Tumor promoting functions of cellular telomerase RNA and viral RNAs in herpesvirus-induced cancer formation

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. MDV encodes a viral telomerase RNA (vTR) that is highly expressed during all stages of the virus life cycle. vTR is crucial for efficient MDV-induced lymphoma formation, however, the mechanism is not completely understood. Despite the high sequence identity between vTR and the cellular telomerase RNA (chTR) of 88%, it remains elusive if the overexpression of the chTR can contribute to cellular transformation. Intriguingly, TRs and Epstein- Barr virus (EBV) encoded RNAs (EBER-1 and EBER-2) have interaction partners in common that are highly conserved in humans and chickens. EBERs are the most abundant viral transcripts in EBV-induced tumor cell. However, their role in tumor development is still controversial. In the first part of the study, we wanted to investigate if the overexpression of cellular TRs (chTR) have tumor-promoting functions using a natural virus-host animal model of herpesvirus tumorigenesis. We initially deleted vTR (vΔvTR) in the RB-1B genome, a very virulent MDV strain, and subsequently inserted chTR at the vTR locus resulting in vchTR, using the Two-step Red-mediated mutagenesis system. The expression levels of vTR and chTR (in vchTR) were confirmed using qRT-PCR. chTR expression levels in vchTR were comparable to vTR in the wild-type. Neither the vTR-deletion nor the chTR insertion effected the MDV replication properties in vitro and in vivo. Intriguingly, the tumor formation was severely impaired in the absence of vTR while, the tumor formation in the chickens infected with vchTR was similar to those infected with the wild-type or revertant virus. Our results provided the first evidence that the overexpression of the cellular TRs can complement the functions of vTR in MDV-induced tumorigenesis. In the second part of this study, we wanted to address if EBERs (EBER-1 and EBER-2) possess tumor promoting functions and can transform chicken T cells using a small animal model for MDV-tumorigenesis. We generated recombinant MDVs expressing either EBER-1 or EBER-2 instead of vTR, termed vEBER-1 and vEBER-2. Expression levels of EBERs were detected during the viral lytic replication in vitro. EBERs were highly expressed and comparable to vTR expression in the wild-type or revertant. Furthermore, the recombinant mutants were replicating efficiently in cell culture and in infected animals. To assess the tumor promoting properties of EBERs, we performed an animal experiment where the infected animals were monitored for tumor development. EBERs partially restored the tumor formation if compared to the vTR-deletion. Tumor incidence with vEBER-2 was higher than with vEBER-1 compared to the wild-type. Our results for this aim displayed the potential tumorigenicity of EBERs their ability to transform different host immune cells. Furthermore, it provided a useful model to investigate the activities of EBERs in the cellular transformation and the underlying mechanism using a small animal model for virus-induced cancer formation.