PDF(Pubmed)
Abstract：
Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.
摘要：
潜伏期的再激活在维持持续的终生爱泼斯坦-巴尔病毒（EBV）感染中起着重要作用。尚未完全了解控制EBV裂解期成功激活和进展的机制。EBV表达多种病毒微小RNA（miRNA）并操纵几种细胞miRNA以支持病毒感染。为了深入了解调节从EBV潜伏期到裂解期的宿主miRNA，我们使用抗Ig抗体在EBV+Burkitt淋巴瘤(BL)细胞中进行了基于CRISPR/Cas9的筛选,以交联B细胞受体(BCR)并诱导再激活.使用针对>1500个注释的人miRNA的gRNA文库，我们确定miR-142是EBV再激活的关键调节因子.miR-142的遗传消融增强了感染的BL细胞中立即早期和早期裂解基因产物的水平。再激活细胞的Ago2-PAR-CLIP实验揭示了与Erk/MAPK信号相关的miR-142靶标，包括直接位于B细胞受体(BCR)下游的组分。与这些发现一致，miR-142的破坏增强了SOS1水平和Mek磷酸化，以响应表面Ig交联。可以通过Mek（考比替尼）或Raf（dabrafenib）的抑制剂来挽救效果。一起来看,这些结果表明miR-142在功能上调节通过BCR启动的SOS1/Ras/Raf/Mek/Erk信号,限制EBV进入裂解周期。
