking cellular components to sustain their lifecycle and replicate. A critical step of infection is the export of viral mRNA from the nucleus to the cytoplasm, where the molecular machinery to produce proteins is located. To provide a link between their mRNA and cellular components of the mRNA export pathway, all herpesviruses use special adaptor proteins. These adaptor proteins specifically select viral mRNAs from the mixture present in the nucleus, and introduce them to cellular mRNA export factors, such as ALYREF. How these viral adaptors manage to trick ALYREF to accept foreign genetic material has not been understood on a molecular level. In this study we reveal how a typical viral adaptor protein ORF57 recognizes specific viral RNA motifs, and also how it binds to the cellular protein ALYREF. We uncover details of how ORF57 transfers the viral RNA to ALYREF, locking it in the cooperative ternary complex. We also describe the atomic-resolution structure of ORF57-ALYREF interaction interface. Together the data provides the first molecular insight of how viral mRNA is transferred between viral and cellular proteins, thus helping virus to hijack a cell.
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影响因子：
10.5
作者：
CALNAN, BJ;BIANCALANA, S;FRANKEL, AD
通讯作者：
FRANKEL, AD
DOI：
10.1038/nsb773
发表时间：
2002-04-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
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作者：
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影响因子：
15
作者：
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通讯作者：
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影响因子：
5.6
作者：
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通讯作者：
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