Transforming Growth Factor β-Induced Reactivation of Epstein-Barr Virus Involves Multiple Smad-Binding Elements Cooperatively Activating Expression of the Latent-Lytic Switch BZLF1 Gene
Transforming Growth Factor β-Induced Reactivation of Epstein-Barr Virus Involves Multiple Smad-Binding Elements Cooperatively Activating Expression of the Latent-Lytic Switch BZLF1 Gene
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DOI：
10.1128/jvi.01197-10
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发表时间：
2011-08-01
影响因子：
5.4
通讯作者：
Mertz, Janet E.
中科院分区：
文献类型：
--
作者：
Iempridee, Tawin;Das, Shreyasi;Mertz, Janet E.
转化生长因子β通过激活EB病毒潜伏裂解开关BZLF1基因的表达，生理性地诱导EB病毒裂解感染。梁等人(J.Biol.化学。23345-23357,2002年)以前在BZLF1启动子ZP中发现了一个Smad结合元件；然而，它只占转化生长因子-β介导的ZP转录激活的20%到30%。在这里，我们确定了导致其余激活的其他因素。将EBV阳性的MutuI细胞与转化生长因子-β中和抗体或转化生长因子-βI型受体(T-βRI)或Smad3的抑制剂孵育，可消除转化生长因子-β诱导的EB病毒的重新激活。在胃癌AGS细胞中，Smad2、Smad3和Smad4的共同表达以及一个结构性的活性形式TβRI可诱导ZP转录15-25倍。通过电泳迁移率改变分析，我们鉴定了另外四个Smad结合元件，命名为SBE2至SBE5。单个SBE中的替换突变使Smad介导的ZP激活减少了20%到60%；这些突变加在一起，基本上消除了它。染色质免疫沉淀分析证实，Smad4在与转化生长因子-β孵育MutuI细胞后，新结合了EBV基因组的ZP区。SBE2与ZP的ZEB结合的ZV沉默元件重叠。根据翻译后修饰的不同，Smad4要么与ZEB1竞争结合，要么在无细胞检测系统中与ZPZV元件上的ZEB1形成复合体。在瞬时转染的细胞中，外源表达的ZEB1抑制了Smad介导的ZP转录激活。我们认为，转化生长因子-β通过多个SBE协同作用激活BZLF1基因表达，通过典型的Smad途径诱导EBV裂解再激活。
Transforming growth factor beta (TGF-beta) physiologically induces Epstein-Barr virus (EBV) lytic infection by activating the expression of EBV's latent-lytic switch BZLF1 gene. Liang et al. (J. Biol. Chem. 277:23345-23357, 2002) previously identified a Smad-binding element (SBE) within the BZLF1 promoter, Zp; however, it accounts for only 20 to 30% of TGF-beta-mediated activation of transcription from Zp. Here, we identified additional factors responsible for the rest of this activation. The incubation of EBV-positive MutuI cells with a TGF-beta neutralizing antibody or inhibitors of the TGF-beta type I receptor (T beta RI) or Smad3 eliminated the TGF-beta-induced reactivation of EBV. The coexpression of Smad2, Smad3, and Smad4 together with a constitutively active form of T beta RI induced 15- to 25-fold transcription from Zp in gastric carcinoma AGS cells. By electrophoretic mobility shift assays, we identified four additional Smad-binding elements, named SBE2 to SBE5. Substitution mutations in individual SBEs reduced Smad-mediated activation of Zp by 20 to 60%; together, these mutations essentially eliminated it. Chromatin immunoprecipitation assays confirmed that Smad4 newly bound the Zp region of the EBV genome following the incubation of MutuI cells with TGF-beta. SBE2 overlaps the ZEB-binding ZV silencing element of Zp. Depending upon posttranslational modifications, Smad4 either competed with ZEB1 for binding or formed a complex with ZEB1 on the Zp ZV element in a cell-free assay system. In transiently transfected cells, exogenously expressed ZEB1 inhibited Smad-mediated transcriptional activation from Zp. We conclude that TGF-beta induces EBV lytic reactivation via the canonical Smad pathway by activating BZLF1 gene expression through multiple SBEs acting in concert.