RNA-sequencing analysis identifies BRLF1-dependent viral and cellular transcriptome during EBV primary infection in B lymphoma cells. RNA-sequencing analysis identifies BRLF1-dependent viral and cellular transcriptome during EBV primary infection in B lymphoma cells

Purpose: Immediate-early protein BRLF1 plays an important role in lytic infection of Epstein-Barr virus (EBV), it activates lytic viral transcription and viral DNA replication, affects cell cycle and suppresses inflammatory responses. However, the understanding of BRLF1 influence on cellular gene expression during early lytic cycle remains limited. Methods:RNA-sequencing analysis identified the differentially expressed genes (DEGs) in B lymphoma cells undergoing wild type and BRLF1-deficient EBV primary infection. The libraries were sequenced on an Illumina HiSeq-Xten platform and 150 bp paired‐end reads were generated. An average read depth of 12G reads per sample was collected and analyzed. Data analysis was performed with Molecule Annotation System 3.0. Results: A total of 159 unigenes were annotated and the major differentially enriched pathways were related to DNA replication and transcription, immune and inflammatory response, cytokine-receptor interaction and chemokine signaling, ECM-receptor interaction and focal adhesion. Further the mass spectrometry identified the BRLF1-binding proteins and showed that BRLF1 employed different strategies to influence RNA polymerase II-dependent viral and cellular transcription, by binding to a particular RREs motif and binding to unique transcription factors which binding to specific motifs. Conclusions: Our study characterized the BRLF1-dependent cellular and viral transcriptional profile during primary infection and then revealed the important virus-cell interaction and alterations of cell activity for EBV primary infection and lytic replication. Overall design: deep RNA sequencing profiles of wild type and BRLF1-deficient EBV primary infected B lymphoma cells

研究目的：即刻早期蛋白BRLF1在EB病毒（Epstein-Barr virus, EBV）的裂解性感染中发挥关键调控作用，可激活病毒裂解性转录程序与病毒DNA复制过程，调控细胞周期并抑制炎症应答。然而，目前对于BRLF1在病毒早期裂解周期中对宿主细胞基因表达的影响机制仍缺乏深入认知。
研究方法：本研究通过RNA测序（RNA-sequencing）分析了野生型与BRLF1缺陷型EBV原代感染的B淋巴瘤细胞中的差异表达基因（differentially expressed genes, DEGs）。测序文库在Illumina HiSeq-Xten测序平台上完成上机测序，生成150 bp双端读段，每个样本的平均测序深度达12G读段，所得数据均进行标准化分析与后续挖掘。数据分析采用Molecule Annotation System 3.0软件完成。
研究结果：共注释得到159个单基因，主要富集的差异通路涉及DNA复制与转录调控、免疫与炎症应答、细胞因子-受体相互作用、趋化因子信号通路、细胞外基质（extracellular matrix, ECM）-受体相互作用以及黏着斑通路。进一步通过质谱分析鉴定得到BRLF1结合蛋白，结果显示BRLF1可通过两种不同策略调控依赖RNA聚合酶II的病毒与宿主细胞转录：一是结合特定的RREs基序，二是结合可识别特定基序的独特转录因子。
研究结论：本研究阐明了EBV原代感染过程中依赖BRLF1的宿主与病毒转录谱特征，并揭示了EBV原代感染与裂解复制过程中关键的病毒-宿主相互作用模式以及细胞活性改变情况。
整体实验设计：野生型与BRLF1缺陷型EBV原代感染的B淋巴瘤细胞的深度RNA测序谱型。