Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Abstract

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV. Author summary EBV is an important cause of human B-cell malignancies and transforms B cells in vitro. Although there are two different types of EBV (T1 and T2), most research has been performed using T1 strains and much less is known about how T2 EBV interacts with B cells. T2 EBV is most frequently found in regions of Africa where malaria is common, and we recently showed that B cells infected with T2 EBV have more lytic infection compared to B cells infected with T1 EBV. Here we used both bulk and single-cell RNA-seq analysis to globally define the differences in cellular gene expression in early passage EBV-transformed lymphoblastoid cell lines (LCLs) infected with T1 versus T2 EBV strains. These studies reveal that hundreds of cellular genes are differentially expressed in T1 versus T2 LCLs, and that most of these differences in gene expression occur in the latently infected cells. We also find that decreased expression of the cellular transcription factor, IRF4, contributes to the both the latent and lytic phenotypes of T2 EBV infected B cells. Together these results suggest that T1 and T2 EBV have distinctly different effects in infected B cells.