Re-localization of cellular protein SRp20 during poliovirus infection: bridging a viral IRES to the host cell translation apparatus.
Re-localization of cellular protein SRp20 during poliovirus infection: bridging a viral IRES to the host cell translation apparatus.
复制标题
DOI：
10.1371/journal.ppat.1002127
复制
发表时间：
2011-07
期刊：
影响因子：
6.7
通讯作者：
Semler BL
中科院分区：
文献类型：
--
作者：
Fitzgerald KD;Semler BL
脊髓灰质炎病毒ires介导的翻译需要某些规范因子和非规范因子的功能来将核糖体募集到病毒RNA上。在脊髓灰质炎病毒感染细胞的提取物中，细胞蛋白PCBP2和SRp20的相互作用已经被描述过，这两种蛋白在病毒翻译中显示出协同作用。为了进一步确定核糖体募集启动脊髓灰质炎病毒ires依赖翻译的机制，我们重点研究了细胞蛋白PCBP2和SRp20之间相互作用的作用。研究表明，SRp20在脊髓灰质炎病毒感染的神经母细胞瘤细胞感染过程中显著地从细胞核重新定位到细胞质。重要的是，SRp20在感染细胞的细胞质中与PCBP2部分共定位，证实了我们之前的体外相互作用数据。此外，这些数据表明这两种蛋白存在于病毒翻译起始复合物中。我们发现，在脊髓灰质炎病毒感染细胞的提取物中，SRp20与与脊髓灰质炎病毒RNA结合的PCBP2相关，表明这种相互作用发生在病毒RNA上。最后，我们生成了一个缺乏RNA识别基序的SRp20突变版本（SRp20ΔRRM），发现该蛋白的定位与全长SRp20相似，并且在脊髓灰质炎病毒感染期间也与PCBP2部分共定位。与野生型SRp20的表达相比，表达这种突变版本的SRp20导致脊髓灰质炎病毒的病毒产量下降~ 100倍，可能是通过显性负作用。综上所述，这些结果与SRp20与结合在病毒RNA上的PCBP2相互作用的模型是一致的，这种相互作用通过其他蛋白质-蛋白质或蛋白质-RNA相互作用的参与，以直接或间接的方式招募核糖体到病毒RNA上。小核糖核酸病毒是一种正感RNA病毒，可引起从普通感冒到脊髓灰质炎等多种疾病。脊髓灰质炎病毒是小核糖核酸病毒科中研究最广泛的成员之一。然而，对病毒RNA基因组指导其蛋白质产物合成的机制尚缺乏完整的了解。脊髓灰质炎病毒篡夺宿主细胞翻译机制，通过不同于细胞帽结合、核糖体扫描翻译模型的机制启动病毒多蛋白合成。这使得病毒可以下调宿主细胞的翻译，同时为其自身的基因表达提供优势。由于其基因组小，脊髓灰质炎病毒利用宿主细胞蛋白促进翻译机制的招募，这一过程仍未完全确定。先前的工作强调了两种特殊的宿主细胞RNA结合蛋白在脊髓灰质炎病毒翻译中的重要性。在这里，我们采用成像技术、分离分析和RNA结合实验来进一步研究这些蛋白质在脊髓灰质炎病毒翻译中发挥的特定作用。我们还生成了其中一种蛋白的截短版本，并观察到其对病毒生长的显著影响，突出了其在脊髓灰质炎病毒感染期间的重要性，并支持了我们将细胞翻译装置连接到病毒RNA的模型。
Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a ∼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions. Picornaviruses are positive-sense RNA viruses that cause diseases ranging from the common cold to poliomyelitis. Poliovirus is one of the most extensively studied members of the Picornaviridae family. However, a complete understanding of the mechanism by which the viral RNA genome directs the synthesis of its protein products is lacking. Poliovirus usurps the host cell translation machinery to initiate viral polyprotein synthesis via a mechanism distinct from the cellular cap-binding, ribosome scanning model of translation. This allows the virus to down-regulate host cell translation while providing an advantage for its own gene expression. Owing to its small genome size, poliovirus utilizes host cell proteins to facilitate the recruitment of the translation machinery, a process that is still not completely defined. Previous work highlighted the importance of two particular host cell RNA binding proteins in poliovirus translation. Here we employ imaging techniques, fractionation assays, and RNA binding experiments to further examine the specific role these proteins play in poliovirus translation. We also generated a truncated version of one of the proteins and observed a dramatic effect on virus growth, highlighting its significance during poliovirus infection and supporting our model for bridging the cellular translation apparatus to viral RNA.
登录
查看更多内容
影响因子：
11.4
作者：
Bedard, Kristin M.;Daijogo, Sarah;Semler, Bert L.
通讯作者：
Semler, Bert L.
影响因子：
5.4
作者：
Kerekatte, V;Keiper, BD;Rhoads, RE
通讯作者：
Rhoads, RE
影响因子：
3.3
作者：
Allemand, E;Dokudovskaya, S;Tazi, J
通讯作者：
Tazi, J
影响因子：
3.7
作者：
BALTIMORE, D;GIRARD, M;DARNELL, JE
通讯作者：
DARNELL, JE
影响因子：
5.4
作者：
Belov, GA;Lidsky, PV;Agol, VI
通讯作者：
Agol, VI