Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection

Journal of Virological Methods 118 (2004) 123–130

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection Morgane Rolland a,b , Cécile Chauvineau b , Stephen Valas b , Robert Z. Mamoun a,c,∗ , Gérard Perrin b a

c

INSERM U577, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France b AFSSA-Niort, Laboratoire d’Etudes et de Recherches Caprines, 79012 Niort, France CNRS UMR 5121 UM1, Institut de Biologie, 4 Bd Henri IV, CS89508, 34960 Montpellier cedex 2, France Received 23 July 2003; received in revised form 3 February 2004; accepted 9 February 2004

Abstract Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15–20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes. © 2004 Elsevier B.V. All rights reserved. Keywords: SRLV; CAEV; MVV; GSM cell line; hTERT

1. Introduction The small ruminant lentiviruses (SRLV) correspond to the ovine maedi visna virus (MVV) and to the caprine arthritis-encephalitis virus (CAEV) and are widely distributed throughout the world. MVV and CAEV cause persistent infections, with diseases including encephalitis, arthritis, progressive pneumonia, and mastitis in goats and sheep, respectively. Like other lentiviruses, CAEV and MVV infect cells of the monocyte/macrophage lineage and dendritic cells (Narayan, 1989; Ryan et al., 2000). MVV and CAEV can replicate in primary fibroblasts derived from choroid plexus or synovial membrane cultures. Thus, these cells are commonly used to detect and study MVV or CAEV infections and to produce these viruses. Even if these cells are more convenient than macrophages, their use presents several drawbacks. Firstly, establishing virus-free primary cell cultures can be tedious, due to the pandemic

∗

Corresponding author. Tel.: +33-467-600-233; fax: +33-467-604420. E-mail address: [email protected] (R.Z. Mamoun).

0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.02.001

infection of goats and sheep by SRLV and to the difficulties in detecting these viruses. Secondly, primary cultured cells have a finite life-span in vitro before entering an irreversible growth arrest, known as senescence. Thirdly, each individual primary culture can exhibit differences in its susceptibility to infection. Moreover, it is well-known that viruses adapt themselves to cells in culture, so it is difficult to compare the biological properties of viruses that have been isolated and grown on cells from various origins. All of these reasons make highly desirable the establishment of a reliable cell line supporting SRLV infection. A permanent cell line, which would be easy to handle and free of viral infection, would be an invaluable tool not only to standardize work on every laboratory but also to set up detection campaigns to know the SRLV status of animals. To date, the establishment of a cell line susceptible to SRLV infection has been reported once: goat embryonic fibroblasts were immortalized by transfection with plasmids containing the simian virus (SV) 40 large T-antigen cDNA (Da Silva Teixeira et al., 1997). However, the presence of simian virus information in this cell line raises concern about the suitability of these cells to detect and study SRLV

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infection. A risk exists that the information from SV40 and from the SRLV of interest may interfere, potentially allowing recombination events, that would represent a harmful threat, particularly when producing virus for vaccination. However, since it was established in 1997, this cell line has not become widely used by the scientific community working on SRLV, maybe due to the potential risks linked to the presence of the SV40 large T-antigen. To our knowledge, no cell line, free of virus genes and harboring all the characteristics listed above, is yet available for veterinary and research laboratory studies on SRLV. Starting from a well characterized primary culture of goat synovial membrane (GSM) cells, we decided to obtain an immortalized cell line by adding only a normal cellular gene. Recent studies demonstrated that the expression of the catalytic subunit of the human telomerase (hTERT) alone is sufficient to immortalize diverse cell types (Bodnar et al., 1998; Vaziri and Benchimol, 1998). Telomerase activity is absent from most normal human somatic cells, but present in over 90% of human tumors and immortalized cell lines (Shay and Bacchetti, 1997; Yi et al., 1999). Telomerase consists of two essential components: an RNA template (TER) and a catalytic subunit (TERT) that acts as a reverse transcriptase on the RNA template. Telomerase serves to maintain the short repetitive sequences (e.g. (TTAGGG)n in vertebrates) at eukaryotic chromosome ends, which constitute the telomeres. Telomeric DNA is lost at a rate of 50–200 bp per cell division (Harley et al., 1990). It was suggested that the progressive shortening of telomeres with each cell division would control entry into senescence (Harley, 1991). The telomerase, by adding (TTAGGG)n repeats, would prevent cells from entering senescence. Here, we report the immortalization of goat synovial membrane (GSM) cells following the transfection of the catalytic subunit of the human telomerase. We next characterized this permanent GSM cell line, notably the status of its telomere, its cell growth properties and finally its ability to be infected and to propagate SRLV.

