Department of Oncology (McArdle Laboratory for Cancer Research), University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
J Virol. 2011 Sep;85(17):8940-53. doi: 10.1128/JVI.00569-11. Epub 2011 Jun 22.
The Epstein-Barr virus (EBV) latent-to-lytic switch is an essential part of the viral life cycle, but the cellular factors that promote viral reactivation are not well defined. In this report, we demonstrate that the cellular transcription factor Oct-1 cooperates with the EBV immediate-early protein BRLF1 (R, Rta) to induce lytic viral reactivation. We show that cotransfected Oct-1 enhances the ability of BRLF1 to activate lytic gene expression in 293 cells stably infected with a BRLF1-defective EBV mutant (BRLF1-stop) and that Oct-1 increases BRLF1-mediated activation of lytic EBV promoters in reporter gene assays. We find that Oct-1 interacts directly with BRLF1 in vitro and that a mutant BRLF1 protein (the M140A mutant) attenuated for the ability to interact with Oct-1 in vitro is also resistant to Oct-1-mediated transcriptional enhancement in 293 BRLF1-stop cells. Furthermore, we show that cotransfected Oct-1 augments BRLF1 binding to a variety of lytic EBV promoters in chromatin immunoprecipitation (ChIP) assays (including the BZLF1, BMRF1, and SM promoters) and that BRLF1 tethers Oct-1 to lytic EBV promoters. In addition, we demonstrate that an Oct-1 mutant defective in DNA binding (the S335D mutant) still retains the ability to enhance BRLF1 transcriptional effects. Finally, we show that knockdown of endogenous Oct-1 expression reduces the level of constitutive lytic EBV gene expression in both EBV-positive B-cell and EBV-positive epithelial cell lines. These results suggest that Oct-1 acts as a positive regulator of EBV lytic gene expression and that this effect is at least partially mediated through its interaction with the viral protein BRLF1.
EBV 潜伏-裂解开关是病毒生命周期的重要组成部分，但促进病毒重新激活的细胞因子尚未得到很好的定义。在本报告中，我们证明了细胞转录因子 Oct-1 与 EBV 早期蛋白 BRLF1（R，Rta）合作诱导裂解病毒重新激活。我们表明，共转染的 Oct-1 增强了 BRLF1 在 293 细胞中激活裂解基因表达的能力，这些细胞稳定感染了 BRLF1 缺陷型 EBV 突变体（BRLF1-stop），并且 Oct-1 增加了 BRLF1 介导的裂解 EBV 启动子在报告基因检测中的激活作用。我们发现 Oct-1 在体外与 BRLF1 直接相互作用，并且体外与 Oct-1 相互作用能力减弱的突变 BRLF1 蛋白（M140A 突变体）也对 293 BRLF1-stop 细胞中 Oct-1 介导的转录增强具有抗性。此外，我们表明共转染的 Oct-1 增强了 BRLF1 在染色质免疫沉淀（ChIP）测定中与多种裂解 EBV 启动子（包括 BZLF1、BMRF1 和 SM 启动子）的结合，并且 BRLF1 将 Oct-1 连接到裂解 EBV 启动子。此外，我们证明了在 DNA 结合中具有缺陷的 Oct-1 突变体（S335D 突变体）仍然保留增强 BRLF1 转录效应的能力。最后，我们表明内源性 Oct-1 表达的敲低降低了 EBV 阳性 B 细胞和 EBV 阳性上皮细胞系中组成性裂解 EBV 基因表达的水平。这些结果表明 Oct-1 作为 EBV 裂解基因表达的正调节剂起作用，并且这种作用至少部分是通过其与病毒蛋白 BRLF1 的相互作用介导的。
