Hiriart Edwige, Bardouillet Lucie, Manet Evelyne, Gruffat Henri, Penin François, Montserret Roland, Farjot Géraldine, Sergeant Alain
Laboratoire de Virologie, Ens-Lyon, INSERM U 412, 46 Allée d'Italie, 69364 Lyon Cedex 07, France.
J Biol Chem. 2003 Sep 26;278(39):37790-8. doi: 10.1074/jbc.M305925200. Epub 2003 Jul 12.
The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because approximately 10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVDeltaEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle.
爱泼斯坦-巴尔病毒（EBV）蛋白EB2（也称为Mta、SM或BMLF1）具有与mRNA输出因子相同的特性，并且对于EBV感染性病毒粒子的产生至关重要。然而，迄今为止，尚未在该蛋白中定位到对EB2介导的mRNA输出必不可少的RNA结合基序。我们通过蛋白质印迹分析表明，从哺乳动物细胞中纯化的EB2蛋白直接与RNA结合。此外，使用重叠的谷胱甘肽S-转移酶（GST）-EB2肽，我们通过RNA电泳迁移率变动分析（REMSA）和蛋白质印迹，在EB2的一个33个氨基酸的片段中定位到一个RNA结合基序，该片段具有在许多RNA结合蛋白中也发现的富含精氨酸的RNA结合基序（ARM）的结构特征。一个包含此EB2 ARM的合成肽（称为Da）在REMSA中与RNA结合。一个GST-Da融合蛋白在REMSA中也与RNA结合，且没有明显的RNA序列特异性，因为大约10个GST-Da分子在一个180个核苷酸的RNA片段上的多个位点结合。重要的是，ARM区域的一个短缺失损害了EB2在体内和体外与RNA的结合以及EB2介导的mRNA输出，而不影响EB2在细胞核和细胞质之间的穿梭。此外，携带EBVDeltaEB2基因组的293细胞中，ARM缺失的EB2的异位表达不能挽救感染性病毒粒子的产生，这表明EB2与RNA的结合在EBV生产周期中起重要作用。
