Herpesvirus Telomerase RNA(vTR)-Dependent Lymphoma Formation Does Not Require Interaction of vTR with Telomerase Reverse Transcriptase (TERT)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)n. Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity

Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma

Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia

Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells

Marek’s disease virus microRNA designated Mdv1-pre-miR-M4 targets both cellular and viral genes

Mdv1-miR-M4 is one of 25 microRNAs (miRNAs) expressed by Marek's disease virus (MDV-1), an oncogenic alphaherpesvirus that induces fatal T-cell lymphoma in chickens. Mdv1-miR-M4 was shown to be the second functional viral ortholog of miR-155, a cellular miRNA that plays a crucial role in several physiological and pathological processes in lymphocyte biology. In this study, we investigated a panel of putative mdv1-miR-M4 targets involved in gene networks affecting both cellular and viral life cycles. Using luciferase reporter assays, we showed that mdv1-miR-M4-5P and miR-155 efficiently targeted a common set of 3′ untranslated regions (3′UTR) of six cellular genes (GPM6B, RREB1, c-Myb, MAP3K7IP2, PU. 1 and C/EBP). In addition, we also investigated the interactions between mdv1-miR-M4-5P and mdv1-miR-M43P and viral mRNAs encoding UL28 and UL32 in both reporter and western blot assays. Mdv1-miR-M4 specifically inhibited the translation of these two viral proteins, which are involved in the cleavage/packaging of herpesvirus DNA. © 2010 Springer-Verlag.</p

Marek's disease virus: from miasma to model

International audienceMarek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens