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

Wille, Coral K.; Nawandar, Dhananjay M.; Henning, Amanda N.; Ma, Shidong; Oetting, Kayla M.; Lee, Dennis; Lambert, Paul F.; Johannsen, Eric; Kenney, Shannon C.

doi:10.1073/pnas.1513432112

Cited by 29 publications

(39 citation statements)

References 49 publications

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“…Importantly, TET2 loss has recently been implicated in the development of EBV ϩ nasopharyngeal carcinoma (NPC) (47). EBV exclusively, lytically infects more differentiated normal epithelial cells (48)(49)(50)(51)(52)(53), and thus, the viral genome remains highly unmethylated (11).…”

Section: Tet2 Promotes Ebv Type III Latency Gene Expressionmentioning

confidence: 99%

“…However, in the case of NPC, EBV maintains a latent infection and the viral genome becomes highly methylated (54). We recently showed that TET2 5hmC modifies the EBV genome to promote lytic gene expression in epithelial cells and that NPC tumors lose TET2 expression, which may enhance oncogenesis (47). Furthermore, TET2 was recently found to be a resistance factor of EBV-induced DNA methylation in gastric carcinoma cells, demonstrating that TET2 may have an important role in protection against EBV ϩ tumor development (55).…”

Section: Tet2 Promotes Ebv Type III Latency Gene Expressionmentioning

confidence: 99%

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

Wille

et al. 2017

J Virol

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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

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Section: Tet2 Promotes Ebv Type III Latency Gene Expressionmentioning

confidence: 99%

Section: Tet2 Promotes Ebv Type III Latency Gene Expressionmentioning

confidence: 99%

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

Wille

et al. 2017

J Virol

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“…In 293T cells stably infected with an EBV genome, high occupancies of Zta were observed in methylated promoters of BALF2, BMRF1, Rta, and BHLF1, whereas these bindings were disrupted once the EBV genome is hypomethylated (54). In addition, during the early stages of the lytic cycle in IgG-induced EBV-positive Akata cells, Zta associated with the promoters of BKRF4, BLLF2, and BRRF1.…”

Section: Discussionmentioning

confidence: 97%

“…It has been demonstrated that a methylated EBV genome is essential for viral lytic reproduction (51,53,72), as the immediate-early protein Zta preferentially binds to methylated viral promoters (54,(73)(74)(75). In 293T cells stably infected with an EBV genome, high occupancies of Zta were observed in methylated promoters of BALF2, BMRF1, Rta, and BHLF1, whereas these bindings were disrupted once the EBV genome is hypomethylated (54).…”

Section: Discussionmentioning

confidence: 99%

“…Evidence to date indicates that the immediate-early protein Zta, also known as the oriLyt-binding protein, preferentially binds to methylated viral DNA and is central to efficient lytic replication (51)(52)(53)(54). In addition, El-Guindy et al demonstrated that binding of Rta onto EBV oriLyt is essential for viral lytic DNA replication (55).…”

Section: Ebv Rta Expression Disrupts the Binding Of Ctcf In The Ebv Gmentioning

confidence: 99%

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Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

Chen

Chang

et al. 2017

J Virol

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Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G 1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.KEYWORDS Epstein-Barr virus, immediate-early protein Rta, lytic cycle reactivation, CTCF, DNA methylation, cell cycle arrest, genome topology, immediate-early protein Rta

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TET2 and LILRB1 mutations are frequent in Epstein–Barr virus‐positive diffuse large B‐cell lymphoma especially in elderly patients

Cho

Kim

Yoon

et al. 2023

Cancer

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Background Diffuse large B‐cell lymphoma (DLBCL) harboring Epstein–Barr virus (EBV) primarily occurs in patients who have underlying immunodeficiency or in elderly patients but is also reported in young, immunocompetent patients. The authors investigated the pathologic differences in EBV‐positive DLBCL in these three groups of patients. Methods In total, 57 patients with EBV‐positive DLBCL were included in the study; of these, 16 patients had associated immunodeficiency, 10 were young (younger than 50 years), and 31 were elderly (aged 50 years or older). Immunostaining for CD8, CD68, PD‐L1, and EBV nuclear antigen 2, and panel‐based next‐generation sequencing was performed on formalin‐fixed, paraffin‐embedded blocks. Results Immunohistochemistry revealed EBV nuclear antigen 2 positivity in 21 of the 49 patients. The degree of CD8‐positive and CD68‐positive immune cell infiltration and PD‐L1 expression did not differ significantly in each group. Extranodal site involvement was more common in young patients (p = .021). In mutational analysis, the genes with the highest mutation frequency were PCLO (n = 14), TET2 (n = 10), and LILRB1 (n = 10). For the TET2 gene, all 10 mutations were found in elderly patients (p = .007). Compared with a validation cohort, both TET2 and LILRB1 showed a higher mutation frequency in EBV‐positive patients than in EBV‐negative patients. Conclusions EBV‐positive DLBCL occurring in three different age and immune status groups showed similar pathologic characteristics. Notably, a high frequency of TET2 and LILRB1 mutations was characteristic of this disease in elderly patients. Further studies are needed to determine the role of TET2 and LILRB1 mutations in the development of EBV‐positive DLBCL along with immune senescence. Plain Language Summary Epstein–Barr virus‐positive diffuse large B‐cell lymphoma occurring in three different groups (immunodeficiency‐associated, young, and elderly) showed similar pathologic characteristics. The frequency of TET2 and LILRB1 mutations was high in elderly patients with Epstein–Barr virus‐positive diffuse large B‐cell lymphoma.

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

Cited by 29 publications

References 49 publications

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

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

Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

TET2 and LILRB1 mutations are frequent in Epstein–Barr virus‐positive diffuse large B‐cell lymphoma especially in elderly patients

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