Dataset Information

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

ABSTRACT: Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten-eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation.

SUBMITTER: Wille CK

PROVIDER: S-EPMC4703011 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

Wille Coral K CK Nawandar Dhananjay M DM Henning Amanda N AN Ma Shidong S Oetting Kayla M KM Lee Dennis D Lambert Paul P Johannsen Eric C EC Kenney Shannon C SC

Proceedings of the National Academy of Sciences of the United States of America 20151209 52

Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, ...[more]

PMID: 26663912

Similar Datasets

Project description:Long term carriers were shown to generate robust polyfunctional T cell (PFC) responses against lytic and latent antigens of Epstein-Barr virus (EBV). However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM]) and asymptomatic (AS) EBV infection. Evaluation of IFN-? secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-?, TNF-?, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs) as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions) concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6-12 months. In conclusion, EBV antigen-specific CD4+ and CD8+ PFC responses emerge during the first year of primary EBV infection, with greatest responses toward immunodominant epitopes in both lytic and latent proteins, correlating to steady decline in PBMC and plasma viral loads.

Project description:Ikaros is a zinc finger DNA-binding protein that regulates chromatin remodeling and the expression of genes involved in the cell cycle, apoptosis, and Notch signaling. It is a master regulator of lymphocyte differentiation and functions as a tumor suppressor in acute lymphoblastic leukemia. Nevertheless, no previous reports described effects of Ikaros on the life cycle of any human lymphotropic virus. Here, we demonstrate that full-length Ikaros (IK-1) functions as a major factor in the maintenance of viral latency in Epstein-Barr virus (EBV)-positive Burkitt's lymphoma Sal and MutuI cell lines. Either silencing of Ikaros expression by small hairpin RNA (shRNA) knockdown or ectopic expression of a non-DNA-binding isoform induced lytic gene expression. These effects synergized with other lytic inducers of EBV, including transforming growth factor ? (TGF-?) and the hypoxia mimic desferrioxamine. Data from chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) and ChIP-sequencing (ChIP-seq) analyses indicated that Ikaros did not bind to either of the EBV immediate early genes BZLF1 and BRLF1. Rather, Ikaros affected the expression of Oct-2 and Bcl-6, other transcription factors that directly inhibit EBV reactivation and plasma cell differentiation, respectively. IK-1 also complexed with the EBV immediate early R protein in coimmunoprecipitation assays and partially colocalized with R within cells. The presence of R alleviated IK-1-mediated transcriptional repression, with IK-1 then cooperating with Z and R to enhance lytic gene expression. Thus, we conclude that Ikaros plays distinct roles at different stages of EBV's life cycle: it contributes to maintaining latency via indirect mechanisms, and it may also synergize with Z and R to enhance lytic replication through direct association with R and/or R-induced alterations in Ikaros' functional activities via cellular signaling pathways.This is the first report showing that the cellular protein Ikaros, a known master regulator of hematopoiesis and critical tumor suppressor in acute lymphoblastic leukemia, also plays important roles in the life cycle of Epstein-Barr virus in B cells.

Dataset Information

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

Publications

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets