Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naive B cells, and facilitates recruitment of transcription factors to the viral genome
Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naive B cells, and facilitates recruitment of transcription factors to the viral genome
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Epstein-Barr 病毒核抗原 EBNA-LP 对于转化幼稚 B 细胞至关重要，并有助于将转录因子招募到病毒基因组中
DOI：
10.1101/176099
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Szymula A
中科院分区：
文献类型：
--
作者：
Szymula A
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。它在B细胞转化中的作用尚不清楚，但据报道，它在体外增强EBV蛋白EBNA 2的基因激活。这些敲除中的第一个中的内含子突变表明EBV sisRNA在转化中的作用。具有完整内含子的LPKO以降低的效率从成人B细胞建立淋巴母细胞样细胞系（LCL），但脐带B细胞和幼稚细胞的LPKO以降低的效率从成人B细胞建立淋巴母细胞样细胞系（LCL）。（IgD+，CD 27-）成人B细胞在感染LPKO后约两周持续死亡，不能建立LCL。EBNA 2调节的病毒基因（LMP 1和LMP 2）和EBNA 2非依赖性EBER基因的转录水平显著降低，特别是在前1-2周。到感染后30天，这些水平已经平衡。相反，EBNA 2调节的宿主基因被LPKO病毒有效诱导。染色质免疫沉淀显示，EBNA 2和宿主因子EBF 1和RBPJ向所有测试的潜伏启动子的募集严重延迟，而这些相同的因子被有效地募集到几个宿主基因中，其中一些表现出增加的EBNA 2募集。而是促进几种转录因子向病毒基因组的募集，以使病毒潜伏基因能够转录。此外，我们的研究结果表明，EBV的不同属性可能有不同的重要性，在转化不同的B细胞subset.Author summaryEpstein-Barr病毒（EBV）感染几乎每个人。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（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。我们已经研究了一种病毒蛋白EBNA-LP的作用，它被认为是支持由病毒必需蛋白EBNA 2激活的基因。这种病毒（LPKO）显示出几种特性。1.它转化成年细胞的能力降低，而未成熟的B细胞（在年轻人中更常见）在LPKO感染后两周死亡。2.有些病毒基因在LPKO感染后不能立即启动。3. EBNA 2与这些基因的结合被延迟，一些细胞因子的结合也是如此。4. EBNA-LP并不以同样的方式影响EBNA 2靶向的细胞基因，这表明EBNA-LP在未成熟细胞中更为重要，而且它比单纯通过EBNA 2更广泛地调节病毒基因，而不是宿主基因。
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 is reported to enhance gene activation by the EBV protein EBNA2 in vitro.We generated two sets of EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of IR1. Intronic mutations in the first of these knockouts suggested a role for the EBV sisRNAs in transformation. LPKOs with intact introns established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but umbilical cord B cells, and naive (IgD+, CD27-) adult B cells consistently died approximately two weeks after infection with LPKO, failing to establish LCLs.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, particularly in the first 1-2 weeks. By 30 days post infection, these levels had equalised. In contrast, EBNA2-regulated host genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that recruitment of EBNA2 and the host factors EBF1 and RBPJ to all latency promoters tested was severely delayed, whereas these same factors were recruited efficiently to several host genes, some of 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 different properties of EBV may have differing importance in transforming different B cell subsets.Author summaryEpstein-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 (mono). EBV is also implicated in several different cancers. EBV infection of B cells (the immune cell that produces antibodies) can drive them to replicate almost indefinitely (‘transformation’), generating cell lines. We have investigated the role of a virus 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 adult cells, while immature B cells (more frequent in the young) die two weeks after LPKO infection. 2. Some virus genes fail to turn on immediately after LPKO infection. 3. Binding of EBNA2 to these genes is delayed, as is binding of some cellular factors. 4. EBNA-LP does not affect EBNA2-targeted cellular genes in the same way.This shows that EBNA-LP is more important in immature cells, and that it regulates virus genes – but not host genes – more widely than simply through EBNA2.
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