Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence virus replication.
Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence virus replication.
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DOI：
10.1016/j.virol.2009.06.006
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发表时间：
2009-09-01
期刊：
影响因子：
3.7
通讯作者：
Almazán F
中科院分区：
文献类型：
--
作者：
Galán C;Sola I;Nogales A;Thomas B;Akoulitchev A;Enjuanes L;Almazán F
冠状病毒RNA合成是由多酶复制酶复合物与细胞因子一起进行的。该过程需要特异性识别位于病毒基因组末端的RNA顺式作用信号。为了鉴定参与冠状病毒RNA合成的细胞蛋白，将携带复制所必需的顺式作用信号的传染性胃肠炎冠状病毒（TGEV）基因组末端用作RNA亲和蛋白纯化的诱饵。通过蛋白质组学分析，10个蛋白质优先被基因组的5′或3′端拉下并被鉴定。其中9个蛋白质，包括异质核糖核蛋白家族（hnRNP）、多聚腺苷酸结合蛋白（PABP）、p100转录共激活蛋白和两个氨酰tRNA合成酶，显示出优先与基因组的3′端结合，而只有多聚嘧啶片段结合蛋白（PTB）优先与基因组的5′端结合。通过使用TGEV衍生的复制子和感染性病毒分析其沉默的效果，研究了3′端相互作用蛋白在病毒复制中的潜在功能。PABP、hnRNP Q和谷氨酰-脯氨酰-tRNA合成酶（EPRS）的基因沉默导致病毒RNA合成显著减少2至3倍。有趣的是，最初用作对照基因的甘油醛3-磷酸脱氢酶（GAPDH）的沉默导致两种系统中病毒RNA合成增加2至3倍。这些数据表明，PABP，hnRNP Q和EPRS在病毒感染中起积极作用，可能是通过与病毒3′端的相互作用介导的，而GAPDH对病毒感染有负面影响。
Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of RNA cis-acting signals located at the ends of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affinity protein purification. Ten proteins were preferentially pulled down with either the 5′ or 3′ ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA synthetases, showed a preferential binding to the 3′ end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5′ end of the genome. The potential function of the 3′ end-interacting proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3′ end, and that GAPDH has a negative effect on viral infection.
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