Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency

Abstract

Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters.IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency. KEYWORDS 5hmC, EBNA2, EBV, Epstein-Barr virus, TET2, germinal center B cell, latency E pstein-Barr virus (EBV) is human gammaherpesvirus that infects over 90% of the world population. EBV is the causative agent of infectious mononucleosis and is also associated with several malignancies of B cell and epithelial cell origin, including Burkitt lymphoma (BL), Hodgkin's disease, posttransplant lymphoproliferative disorder, diffuse large B cell lymphoma (DLBCL), nasopharyngeal carcinoma (NPC), and gastric carcinoma (1, 2). EBV establishes latent infection in normal B cells, in which the virus

TET enzymes control antibody production and shape the mutational landscape in germinal centre B cells

“…1A) and TET3 (Fig. As compared to mature na€ ıve follicular (FO) B cells, TET2 and TET3 are substantially down- in antigen-experienced GC B cells and plasma cells, a result in agreement with a recent report in human GC B cells [37] (compare Fig. Such a pattern indicates that the two proteins may have partially overlapping functions in developmental transitions or the selection of immature B cells, potentially controlling the appearance of autoimmune or nonfunctional mature B cells.…”

“…However, a robust understanding of TET-function in peripheral immature and mature na€ ıve B-cell subsets could only be achieved using conditional Cre transgenes with selective activity in the cell type of interest. As compared to mature na€ ıve follicular (FO) B cells, TET2 and TET3 are substantially down- in antigen-experienced GC B cells and plasma cells, a result in agreement with a recent report in human GC B cells [37] (compare Fig. 1A and B; FO vs. GC vs. PC).…”

“…TET2 and TET3 are expressed throughout B-cell development and terminal differentiation Human and mouse B cells express TET2 and TET3, whereas TET1 is barely detectable throughout development and upon activation [37][38][39][40]. To analyse the relative mRNA expression of TET2 and TET3 in cell populations spanning the entire B-cell lineage, we FACS-sorted developing and mature na€ ıve B-cell subsets from unchallenged wild-type mice and antigen-experienced B cells from mice that were immunized with sheep red blood cells (RBC), and performed quantitative real-time PCR (qRT-PCR).…”

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

“…Accordingly, EBNA1 expression in murine B cells induces tumors with some similarities to Burkitt's lymphoma 40 . EBNA2 induces the transcription of the cellular oncogene MYC and compromises lytic EBV replication by inducing Tet methylcytosine dioxygenase 2 (TET2) expression, thereby blocking methylation sites for BZLF1 binding 16,17,41 .…”

“…Importantly, this epigenetic modification might strongly depend on the cellular context. Lytic cycle reactivation might be less dependent on DNA methylation in epithelial cells and this epigenetic modification of the viral genome could also differ in its kinetics from B cells in this cell type [14][15][16][17] . Nevertheless, transcytosis of EBV across polarized oral epithelia cell cultures has been demonstrated 18 .…”