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Comparative Study
. 2006 May 15;203(5):1307-17.
doi: 10.1084/jem.20052240. Epub 2006 May 1.

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis

Sascha Trapp  1 , Mark S Parcells, Jeremy P Kamil, Daniel Schumacher, B Karsten Tischer, Pankaj M Kumar, Venugopal K Nair, Nikolaus Osterrieder
Affiliations
Comparative Study

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis

Sascha Trapp et al. J Exp Med. .

Abstract

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.

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Figures

Figure 1.
Figure 1.
Genomic structure of vTR deletion and revertant viruses. (A) Schematic presentation of the genomic organization and the BamHI restriction map of MDV. The terminal and internal repeat long regions (TRL, IRL), the unique long region (UL), the internal and terminal repeat short regions (IRS, TRS), and the unique short region (US) are shown. (B) Schematic presentation of the MDV BamHI-L fragment and the genes located therein. The parental (vRB-1B) and mutant viruses lacking either one (vCR1-2+/−, vCR1-4+/−) or both copies of vTR (vCR1-2−/−, vCR1-4−/−) are shown. BamHI restriction sites are given. (C) Detailed schematic presentation of the vTR gene and the putative ICP0 ortholog. The eight CRs of vTR are given in black. Deletion of CRs 1 and 2 (ΔCR1-2) or 1 to 4 (ΔCR1-4) in the genome of vTR mutant viruses is indicated. Also shown is the location of sequences contained in the Southern blot probe. (D) Southern blot analysis of mutant MDV BACs. DNA of pRB-1B and mutant MDV BACs (pCR1-2+/−, pCR1-2−/−, pCR1-4+/−, pCR1-4−/−, and pCR1-2−/−R1) was prepared, digested with BamHI, and separated on a 0.8% agarose gel. Southern blot analysis was performed using a PCR-generated digoxigenin-labeled probe using oligonucleotide primers vTRfw 5′-TGGCGGGTGGAAGGC-3′ and vTRrv 5′-CTGCGGGCGAGGACC-3′. Fragments detected by the vTR probe are indicated by asterisks, and sizes are given. (E) PCR analysis of the mutant BACs. vTR-specific sequences were amplified by using oligonucleotide primers vTRfw and vTRrv, and amplification products were separated on a 1% agarose gel. The size of the specific PCR product is given.
Figure 2.
Figure 2.
Expression of vTR in lytically infected CECs and latently infected and transformed lymphoma cells. An RT-PCR analysis of total RNA (2 μg) extracted from RB-1B–infected CECs or uninfected cells, as well as from lymphoblastoid cell lines RECC-CU91 and MDCC-UD14, is shown. RECC-CU91 is a lymphoblastoid cell line transformed by a retrovirus, reticuloendotheliosis virus. MDCC-UD14 is a T cell lymphoma cell line isolated from an RB-1B–infected bird. RT-PCR was performed using oligonucleotide primers, vTR(exp)fw 5′-GGCACACGTGGCGGGTGGAAGG-3′ and vTR(exp)Arv 5′-CAGTGCTGCGCCGATTCTAC-3′.
Figure 3.
Figure 3.
Growth properties in vitro of mutant viruses demonstrating that vTR is dispensable for MDV replication in vitro. (A) Growth kinetics of the indicated viruses. 106 CECs were infected with 100 PFU of vRB-1B or vTR mutant viruses. At the indicated times after infection, infected cells were dissociated by trypsinization and virus titers were determined by plating serial 10-fold dilutions of infected cells onto fresh CECs. Means and standard deviations (error bars) of three independent experiments are given. (B) Relative plaque sizes induced by the indicated viruses. Plaque areas were measured 7 d after infection after fixing cells with 90% acetone and performing indirect immunofluorescence using a convalescent MDV-specific chicken serum and Alexa Fluor 488 goat anti–chicken IgG (Invitrogen). For each virus, 100 randomly selected plaques were photographed with a digital camera (Zeiss Axiovert 25 and Axiocam; Carl Zeiss MicroImaging, Inc.), and plaque areas were determined using ImageJ software. Means and standard deviations (error bars) are given.
