EBV epigenetically suppresses the B cell-to-plasma cell differentiation pathway while establishing long-term latency

Abstract

Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes, express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in which EBV persistence is established. Three related latency-associated viral proteins EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are required to sustain proliferation, in part, by repressing the expression of tumour suppressor genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—after a phase of rapid proliferation—infected primary B cells express elevated levels of factors associated with plasma cell (PC) differentiation. These include the cyclin-dependent kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38 and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming. Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic changes, cells become committed to a B-blast fate <12 days post-infection and are unable to de-repress p18INK4c or BLIMP-1—in either newly infected cells or conditional LCLs—by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype. Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic persistence.

Latency and lytic replication in Epstein–Barr virus-associated oncogenesis

“…EBNA3A and EBNA3C rescue infected cells that are driven into a proliferative state by EBNA2-dependent MYC expression via the down-regulation of the proapoptotic BIM and p16 INK4a proteins that respond to the hyperproliferation of the infected cells 43 . Furthermore, they prevent transition into lytic replication by suppression of BLIMP1 expression 44 . By contrast,…”

Epstein-Barr Virus LMP1-Mediated Oncogenicity

“…To overcome this barrier, LMP1, EBNA3, and EBV miRNAs each downmodulate Blimp1 (40,55,56). LMP1 upregulates IRF2 and its corepressor IRF2BP2 via EBV SE, which may form repressor complexes at a Blimp1 promoter interferon response element to counteract IRF4 effects on Blimp1 (40).…”

“…LMP1 upregulates IRF2 and its corepressor IRF2BP2 via EBV SE, which may form repressor complexes at a Blimp1 promoter interferon response element to counteract IRF4 effects on Blimp1 (40). EBNA3A and EBNA3C also block LCL Blimp1 upregulation, and withdrawal of EBNA3A and EBNA3C expression triggered plasma cell differentiation (56). Further studies are required to determine whether LMP1-activated IRF4, BATF4, and perhaps IRF2 tether EBNA3 corepressor complexes at the Blimp1 promoter.…”

Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

“…It is known that in order to escape the immune surveillance of the host and establish a chronic infection, EBV has evolved different mechanisms to maintain B cells in a status of long-lived circulating memory B cells and prevent them from differentiating into antibody-secreting plasma cells. A recent study showed that EBNA3A and EBNA3C block the terminal differentiation of memory B cells to plasma cells by epigenetically repressing the gene encoding BLIMP-1, a master regulator in B-cell differentiation (33). Notably, a previous study showed that KDM2B plays a key role in the differentiation of hematological stem cells (9).…”