Farrell Christopher J, Lee Jae Myun, Shin Eui-Cheol, Cebrat Marek, Cole Philip A, Hayward S Diane
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 1650 Orleans Street, Baltimore, MD 21231, USA.
Proc Natl Acad Sci U S A. 2004 Mar 30;101(13):4625-30. doi: 10.1073/pnas.0306482101. Epub 2004 Mar 19.
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. Antiviral drugs targeted against lytic viral replication have limited efficacy in these disease settings. EBV infection of peripheral blood mononuclear cells induces growth proliferation and the EBV latency Epstein-Barr virus-encoded nuclear antigen (EBNA)2 transcriptional transactivator (TAT) is essential for this response. EBNA2 targets the cellular DNA-binding protein CBF1 to mimic activated Notch signaling. A 10-aa peptide from the CBF1 interaction domain of EBNA2 was synthesized as a fusion with the protein transduction domain of HIV-1 TAT. The EBNA2-TAT peptide blocked EBNA2-CBF1 interaction in an in vitro GST affinity assay and labeling with fluorescein confirmed that the EBNA2-TAT peptide efficiently entered cultured B cells. Neither EBNA2-TAT, nor a mutant peptide with a 2-aa substitution that was unable to block the EBNA2-CBF1 interaction, significantly affected the growth of non-EBNA2-expressing EBV(-) B cells or Burkitt's lymphoma Akata cells. However, treatment of an EBV-immortalized lymphoblastoid cell line with the EBNA2-TAT peptide stopped cell growth and reduced cell viability. RT-PCR analyses of gene expression in the peptide-treated lymphoblastoid cell line cultures revealed that EBNA2-TAT treatment down-regulated the EBNA2-responsive viral LMP1 and LMP2 genes and cellular CD23, intercellular adhesion molecule 1, BATF, and Cdk1 genes while up-regulating expression of the cyclin-dependent kinase inhibitor p21. EBV-induced outgrowth of B cells from cultured peripheral blood mononuclear cells was also blocked in a dose-responsive manner by the EBNA2-TAT peptide. This study suggests that cell-permeable EBNA2 peptides may have potential as novel anti-EBV therapeutics.
爱泼斯坦-巴尔病毒（EBV）可引起传染性单核细胞增多症，并与免疫功能低下人群的癌症有关。针对病毒裂解复制的抗病毒药物在这些疾病环境中的疗效有限。外周血单核细胞的EBV感染会诱导生长增殖，而EBV潜伏感染时爱泼斯坦-巴尔病毒编码的核抗原（EBNA）2转录反式激活因子（TAT）对这种反应至关重要。EBNA2靶向细胞DNA结合蛋白CBF1以模拟活化的Notch信号。合成了一种来自EBNA2的CBF1相互作用结构域的10个氨基酸的肽，并与HIV-1 TAT的蛋白质转导结构域融合。在体外GST亲和试验中，EBNA2-TAT肽阻断了EBNA2-CBF1相互作用，用荧光素标记证实EBNA2-TAT肽能有效进入培养的B细胞。EBNA2-TAT和一种具有2个氨基酸替代的突变肽（无法阻断EBNA2-CBF1相互作用）均未显著影响不表达EBNA2的EBV（-）B细胞或伯基特淋巴瘤Akata细胞的生长。然而，用EBNA2-TAT肽处理EBV永生化淋巴母细胞系会停止细胞生长并降低细胞活力。对肽处理的淋巴母细胞系培养物中的基因表达进行RT-PCR分析表明，EBNA2-TAT处理下调了EBNA2反应性病毒LMP1和LMP2基因以及细胞CD23、细胞间粘附分子1、BATF和Cdk1基因，同时上调了细胞周期蛋白依赖性激酶抑制剂p21的表达。EBNA2-TAT肽也以剂量反应方式阻断了EBV诱导的培养外周血单核细胞中B细胞的生长。这项研究表明，细胞可渗透的EBNA2肽可能具有作为新型抗EBV治疗药物的潜力。