Figure 4.
Figure 4.
vTR is dispensable for lytic MDV replication in vivo and for the establishment of latency. A qPCR analysis of the viral gD gene and a host gene (iNOS) in total DNA isolated from whole blood samples, which were taken at days 4, 7, 12, 15, 19, and 30 after infection from virus-infected chickens, is shown. Mean viral load values and standard deviations (error bars) are given as copies of gD per 106 copies of chicken iNOS. No statistically significant differences in viremia levels were observed between groups infected with vTR single and double deletion viruses or parental vRB-1B.
Figure 5.
Figure 5.
Mutant viruses lacking both copies of vTR are attenuated and severely impaired in their ability to induce lymphoma. In three independent experiments, chickens were inoculated with 500 PFUs of vRB-1B or vTR mutant viruses. During the course of the experiments, moribund birds were killed and necropsied. After a 9-wk observation period, all surviving birds were necropsied and evaluated for MD. Morbidity and lymphoma incidences were recorded. (A) MD incidence (percentage of birds showing clinical symptoms and/or lymphomatous lesions) in animal groups infected with the indicated viruses. Means and standard deviations (error bars) are given. The decreased MD incidences observed in animal groups infected with vCR1-2−/− and vCR1-4−/− relative to groups infected with vRB-1B, vCR1-2+/−, or vCR1-4+/− were statistically significant (*) as follows: vRB-1B versus vCR1-2−/−, P = 0.0117; vRB-1B versus vCR1-4−/−, P = 0.0117; vCR1-2+/− versus vCR1-2−/−, P = 0.0055; vCR1-2+/− versus vCR1-4−/−, P = 0.0055; vCR1-4+/− versus vCR1-2−/−, P = 0.0055; vCR1-4+/− versus vCR1-4−/−, P = 0.0055. (B) Lymphoma incidences (percentage of birds showing lymphomatous lesions) in groups infected with the indicated viruses are given as means and standard deviations (error bars). The decreased lymphoma incidences detected in groups infected with vCR1-2−/− and vCR1-4−/− relative to groups infected with vRB-1B, vCR1-2+/−, or vCR1-4+/− were statistically significant (*) as follows: vRB-1B versus vCR1-2−/−, P = 0.0117; vRB-1B versus vCR1-4−/−, P = 0.0117; vCR1-2+/− versus vCR1-2−/−, P = 0.0055; vCR1-2+/− versus vCR1-4−/−, P = 0.0055; vCR1-4+/− versus vCR1-2−/−, P = 0.0055; vCR1-4+/− versus vCR1-4−/−, P = 0.0055. (C) Lymphomas induced by mutant viruses lacking both copies of vTR have an altered dissemination pattern within individual chickens. Organ manifestations of lymphomatous lesions in individual birds were recorded in three independent animal experiments, and percentages are given of birds infected with the indicated viruses, which failed to develop lymphoma (0) or in which less than or more than two sites of lymphoma manifestation were observed. Means and standard deviations (error bars) are given. The reduced disseminations of lymphomas detected in groups infected with vCR1-2−/− and vCR1-4−/− relative to groups infected with vRB-1B, vCR1-2+/− or vCR1-4+/− were statistically significant (*) as follows: vRB-1B versus vCR1-2−/−, P = 0.0025; vRB-1B versus vCR1-4−/−, P = 0.0033; vCR1-2+/− versus vCR1-2−/−, P = 0.0022; vCR1-2+/− versus vCR1-4−/−, P = 0.0029; vCR1-4+/− versus vCR1-2−/−, P = 0.0143; vCR1-4+/− versus vCR1-4−/−, P = 0.0188.
Figure 6.
Figure 6.
