Importance of vTR-TERT interaction in MDV-induced tumor formation and dissemination

Marek’s disease virus (MDV) is a cell associated alphaherpesvirus mostly renowned for its oncogenic potential. Several viral factors involved in tumor formation and dissemination have been identified. The underlying mechanism of transformation and lymphoma formation, however, is poorly understood. One of the factors is vTR, a viral homologue of the cellular telomerase RNA (TR), which is part of the telomerase complex that also contains telomerase reverse transcriptase (TERT). Previously, recombinant viruses lacking both copies of vTR were found to have a highly reduced tumor incidence without affecting primary lytic replication. To determine whether the function of vTR is dependent on its recruitment into the telomerase complex, we generated mutant viruses containing a mutation within the P6.1 stem-loop (vRB-1B?P6.1). This mutation was previously shown in other systems to disrupt the stem-loop and therefore abrogate formation of the telomerase complex and its enzymatic activity. As expected, neither growth in vitro nor lytic replication in vivo of a mutant virus was affected by the altering the P6.1 stem loop. Growth kinetics were comparable to wild-type and revertant viruses. In order to investigate the oncogenic potential of vTR independent of its enzyme complex formation, we inoculated 1-day-old chickens. In contrast to viruses lacking vTR, vRB-1B?P6.1 induced tumors very efficiently, even though the survival of infected animals was slightly prolonged. Our study provides conclusive evidence that viral telomerase RNA functions in tumor formation independently of the formation of an enzymatically active telomerase complex.

The role of telomeric repeats in Marek’s disease virus (MDV) tumorgenesis

Marek’s disease virus (MDV) causes a malignant T cell lymphoma in its natural host, the chicken. MDV integration into the host cell genome of infected T lymphocytes is believed to play an important role in lymphomagenesis. Previously, analyses of malignantly transformed T cells have shown that MDV genomic sequences are mainly present in chromosome termini. These regions contain telomere sequences, which are of varying length (5 kbp to 2 Mbp) and play a crucial role for genome maintenance and stability. Surprisingly, telomeric repeat sequences (TMRs) are also present at both termini of the MDV genome. Therefore, we hypothesize that MDV telomeric repeats could facilitate genome maintenance and integration into the host genome via homologuous recombination. Therefore, TMRs (TTAGGG) in the MDV genome were replaced by synthetic repeat sequences (TAAGGC). One or both repeat regions were replaced in an infectious RB-1B genome (pRB-1B), a highly oncogenic MDV strain, via two-step Red-mediated recombination. Recombinant virus grew with kinetics in tissue culture that were comparable to that of parental virus indicating that the telomeric repeats are not essential for replication in vitro. Currently, we are investigating the effect of the absence of TMRs on growth and tumorigenesis in vivo.

Mutational analysis of Varicella zoster virus (VZV) ORFS/L