Isaksson Asa, Berggren Malin, Ekeland-Sjöberg Kerstin, Samuelsson Tore, Ricksten Anne
Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, Gothenburg, Sweden.
J Med Virol. 2007 May;79(5):573-81. doi: 10.1002/jmv.20854.
The U leader exon in the 5' untranslated region of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site, the EBNA internal ribosome entry segment (IRES), which promotes cap-independent translation and increases the expression level of the EBNA1 protein. It was previously reported that immunosuppressed organ transplanted patients showed an alternatively spliced EBNA1 transcript, excluding the EBNA IRES element. To further investigate the function of the EBNA IRES, sequence analysis of the EBNA IRES mRNA was performed in samples from seven organ transplant patients. Two nucleotide changes, G --> A at position 67531 and C --> U at position 67585 were found in the EBNA IRES mRNA, relative to the corresponding genomic Epstein-Barr virus (EBV) sequence in all patients. Moreover, the patient derived EBNA IRES mRNA was shown to differ from the IRES mRNA derived from the cell line B95.8 at position 67531 and from the cell lines Rael and P3HR1 at positions 67531 and 67585. cDNA from the various EBNA IRES sequences were cloned into bicistronic vectors, respectively, and used in transient transfection experiments in six human cell lines. The patient specific sequence significantly decreased the IRES activity in T-cells, while the base changes had no significant impact on the activity in B- or in epithelial cells. The genetic mechanisms behind EBV-associated diseases are complex, involving gene regulation by alternative promoters, alternative splicing, and translational control. The nucleotide changes in the patient specific EBNA IRES transcript and its influence on the translational activity, might illustrate new strategies utilised by the EBV to adapt to the immune control in patients with EBV associated diseases.
爱泼斯坦-巴尔病毒核抗原1（EBNA1）基因5'非翻译区的上游主要外显子包含一个内部核糖体进入位点，即EBNA内部核糖体进入片段（IRES），它促进不依赖帽子结构的翻译并增加EBNA1蛋白的表达水平。此前有报道称，免疫抑制的器官移植患者显示出一种可变剪接的EBNA1转录本，其中不包含EBNA IRES元件。为了进一步研究EBNA IRES的功能，对7名器官移植患者样本中的EBNA IRES mRNA进行了序列分析。相对于所有患者中相应的基因组爱泼斯坦-巴尔病毒（EBV）序列，在EBNA IRES mRNA中发现了两个核苷酸变化，第67531位的G→A和第67585位的C→U。此外，患者来源的EBNA IRES mRNA在第67531位与细胞系B95.8来源的IRES mRNA不同，在第67531位和第67585位与细胞系Rael和P3HR1来源的IRES mRNA不同。分别将来自各种EBNA IRES序列的cDNA克隆到双顺反子载体中，并用于6种人类细胞系的瞬时转染实验。患者特异性序列显著降低了T细胞中的IRES活性，而碱基变化对B细胞或上皮细胞中的活性没有显著影响。EBV相关疾病背后的遗传机制很复杂，涉及由可变启动子、可变剪接和翻译控制进行的基因调控。患者特异性EBNA IRES转录本中的核苷酸变化及其对翻译活性的影响，可能说明了EBV用于适应EBV相关疾病患者免疫控制的新策略。