Lymphoma incidences (A) and dissemination patterns (B) in chickens infected with parental, vTR mutant, and vTR revertant viruses. Lymphoma incidences are given as the percentage of birds having lymphomatous lesions. Dissemination patterns are shown as the percentages of birds that failed to develop lymphoma (0) or in which less than or more than two sites of lymphoma manifestation were observed. The decreased lymphoma incidence detected in groups infected with vCR1-2−/− relative to groups infected with vRB-1B, vCR1-2+/−, vCR1-2−/−R1, or vCR1-2−/−R2 were statistically significant (*) as follows: vCR1-2−/− versus vRB-1B, P = 0.0361; vCR1-2−/− versus vCR1-2+/−, P = 0.0453; vCR1-2−/− versus vCR1-2−/−R1, P = 0.0361; vCR1-2−/− versus CR1-2−/−R1, P = 0.0094.
Figure 7.
Figure 7.
DF-1 cells overexpressing vTR exhibit a partially transformed phenotype and elevated integrin αv expression levels. (A) Growth rates of DF-1vector, DF-1vTR, and DF-1Meq cell lines. Cells were seeded in duplicate at a density of 105 in 12-well dishes. At 8, 24, 48, 72, 96, 120, and 144 h after plating, cells were trypsinized and viable cells were counted using a hemocytometer. Means and standard deviations (error bars) of three independent experiments are given. At 144 h after plating, the average numbers of viable cells of the different recombinant cell lines were statistically significantly different as follows: DF-1vector versus DF-1vTR, P < 0.0001; DF-1vector versus DF-1Meq, P < 0.0001; DF-1vTR versus DF-1Meq, P = 0.0155. (B) Anchorage-independent growth of DF-1vector, DF-1vTR, and DF-1Meq cells in soft agar as determined by colony formation after 4 wk of incubation at 37°C. The cloning efficiencies (percentages of cells forming colonies after 3 wk) of the indicated cell lines are shown. Means and standard deviations (error bars) of three independent experiments are given. No statistically significant differences in cloning efficiencies between the cell lines were observed. (C) Relative sizes of soft agar colonies formed by DF-1vector, DF-1vTR, and DF-1Meq. For each of the indicated cell lines, 100 randomly selected colonies were photographed with a digital camera and colony diameters were determined using ImageJ software. Means and standard deviations (error bars) are given. The average colony sizes formed by DF-1vector, DF-1vTR, and DF-1Meq cells were statistically significantly different as follows: DF-1vector versus DF-1vTR, P = 0.0021; DF-1vector versus DF-1Meq, P < 0.0001; DF-1vTR versus DF-1Meq, P = 0.0007. (D) Morphological phenotypes of DF-1vector, DF-1vTR, and DF-1Meq cells photographed under an inverted microscope (Zeiss Axiovert 25; Carl Zeiss MicroImaging, Inc.). Bar, 50 μm. (E) Integrin αv expression was higher in DF-1vTR relative to DF-1vector or DF-1Meq cells, whereas MHC class I expression was up-regulated in DF-1Meq cells. Cell lines were trypsinized and incubated with a mixture of a 1:2,000 dilution of a rabbit polyclonal antibody directed against integrin αv (Chemicon) and a 1:200 dilution of anti–chicken MHC class I monoclonal antibody C6B12 (Developmental Study Hybridoma Bank, University of Iowa). The secondary antibodies used were anti–rabbit IgG Alexa 488 (Invitrogen) and anti–mouse IgG Cy5 (Jackson ImmunoResearch Laboratories), used at 1:200 dilutions, respectively. Cells were examined using FACScan (Becton Dickinson), and data were analyzed using FlowJo, version 5.7.2 for Windows (Tree Star). A representative experiment of integrin αv expression (left) and MHC class I expression (middle) is shown. Means and standard deviations of mean channel of fluorescence ratios of integrin αv and MHC class I expression of DF-1vTR and DF-1Meq cells relative to DF-1 vector cells of three independent experiments are also given (right).
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References

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