Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis
Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis
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DOI：
10.1128/jvi.06303-11
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发表时间：
2012-05-01
影响因子：
5.4
通讯作者：
Marechal, Vincent
中科院分区：
文献类型：
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作者：
Jourdan, Nathalie;Jobart-Malfait, Aude;Marechal, Vincent
爱泼斯坦-巴尔病毒(EBV)在人类中建立了一种终身潜伏感染。在增殖的潜伏感染细胞中，EBV基因组以多个附着体的形式存在，每个细胞周期经历一次DNA复制事件，并保持附着在有丝分裂染色体上。EB病毒核抗原1(EBNA-1)与EB病毒和细胞基因组结合是确保EB病毒复制和分离的关键。然而，EBNA-1与染色质相互作用的性质和调控还没有清楚地阐明。这一活性被认为涉及EBNA-1与DNA、双链RNA和/或蛋白质的结合。EBNA-1结合蛋白2(EBP2)是一种核仁蛋白，被认为是EBNA-1在有丝分裂染色体上的对接蛋白。然而，到目前为止，还没有直接证据表明EBP2在有丝分裂过程中与EBNA-1有关。通过结合视频显微镜和Forster共振能量转移(FRET)显微镜，我们首次证明了EBNA-1和EBP2在核浆中相互作用，以及在间期在核仁中相互作用。然而，与目前提出的模型形成强烈对比的是，我们无法在有丝分裂染色体上观察到EBNA-1和EBP2之间的任何相互作用。我们还进行了酵母双杂交筛选，随后进行了FRET分析，这导致我们确定众所周知的染色质成分HMGB2(高迁移率族蛋白2)是EBNA-1在间期和有丝分裂期间染色质上的新合作伙伴。尽管HMGB2的缺失部分改变了HeLa细胞间期和有丝分裂过程中EBNA-1与染色质的结合，但对Raji细胞中EBV表型的维持没有显著影响。
Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Forster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.