title: 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.
authors.affiliation: Department of Medical Microbiology and Immunology, Wisconsin Institutes for Medical Research (WIMR) II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Coral K
authors.initials: CK
authors.lastname: Wille
authors.affiliation: Cellular and Molecular Biology Graduate Program, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Dhananjay M
authors.initials: DM
authors.lastname: Nawandar
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Amanda N
authors.initials: AN
authors.lastname: Henning
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Shidong
authors.initials: S
authors.lastname: Ma
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Kayla M
authors.initials: KM
authors.lastname: Oetting
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Dennis
authors.initials: D
authors.lastname: Lee
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705;
authors.forename: Paul
authors.initials: P
authors.lastname: Lambert
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705; Department of Medicine, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Johannsen
authors.affiliation: Department of Oncology, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705; Department of Medicine, WIMR II, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705 skenney@wisc.edu.
authors.forename: Shannon C
authors.initials: SC
authors.lastname: Kenney
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002277:Carcinoma; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049308:Dioxygenases; D016679:Genome, Viral / Q000235:genetics*; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D015603:Keratinocytes / Q000378:metabolism / Q000821:virology; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000473:pathology / Q000821:virology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014775:Virus Activation / Q000235:genetics*; D017735:Virus Latency / Q000235:genetics*
chemical_list: C068382:BRLF1 protein, Human herpesvirus 4; C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D049308:Dioxygenases; C541081:TET2 protein, human
keywords: 5hmC; EBV; NPC; lytic reactivation