Nakayama Sanae, Murata Takayuki, Murayama Kazutaka, Yasui Yoshihiro, Sato Yoshitaka, Kudoh Ayumi, Iwahori Satoko, Isomura Hiroki, Kanda Teru, Tsurumi Tatsuya
Division of Virology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681, Japan.
J Biol Chem. 2009 Aug 7;284(32):21557-68. doi: 10.1074/jbc.M109.015685. Epub 2009 Jun 2.
The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, whereas the BALF2 protein is a single-stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.
爱泼斯坦-巴尔病毒（EBV）的BMRF1蛋白是一种必需的复制蛋白，作为病毒DNA聚合酶持续性因子在病毒复制叉处发挥作用，而BALF2蛋白是一种单链DNA结合蛋白，也在复制叉处发挥作用，并且在病毒生产性复制期间表达最为丰富。在此我们证明，BMRF1蛋白明显增强了病毒BZLF1转录因子介导的BALF2基因启动子的反式激活。诱变和电泳迁移率变动分析表明，BALF2启动子含有两个BZLF1蛋白结合位点（BZLF1反应元件）。BZLF1蛋白与BZLF1反应元件的直接结合以及BZLF1与BMRF1蛋白之间的物理相互作用是BMRF1蛋白上调BALF2基因启动子的先决条件。一种在同源二聚化方面有缺陷的单体突变体C95E，仍然可以相互作用并增强BZLF1介导的反式激活。此外，尽管EBV蛋白激酶广泛磷酸化BMRF1蛋白，但结果表明，该激酶对该蛋白的磷酸化抑制了BZLF1介导的BALF2启动子反式激活的增强。BMRF1蛋白的外源表达增强了携带EBV基因组但缺乏BMRF1和BALF5基因的HEK293细胞中BALF2的表达，证明其在病毒感染情况下作为转录调节因子的功能。总体而言，BMRF1蛋白是一种多功能蛋白，它不仅可以作为DNA聚合酶持续性因子发挥作用，还可以作为BZLF1蛋白的共激活因子增强BALF2启动子转录，调节病毒单链DNA结合蛋白的表达水平。
