Davenport M G, Pagano J S
Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.
J Virol. 1999 Apr;73(4):3154-61. doi: 10.1128/JVI.73.4.3154-3161.1999.
Expression of EBNA-1 protein is required for the establishment and maintenance of the Epstein-Barr virus (EBV) genome during latent infection. During type I latency, the BamHI Q promoter (Qp) gives rise to EBNA-1 expression. The dominant regulatory mechanism for Qp appears to be mediated through the Q locus, located immediately downstream of the transcription start site. Binding of EBNA-1 to the Q locus represses Qp constitutive activity, and repression has been reported to be overcome by an E2F family member that binds to the Q locus and displaces EBNA-1 (N. S. Sung, J. Wilson, M. Davenport, N. D. Sista, and J. S. Pagano, Mol. Cell. Biol. 14:7144-7152, 1994). These data suggest that the final outcome of Qp activity is reciprocally controlled by EBNA-1 and E2F. Since E2F activity is cell cycle regulated, Qp activity and EBNA-1 expression are predicted to be regulated in a cell cycle-dependent manner. Proliferation of the type I latently infected cell line, Akata, was synchronized with the use of the G2/M blocking agent nocodazole. From 65 to 75% of cells could be made to peak in S phase without evidence of viral reactivation. Following release from G2/M block, EBNA-1 mRNA levels declined as the synchronized cells entered the G1 phase of the cell cycle. As cells proceeded into S phase, EBNA-1 mRNA levels increased parallel to the peak in cell numbers in S phase. However, EBNA-1 protein levels showed no detectable change during the cell cycle, most likely due to the protein's long half-life as estimated by inhibition of protein synthesis by cycloheximide. Finally, in Qp luciferase reporter assays, the activity of Qp was shown to be regulated by cell cycle and to be dependent on the E2F sites within the Q locus. These findings demonstrate that transcriptional activity of Qp is cell cycle regulated and indicated that E2F serves as the stimulus for this regulation.
EBNA-1蛋白的表达是潜伏感染期间爱泼斯坦-巴尔病毒（EBV）基因组建立和维持所必需的。在I型潜伏期间，BamHI Q启动子（Qp）可导致EBNA-1的表达。Qp的主要调控机制似乎是通过位于转录起始位点下游紧邻的Q位点介导的。EBNA-1与Q位点的结合会抑制Qp的组成型活性，据报道，一种与Q位点结合并取代EBNA-1的E2F家族成员可克服这种抑制作用（N.S. Sung、J. Wilson、M. Davenport、N.D. Sista和J.S. Pagano，《分子细胞生物学》14:7144 - 7152，1994年）。这些数据表明，Qp活性的最终结果受到EBNA-1和E2F的相互控制。由于E2F活性受细胞周期调控，因此预计Qp活性和EBNA-1表达会以细胞周期依赖性方式受到调控。使用G2/M阻断剂诺考达唑使I型潜伏感染细胞系Akata的增殖同步化。65%至75%的细胞可在S期达到峰值，且无病毒重新激活的迹象。从G2/M阻断中释放后，随着同步化细胞进入细胞周期的G1期，EBNA-1 mRNA水平下降。当细胞进入S期时，EBNA-1 mRNA水平随着S期细胞数量的峰值平行增加。然而，EBNA-1蛋白水平在细胞周期中未显示出可检测到的变化，这很可能是由于通过环己酰亚胺抑制蛋白质合成所估计的该蛋白的长半衰期所致。最后，在Qp荧光素酶报告基因测定中，Qp的活性显示受细胞周期调控且依赖于Q位点内的E2F位点。这些发现表明Qp的转录活性受细胞周期调控，并表明E2F是这种调控的刺激因素。
