Comparative Functional Study of the Viral Telomerase RNA Based on Natural Mutations

Abstract

Telomerase activity is present in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. We previously characterized the first identified viral telomerase RNA (vTR) encoded by the Marek's disease virus (MDV) (Fragnet, L., Blasco, M. A., Klapper, W., and Rasschaert, D. (2003) J. Virol. 77, 5985-5996). This avian herpesvirus induces T-lymphomas. We demonstrated that the vTR subunit of the oncogenic MDV-RB1B strain is functional and would be more efficient than its chicken counterpart, cTR, which is 88% homologous. We take advantage of the functionality of those natural mutant TRs to investigate the involvement of the mutations of vTR on its efficiency in a heterologous murine cell system and in a homologous in vitro system using the recombinant chicken telomerase reverse transcriptase. The P2 helix of the pseudoknot seems to be more stable in vTR than in cTR, and this may account for the higher activity of vTR than cTR. Moreover, the five adenines just upstream from the P3 helix of vTR may also play an important role in its efficiency. We also established that the substitution of a single nucleotide at the 3-extremity of the H-box of the vaccine MDV-Rispens strain vTR resulted in a lack of its accumulation within the cell, especially in the nucleus, correlated with a decrease in telomerase activity.Telomeres are nucleoprotein structures found at the extremities of eukaryotic linear chromosomes (1). They are essential for chromosome stability, and thus, genome rearrangements, the cell cycle, and carcinogenesis depend directly on the integrity of these structures (2). Telomeres are composed of tandemly repeated DNA sequences, which consist of 5Ј-GGT-TAG-3Ј hexamers in vertebrates. Telomeres replicate by a specific mechanism involving telomerase, a ribonucleoprotein complex that serves as the preferential system for compensating the erosion of telomeres at each round of DNA replication (3). The minimal telomerase complex sufficient for in vitro activity comprises two major components: 1) a protein subunit (TERT) 1 with reverse transcriptase activity associated with 2)an RNA subunit (TR) containing a short template sequence specific for telomere repeats (4). Telomerase activity is tightly regulated in cells. According to the constitution of the minimal telomerase complex, the regulation of the telomerase activity would rest on either the expression of the TERT or the TR subunits, as described in human or mouse, respectively. In addition, telomerase is not detectable in normal somatic cells but is strongly expressed during fetal development and constitutively expressed in highly proliferative postnatal cells such as germ line cells, stem cells, and lymphocytes (5, 6). Furthermore, high levels of telomerase activity are also detected in a large range of cancers (at least 85%) and are closely associated with immortalization processes (7, 8).In the last three years, a correlation has been established between telomerase activity and viral infections with the oncogenic p...

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

“…Moreover, deletion of the two copies of the vTR gene induces a decrease of about 60% in tumor incidence in chickens [30]. In vitro studies established the function of vTR with its natural partner, chicken TERT (chTERT), and further demonstrated that vTR and chTERT reconstitute a more active complex than the native one (chTR/chTERT) [33]. Although the alignment of the primary sequences of vTR and hTR reveals only a weak sequence homology (30%; data not shown), the overall secondary structure predicted for vTR shows high similarity to the consensus functional structures described for hTR, including the conserved regions CR1 to CR8 [28,34,35].…”

“…It was then subcloned into the pUC-119 vector, which had been previously digested with XbaI/PstI. The chTR gene under the control of the T7 promoter was obtained from the XbaI/PstI digestion of pBS-T7chTR [33], and directly cloned in the pUC-119 vector. All intermediate and final constructs were sequenced with appropriate primers to check the integrity of the T7 promoter and genes of interest.…”

“…The plasmids phTR+1, pCR3.1-Flag-hTERT, pET-14b-chTERT and pCDNA3-vTR were previously described [33,36,37]. The vTR gene was cloned in the pUC-119 vector, similarly to the cloning of hTR [36].…”

“…Assemblies of hTERT or chTERT with the different TRs in a cell-free environment were performed as previously described [33]. Briefly, 0.75 µl of TERT proteins expressed in RRL was incubated with 1 µg of in vitro transcribed TR at 30ºC for 2 h in 1X buffer (50 mM NaCl, 20 mM HEPES (pH 8), 2 mM MgCl 2 , 0.2 mM EGTA, 2 mM DTT and 10% glycerol).…”

“…The DNA templates used for in vitro T7 transcription of vTR, hTR and chTR consist of the pUC-119 constructs encompassing the genes of interest, linearized at the 3'-end of each gene either with an FspI digestion (vTR and hTR), or with an XbaI digestion (chTR). TRs were synthesized from 10 µg of template DNA, as previously described [33]. The resulting RNAs were treated with 10 units of RNase-free DNase I (Amersham Pharmacia Biotech) at 37ºC for 15 min.…”