Institute of Virology, Saarland University Medical School, Homburg/Saar, Germany.
PLoS One. 2012;7(8):e42106. doi: 10.1371/journal.pone.0042106. Epub 2012 Aug 3.
The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.
EB 病毒（EBV）编码的 EBNA2 蛋白对于体外转化 B 淋巴细胞至关重要，它通过结合蛋白质上的保守序列基序来干扰细胞过程。其精氨酸-甘氨酸（RG）重复元件包含对称或非对称二甲基化的精氨酸残基（SDMA 和 ADMA，分别）。EBNA2 通过其 SDMA 修饰的 RG 重复与存活运动神经元蛋白（SMN）结合，通过 ADMA-RG 重复与人类内源性逆转录病毒 K 的 NP9 蛋白（HERV-K（HML-2）Type 1）结合。这项工作的假设是，甲基化 RG 重复模拟与细胞蛋白共享的表位，用于与靶结构相互作用。使用针对修饰 RG 重复的单克隆抗体，我们确实鉴定了细胞同源物，这些同源物显然具有与甲基化 EBNA2 相同的表面结构。使用 SDMA 特异性抗体，我们沉淀了 Sm 蛋白 D3（SmD3），与 EBNA2 一样，SmD3 通过其 SDMA 修饰的 RG 重复与 SMN 结合。使用 ADMA 特异性抗体，我们沉淀了异质核糖核蛋白 K（hnRNP K）。使用在大肠杆菌中表达/ADMA 甲基化 hnRNP K 证明了 ADMA 抗体与 hnRNP K 的特异性结合。此外，我们还表明 EBNA2 和 hnRNP K 在 EBV 感染的 B 细胞中形成复合物。最后，hnRNP K 与 EBNA2 共表达时，通过未知机制强烈增强病毒潜伏膜蛋白 2A（LMP2A）的表达，因为我们在凝胶迁移实验中没有检测到 hnRNP K 与 DNA 结合的 EBNA2 之间的直接关联。我们的数据支持这样的观点，即 EBNA2 甲基化表面模拟细胞蛋白的表面结构，以干扰或篡夺其功能特性。
