Gorrill Timothy S, Khalili Kamel
Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 North 12th Street, 015-96, Room 203, Philadelphia, PA 19122, USA.
Virology. 2005 Apr 25;335(1):1-9. doi: 10.1016/j.virol.2005.02.006.
Reactivation of the human polyomavirus BK (BKV) has emerged as an important cause of allograft rejection in renal transplant recipients. Expression of the viral early promoter that leads to production of T-antigen is the first event in viral lytic infection. In an effort to understand the mechanism involved in the activation of BKV early gene (BKV(E)) expression, we analyzed the promoter/enhancer region of the virus and identified binding motifs for the inducible transcription factors NF-kappaB and C/EBPbeta, which are in juxtaposition to each other downstream from the early gene transcription initiation site. Results from transfection studies demonstrate that overexpression of the p65 subunit of NF-kappaB, but not C/EBPbeta stimulates transcription of the BKV(E) promoter in CV-1 cells. Interestingly, low level expression of C/EBPbeta showed a synergistic effect on p65 activation of the BKV(E) promoter, suggesting a functional cooperativity between these two regulators upon viral gene transcription. Results from DNA-binding studies showed the ability of p65 and C/EBPbeta to bind independently with BKV DNA as removal of the binding site for p65 or C/EBPbeta had no significant effect on the interaction of p65 and C/EBPbeta with their motifs, respectively. Functional evaluation of the mutant promoter with no binding sites for either NF-kappaB or C/EBPbeta showed that the observed synergism requires the p65 but not the C/EBPbeta binding site, suggesting cross-talk between C/EBPbeta and p65 in this event. Results from the co-expression of p65 and C/EBPbeta showed no evidence for the formation of a DNA-protein complex containing both p65 and C/EBPbeta, although results from protein-protein interaction studies verified the ability of C/EBPbeta to interact with p65. A dominant-negative isoform of C/EBPbeta which contains the DNA binding but not activation domain of full-length C/EBPbeta cooperated with p65 in activating the BKV(E) promoter, suggesting a functional interaction between the b-ZIP domain of C/EBPbeta and NF-kappaB.
人多瘤病毒BK（BKV）的重新激活已成为肾移植受者同种异体移植排斥反应的一个重要原因。导致T抗原产生的病毒早期启动子的表达是病毒裂解感染的首个事件。为了了解参与BKV早期基因（BKV(E)）表达激活的机制，我们分析了病毒的启动子/增强子区域，并确定了诱导型转录因子NF-κB和C/EBPβ的结合基序，它们在早期基因转录起始位点下游彼此相邻。转染研究结果表明，NF-κB的p65亚基过表达可刺激CV-1细胞中BKV(E)启动子的转录，但C/EBPβ则无此作用。有趣的是，C/EBPβ的低水平表达对BKV(E)启动子的p65激活具有协同作用，表明这两种调节因子在病毒基因转录时存在功能协同性。DNA结合研究结果显示，p65和C/EBPβ能够分别独立与BKV DNA结合，因为去除p65或C/EBPβ的结合位点对p65和C/EBPβ与其基序的相互作用均无显著影响。对既无NF-κB也无C/EBPβ结合位点的突变启动子进行功能评估表明，观察到的协同作用需要p65结合位点而非C/EBPβ结合位点，提示在此过程中C/EBPβ与p65之间存在相互作用。p65和C/EBPβ共表达的结果未显示形成同时包含p65和C/EBPβ的DNA-蛋白质复合物的证据，尽管蛋白质-蛋白质相互作用研究结果证实了C/EBPβ与p65相互作用的能力。一种包含全长C/EBPβ的DNA结合但无激活结构域的C/EBPβ显性负性异构体与p65协同激活BKV(E)启动子，提示C/EBPβ的b-ZIP结构域与NF-κB之间存在功能相互作用。
