RNA-seq analyses of gene expression during Epstein-Barr virus infection of primary B lymphocytes.

Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro. EBV transforms RBLs through expression of viral latency genes and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and polyA plus RNAs were analyzed by RNA-seq. ANOVA analyses found 3669 protein coding genes that were differentially expressed (FDR<0.01). 94% of LCL genes that are essential for LCL growth and survival were differentially expressed. Pathway analyses identified significant enrichment of pathways involved in cell proliferation, DNA repair, metabolism, and anti-viral responses. RNA-seq also identified lncRNAs differentially expressed during EBV infection. CRISPRi and CRISPRa found that CYTOR and NORAD lncRNAs were important for LCL growth. During EBV infection, type III EBV latency genes were expressed rapidly after infection. Immediately after LCL establishment, EBV lytic genes were also expressed in LCLs and ∼4% of the LCLs express gp350. ChIP-seq and POLR2A ChIA-PET data linked EBV enhancers to 90% of EBV-regulated genes. Many genes were linked to enhancers occupied by multiple EBNAs or NF-kB subunits. Incorporating these assays, we generated a comprehensive EBV regulome in LCLs. IMPORTANCE EBV immortalization of RBL is a useful model system to study EBV oncogenesis. By incorporating RNA-seq, ChIP-seq, ChIA-PET, and genome-wide CRISPR screen, we identified key pathways that EBV usurps to enable B cell growth and transformation. Multiple layers of regulation could be achieved by co-operations between multiple EBV transcription factors binding to the same enhancers. EBV manipulated the expression of most cell genes essential for LCL growth and survival. In addition to proteins, lncRNAs regulated by EBV also contributed to LCL growth and survival. The data presented in this paper not only allowed us to further define the molecular pathogenesis of EBV, but also serve as a useful resource to the EBV research community.