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

PhD thesis defended by Srdan PEJAKOVIC (Prof. Benoît MUYLKENS) - 29/09/2020

Prof. Benoît MUYLKENS, UNamur, Integrated Veterinary Research Unit (URVI)


Thierry ARNOULD (UNamur), President; Benoît MUYLKENS (UNamur), Supervisor and Secretary; Luca BERTZBACH (Freie Universität von Berlin); Anabelle DECOTTIGNIES (UCLouvain); Damien HERMAND (UNamur); Olivier DEBACKER (UNamur); Jean-Pierre GILLET (UNamur)


Gallid herpesvirus 2 (GaHV-2) is an avian alphaherpesvirus that causes highly malignant T-cell lymphoma considered to be the most prevalent cancer in the animal kingdom. In susceptible chickens, the ultimate consequence of the host-virus interactions is the transformation of the CD4+ T-cells, which eventually proliferate to form visceral lymphomas, causing high mortality. During GaHV-2 infection, several viral factors, proteins and diverse RNAs, including the major oncoprotein Meq, the viral interleukin-8 and GaHV-2-encoded miRNAs, contribute to lymphomagenesis. In addition, GaHV-2 encodes two copies of viral telomerase RNA subunit (vTR), which is expressed both during productive infection and in virus-transformed T-cell lines. vTR interacts with the chicken telomerase reverse transcriptase subunit (TERT) enhancing telomerase activity and contributing to the efficient and rapid onset of lymphoma. It is the most abundant viral transcript detected in GaHV-2-induced tumour cells with higher expression than chTR in infected cells, consequence likely due to differences in their promoters. The vTR promoter has additional AP-1 sites, c-Myc transcription response elements (namely E-box 1, E-box 2 and E-box 3) and EBS transcription factor binding sites. It was shown that the c-Myc oncoprotein is involved in the regulation of vTR during GaHV-2-induced lymphomagenesis and that increased expression of vTR is essential for the oncogenic function of the virus. During the viral life cycle, transcriptional modifications and epigenetic changes, together with post-transcriptional and post-translational modifications, regulate expression of cellular and viral genes. Altogether, they allow GaHV-2 to switch between the productive and latent phases, and to induce infected cell transformation.

The focus of this study was on the epigenetic mechanisms involved in the switch between the productive and latent phase of GaHV-2 life cycle. We established DNA methylation/hydroxymethylation patterns of vTR promoter in vitro and in vivo and measured the impact of methylation on the telomerase activity and c-Myc response elements (c-Myc REs) of the vTR promoter. Furthermore, to study the importance of the c-Myc binding sites in virus-induced tumorigenesis, a recombinant virus bearing mutations in functional c-Myc REs, as well as revertant, were produced using the bacterial artificial chromosome of a highly oncogenic strain (pRB-1BΔIRL) using two-step Red-mediated mutagenesis. Susceptible (B13B13) chickens were infected with the recombinant viruses to assess the impact of c-Myc REs mutations and to investigate GaHV-2 replication and telomerase activity during infection.

We demonstrated that telomerase activity was considerably increased following viral reactivation. Furthermore, CpG sites within functional c-Myc REs showed specific changes in methylation after in vitro reactivation and infected animals over time. Promoter reporter assays indicated that c-Myc RE located two nucleotides downstream of transcription start site 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, we introduced mutation in vTR promoter that did not affect the replication properties of the recombinant viruses in vitro. In contrast, replication and 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.