Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.
Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.
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DOI：
10.1371/journal.ppat.1006890
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发表时间：
2018-03
期刊：
影响因子：
6.7
通讯作者：
White RE
中科院分区：
文献类型：
--
作者：
Szymula A;Palermo RD;Bayoumy A;Groves IJ;Ba Abdullah M;Holder B;White RE
EB病毒（EBV）核抗原前导蛋白（EBNA-LP）是EBV感染静息B细胞后产生的第一种病毒潜伏相关蛋白。其在B细胞转化中的作用尚不清楚，但据报道其在体外可增强EBV蛋白EBNA 2的基因激活。我们产生了EBNA-LP敲除（LPKO）EBV，其在内部重复序列1（IR1）的每个重复单元内含有终止密码子。EBNA-LP突变型EB病毒以较低的效率从成人B细胞中建立了淋巴母细胞样细胞系（LCL），但从脐带B细胞中却没有建立，脐带B细胞在感染后大约两周死亡。成人B细胞仅建立具有记忆（CD27+）表型的EBNA-LP-无效LCL。感染后病毒基因表达的定量PCR分析鉴定了EBNA基因的改变比率，以及EBNA2调节的病毒基因（LMP1和LMP2）和EBNA2非依赖性EBER基因在前2周的转录水平的急剧降低。感染后30天，LPKO转录与野生型EB病毒相同。相反，EBNA2调节的细胞基因被LPKO病毒有效诱导。染色质免疫沉淀显示，EBNA2和宿主转录因子EBF1和RBPJ延迟招募到所有测试的病毒潜伏启动子，而这些相同的因子被有效地招募到几个宿主基因，表现出增加EBNA2的招募。我们的结论是，EBNA-LP并不简单地合作与EBNA2在激活基因转录，而是促进招聘的几个转录因子的病毒基因组，使病毒潜伏基因的转录。此外，我们的研究结果表明，EBNA-LP是EBV感染的幼稚B细胞的生存所必需的。EB病毒（EBV）几乎感染所有人。一旦被感染，人们会终生携带病毒，通过唾液传播。儿童感染无症状，但在青春期或成年期首次感染可引起腺热（传染性单核细胞增多症）。EBV还涉及几种不同的癌症。EBV感染B细胞（产生抗体的免疫细胞）可以驱使它们几乎无限地复制（"转化"），产生细胞系。我们已经研究了EBV蛋白（EBNA-LP）的作用，该蛋白被认为支持由必需病毒蛋白EBNA2进行的基因激活。我们已经制造了EBNA-LP基因被破坏的EBV。这种病毒（LPKO）显示出几种特性。1.其转化B细胞的能力降低; 2. "幼稚" B细胞（其抗体尚未适应对抗感染的细胞）在LPKO感染后两周死亡; 3.有些病毒基因在LPKO感染后不能立即启动。4. EBNA2和各种细胞因子与这些基因的结合被延迟。5. EBNA-LP不会以相同的方式影响EBNA2靶向细胞基因。这表明EBNA-LP在幼稚B细胞中更重要，它有助于打开病毒基因，而不是细胞基因。
The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells. Epstein-Barr virus (EBV) infects almost everyone. Once infected, people harbor the virus for life, shedding it in saliva. Infection of children is asymptomatic, but a first infection during adolescence or adulthood can cause glandular fever (infectious mononucleosis). EBV is also implicated in several different cancers. EBV infection of B cells (antibody-producing immune cells) can drive them to replicate almost indefinitely (‘transformation’), generating cell lines. We have investigated the role of an EBV protein (EBNA-LP) which is thought to support gene activation by the essential virus protein EBNA2. We have made an EBV in which the EBNA-LP gene has been disrupted. This virus (LPKO) shows several properties. 1. It is reduced in its ability to transform B cells; 2. ‘Naïve’ B cells (those whose antibodies have not adapted to fight infections) die two weeks after LPKO infection; 3. Some virus genes fail to turn on immediately after LPKO infection. 4. Binding of EBNA2 and various cellular factors to these genes is delayed. 5. EBNA-LP does not affect EBNA2-targeted cellular genes in the same way. This shows that EBNA-LP is more important in naïve B cells, and that it helps to turn on virus genes, but not cell genes.
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