Department of Life Sciences, Tzu-Chi University, Hualien 97004, Taiwan.
Proc Natl Acad Sci U S A. 2014 Jan 7;111(1):243-8. doi: 10.1073/pnas.1321800111. Epub 2013 Dec 16.
Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.
EB 病毒（EBV）核抗原 1（EBNA1）对于 EBV 病毒基因组的维持、复制和转录是必需的。这些作用是通过 EBNA1 与同源 oriP DNA 的结合来介导的，oriP DNA 包含 20 个不完美的 30 碱基对二联体对称增强子拷贝和一个 DNA 复制起始点。为了鉴定对这些 EBNA1 功能至关重要的细胞蛋白，用免疫沉淀法沉淀与 EBNA1 相关的细胞蛋白，并通过液相色谱-串联质谱分析法进行分析。鉴定出核仁磷蛋白（NCL）与 EBNA1 相关。EBNA1 的 N 端 100 个氨基酸和 NCL 的 RNA 结合结构域对于 EBNA1/NCL 相互作用是至关重要的。慢病毒 shRNA 介导的 NCL 耗竭显著降低了 EBNA1 向 oriP DNA 的募集、EBNA1 依赖的 EBV oriP 荧光素酶报告基因的转录以及淋巴母细胞系中 EBV 基因组的维持。NCL 的 RNA 结合结构域 K429 对于 ATP 和 EBNA1 的结合是至关重要的。NCL 的过表达增加了 EBNA1 与 oriP 的结合和转录，而 NCL K429A 则缺失。此外，NCL 的沉默会损害淋巴母细胞系的生长。这些实验揭示了 NCL K429 在 EBNA1 病毒基因组维持和转录中的一个惊人的关键作用，这可能是治疗干预的一个靶点。
