AbstractGallid 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, resulting in high mortality.
The GaHV-2 genome belongs to a class E genome with a size of 175 to 180 kilobase pairs. GaHV-2 genome consists of a unique long (UL), and a unique short (US) segments bracketed by inverted repeats known as terminal and internal repeats long (TRL and IRL) and terminal and internal repeats short (TRS and IRS). GaHV-2 genes, similar to those of other herpesviruses, also belong to three kinetic classes of immediate-early, early and late genes based on the requirements for viral protein synthesis and DNA replication.
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, a non-coding RNA, shares 88% sequence homology with chicken TR (chTR) and it was likely acquired from the chicken genome during virus-host co-evolution. vTR interacts with the chicken telomerase reverse transcriptase subunit (TERT) enhancing telomerase activity and contributing to the efficient and rapid onset of lymphoma. Furthermore, vTR re-localises ribosomal protein L22 that plays an essential role in T-cell development and transformation. Moreover, vTR functions independent of the telomerase complex are responsible for tumour progression and dissemination. 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. However, it was demonstrated that E-box 1 was not functional. 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 and on the importance of functional c-Myc response elements during virus-induced lymphomagenesis.
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) by two-step Red-mediated mutagenesis. Susceptible (B13B13) chickens were infected with the recombinant viruses to assess the impact of c-Myc REs mutations. To investigate GaHV-2 replication and telomerase activity during infection, blood and feather follicle epithelium were collected at specific time points from infected chicken. Animals were daily monitored for the clinical symptoms of the disease and euthanised 55 days-post infections to assess the number of tumours developed in visceral organs.
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 in infected animals over time. Promoter reporter assays indicated that c-Myc RE, located two nucleotides downstream of the 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 in virus-induced tumorigenesis, we generated a recombinant virus containing mutations in both CpG sites of c-Myc REs as well as revertant. 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 animals was severely impaired and tumour formation completely abrogated. Our data provide a more in-depth characterisation of c-Myc oncoprotein REs and DNA methylation involvement in transcriptional regulation of vTR.
|Date of Award||29 Sept 2020|
|Sponsors||University of Namur|
|Supervisor||Benoit Muylkens (Supervisor), Thierry Arnould (President), Damien Hermand (Jury), Olivier De Backer (Jury), Jean-Pierre Gillet (Jury), Anabelle Decottignies (Jury) & Luca Bertzbach (Jury)|
- Gallid herpesvirus type 2
- viral telomerase RNA subunit
- epigenetic regulation
- DNA methylation
- telomerase activity
- virus-induced oncogenesis