Kagele Dominique, Gao Yang, Smallenburg Kate, Pari Gregory S
University of Nevada-Reno School of Medicine, Department of Microbiology and Immunology and the Cell and Molecular Biology Graduate Program, Howard Bldg. 210, Reno, NV 89557, USA.
Virology. 2009 Sep 15;392(1):16-23. doi: 10.1016/j.virol.2009.06.035. Epub 2009 Jul 23.
Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the cis-acting oriLyt region and requires six core replication proteins along with UL84 and IE2. Although UL84 is thought to be the replication initiator protein, little is known about its interaction with oriLyt. We have now performed chromatin immunoprecipitation assays (ChIP) using antibodies specific to UL84, IE2, UL44, CCAAT/enhancer binding protein (C/EBPalpha) and PCR primers that span the entire oriLyt region to reveal an evaluation of specific protein binding across oriLyt. UL84 interacted with several regions of oriLyt that contain C/EBPalpha transcription factor binding sites. Mutation of either of one of C/EBPalpha (92,526 or 92,535) sites inactivated oriLyt and resulted in the loss of binding of UL84. These data reveal the regions of interaction within oriLyt for several key replication proteins and show that the interaction between UL84 and C/EBPalpha sites within oriLyt is essential for lytic DNA replication.
人巨细胞病毒（HCMV）的裂解性DNA复制起始于顺式作用的oriLyt区域，需要六种核心复制蛋白以及UL84和IE2。尽管UL84被认为是复制起始蛋白，但其与oriLyt的相互作用却知之甚少。我们现在使用针对UL84、IE2、UL44、CCAAT/增强子结合蛋白（C/EBPα）的特异性抗体以及跨越整个oriLyt区域的PCR引物进行了染色质免疫沉淀分析（ChIP），以揭示对oriLyt上特定蛋白结合的评估。UL84与oriLyt的几个区域相互作用，这些区域包含C/EBPα转录因子结合位点。C/EBPα（92,526或92,535）位点之一的突变使oriLyt失活，并导致UL84结合丧失。这些数据揭示了oriLyt内几种关键复制蛋白的相互作用区域，并表明oriLyt内UL84与C/EBPα位点之间的相互作用对于裂解性DNA复制至关重要。
