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.

A comprehensive single cell data analysis of in lymphoblastoid cells reveals the role of Super-enhancers in maintaining EBV latency

“…1e, S1f). Consistent with previous reports (33,34), GP350 + LMP1 hi cells highly expressed host NFATC1, MIER2, SFN and SGK1 genes and were the highest expressors of host box-dependent myc-interacting protein 1 (BIN1). Conversely, GP350 -LMP1 hi cells had the highest expression of host genes HSPB1, ABCB10, MALAT1 and CD44 (Fig.…”

“…Consistently, a recent study has found that IRF4 knockdown in LCLs induces EBV lytic reactivation in LCLs and lytically infected cells have increased NFATc1 and decreased IRF4 expression (34). Collectively, our data suggest that SE-regulated genes are less highly expressed in GP350 + LMP1 hi LCLs, which show evidence of lytic EBV reactivation, and that experimental induction of EBV lytic cycle also represses expression of these genes.…”

“…Recent advances in single-cell transcriptomics have enabled successful resolution of tissue/cell heterogeneity in several species. Since these technologies agnostically capture both host and infecting viral sequences, they have also been utilized to explore host-virus interactions at a single cell level (32)(33)(34). Leveraging this feature here, we sought to identify the determinants of EBV latency in lymphoblastoid cells.…”

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes

“…As shown in Table 1 , comparison of cellular gene expression in the T1 Akata virus-infected NOKs versus T2 AG876 virus-infected NOKs reveals 85 genes were significantly upregulated, and 36 genes significantly downregulated, in the Akata versus AG876 virus infected cells. In contrast, we previously found that close to 600 cellular genes were expressed at significantly different levels in T1 versus T2 EBV-infected lymphoblastoid B cell lines [ 29 ]. The AG876 virus-infected and Akata virus-infected NOKs are much more similar to each other compared to the uninfected NOKs.…”

“…The EBV transcript analysis of the RNA-seq results does not suggest that AG876 virus-infected and Akata virus-infected NOKs are significantly different in their ability to spontaneously lytically reactivate. However, given our recent finding that T2 EBV is more lytic than T1 EBV in lymphoblastoid B cell lines and in lymphomas of humanized mice [ 27 , 29 ], we explored whether T1 Akata virus-infected versus T2 AG876 virus-infected NOKs differ in their ability to switch to the lytic form of viral reactivation when treated with the phorbol ester TPA, suspended in methylcellulose, or rafted. The Kenney and Mertz labs have previously shown that the ability of TPA to induce lytic EBV reactivation of Akata virus-infected NOKs is at least partially dependent upon differentiation activated transcription factors such as BLIMP1 and KLF4 [ 12 , 44 ].…”