Role of DNA methylation and CpG sites in the viral telomerase RNA promoter during Gallid herpesvirus 2 pathogenesis

Srđan Pejaković, André Claude Mbouombouo Mfossa, Laëtitia Wiggers, Ahmed Kheimar, Damien Coupeau, Benedikt B Kaufer, Benoît Muylkens
2020 Journal of Virology  
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
more » ... ontrolling 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.
doi:10.1128/jvi.01488-20 pmid:32967954 fatcat:m7dqx3efhfgtlke5bixxxd2m3q