Role of DNA Methylation and CpG Sites in the Viral Telomerase RNA Promoter during Gallid Herpesvirus 2 Pathogenesis

Abstract

Gallid herpesvirus type 2 (GaHV-2) is an oncogenic alphaherpesvirus that induces malignant T-cell lymphoma in chicken. GaHV-2 encodes a viral telomerase RNA subunit (vTR) that plays a crucial role in virus-induced tumorigenesis, enhances telomerase activity and possesses function independent of the telomerase complex. vTR is driven by a robust viral promoter, highly expressed in virus-infected cells and regulated by two c-Myc response elements (c-Myc REs). The regulatory mechanisms involved in controlling vTR and other genes during viral replication and latency remain poorly understood but are crucial to understand this oncogenic herpesvirus. Therefore, we investigated DNA methylation patterns of CpG dinucleotides found in the vTR promoter and measured the impact of methylation on the telomerase activity. We demonstrated that telomerase activity was considerably increased following viral reactivation. Furthermore, CpG sites within c-Myc REs showed specific changes in methylation after in vitro reactivation and in infected animals over time. Promoter reporter assays indicated that one of the c-Myc RE is involved in regulating vTR transcription, and that methylation strongly influenced vTR promoter activity. To study the importance of the CpG sites found in c-Myc REs in virus-induced tumorigenesis, we generated a recombinant virus containing mutations in both CpG sites of c-Myc REs as well as revertant, by two-step Red-mediated mutagenesis. Introduced mutation in vTR promoter did not affect the replication properties of the recombinant viruses in vitro. In contrast, replication of the mutant virus in infected chickens was severely impaired, and tumour formation completely abrogated. Our data provided a more in-depth characterisation of c-Myc oncoprotein REs and DNA methylation involvement in transcriptional regulation of vTR. IMPORTANCE Previous studies demonstrated that telomerase RNAs possess functions that promote tumour development independent of the telomerase complex. vTR is a herpesvirus-encoded telomerase RNA subunit that plays a crucial role in virus-induced tumorigenesis and is expressed by a robust viral promoter that is highly regulated by the c-Myc oncoprotein binding to the E-boxes. Here we demonstrated that the DNA methylation patterns in the functional c-Myc response elements of the vTR promoter change upon reactivation from latency and that demethylation strongly increases telomerase activity in virus-infected cells. Moreover, the introduction of mutation in the CpG dinucleotides of the c-Myc binding sites resulted in decreased vTR expression and complete abrogation of tumour formation. Our study provides further confirmation of the involvement of specific DNA methylation patterns in the regulation of vTR expression and vTR importance for virus-induced tumorigenesis.

Marek’s Disease Virus Virulence Genes Encode Circular RNAs

“…An RT-PCR approach, based on divergent primers that exclusively amplify circRNAs, was applied to total RNA extracts depleted of linear transcripts. This method was carried out on several relevant biological samples associated with the different phases of the viral cycle: (i) two commonly used permissive cell types (chicken embryonic fibroblasts, CEF, and ESCDL-1 cells) infected with the very virulent RB-1B strain as a model for the productive phases of the virus; (ii) a lymphoma-derived cell line (MSB1) as a model for transformed T cells infected with MDV; (iii) MSB1 cells treated with sodium butyrate (NaBu) or 5’azacytidine as a model for virus reactivation as demonstrated previously ( 51 ); (iv) three samples harvested from RB-1B infected chickens (peripheral blood lymphocytes, spleen, and feather follicle epithelium) ex vivo RB-1B-infected T and B lymphocytes ( 52 ).…”

“…They were grown at 41°C with 5% of CO 2 . Reactivation was induced by treatment of either 3 mM NaBu or 5 μM 5’azacytidine (Merck) and was verified by the expression of VP5 as previously described ( 51 , 80 , 81 ).…”

“…Ex vivo infections of B and T lymphocytes were carried out using the RB-1B strain and following the guidelines described by Schermuly and collaborators ( 52 ) with small adaptations of the infection procedure ( 63 , 83 ). In vivo samples used in this study (spleen, feather follicles (FFE), and peripheral blood lymphocytes (PBL)) were obtained from previous experiments ( 51 ). They were conducted following Belgian and European laws, notably Directive 2010/63/EU.…”

Assessment of the Epidemic Potential of the Marek’s Disease Virus in the Russian Federation

“…i.e. disruption of the folate cycle in humans which increases the risk of manifestation of diseases associated with DNA viruses, including an increased risk of infection with animal viruses [12,13];…”

Chicken Hypothalamic and Ovarian DNA Methylome Alteration in Response to Forced Molting

“…DNA methylation is a form of epigenetics that plays an important role in cell differentiation, development, aging, and disease by regulating gene expression. DNA methylation has been widely studied in chickens, mainly involving embryonic development [ 12 ], meat quality traits [ 13 , 14 , 15 ], fat deposition [ 16 , 17 , 18 ], environmental adaptation [ 19 , 20 ], nutritional regulation [ 21 , 22 ], disease resistance [ 23 , 24 , 25 ], and reproductive traits [ 26 , 27 , 28 ]. However, these complex biological phenomena also occur during FM.…”