2. Material and methods 2.1. Primary goat synovial membrane cell culture Explants of synovial membrane were obtained aseptically from a SRLV-seronegative newborn goat originating from a SRLV-seronegative flock. Small tissue fragments were cut from the synovial membrane and cultured at 37 ◦ C, at 5% CO2 in a humidified atmosphere. The standard culture medium was Dulbecco’s minimum essential medium (DMEM) supplemented with 2 mM glutamine, gentamicin (25 ␮g/ml) and 10% decomplemented foetal calf serum (FCS). Medium was changed 24 h after plating, and then every other day until a confluent monolayer was reached. Confluent GSM cells were dissociated with a phosphate-buffered saline (PBS) solution containing

0.1% trypsin–0.025% EDTA and maintained in standard medium. 2.2. Transfection of GSM cells with the pLPChTERT vector pLPChTERT (Geron Corporation) is designed for gene delivery and expression of human telomerase reverse transcriptase. Upon transfection, pLPChTERT expresses a transcript containing the puromycin resistance gene and hTERT. GSM cells were transfected with the hTERT vector at passage 11, using the FuGENE 6TM transfection reagent (Roche) according to the supplier’s recommendation. Briefly, cells were transfected with 10 ␮g of hTERT vector mixed with 30 ␮l of FuGENE 6TM transfection reagent in a 35 mm Petri dish containing a monolayer of cells that were 50% confluent. Cells transfected with the hTERT vector were cultured in standard medium supplemented with 1 ␮g/ml of puromycin. After 2 weeks of culture, puromycin resistant clones were transferred to 100 cm2 flasks and expanded in standard medium. Confluent cells were dissociated with a PBS solution of trypsin (0.1%)–EDTA (0.025%), passaged at a 1:4 split ratio and cultured in standard medium. 2.3. Growth characterization 2.3.1. Cell proliferation The proliferation rate of the cell line was measured with a tetrazolium-based assay (Mosmann, 1983). Briefly, 1000 and 8000 cells (primary GSM and hTERT-transfected GSM) were seeded in 24 wells of eight culture plates (96-well flat-bottom plates) in 200 ␮l of standard medium and cultured for 2–14 days. Every 2 days, each well of one plate was stained with 125 ␮l of a 1 ␮g/␮l solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; thiazolyl blue) and incubated 4 h at 37 ◦ C. After discarding the culture medium, blue formazan crystals were solubilized in 100 ␮l of dimethyl sulfoxyde. Absorbance was measured at 540 nm with an ELISA (enzyme-linked immunoadsorbent assay) reader. For every assay, each OD540 value is the mean of the 24 OD540 values obtained for each category. Results shown represent the mean values of five MTT assays with hTERT-transfected GSM cells ranging from the 26th to the 73rd passage. Growth index is expressed as a percentage ratio of the OD540 value for the hTERT-transfected GSM cell line to that of the control primary GSM cell line. 2.3.2. Plating efficiency The ability to form colonies of the primary GSM and hTERT-transfected GSM cells was assessed in two ways: cells were seeded, either individually in multiwell plates, or in different quantities in flasks, where the plating efficiency could be influenced positively by the presence of other cells in the culture medium and negatively by the

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volume of medium. On the one hand, the plating efficiency of the primary and hTERT-transfected GSM cells was compared by plating serial dilutions of 0.1 cell per well in 96-well flat-bottom plates. Cells were allowed to grow over a 3-week-period. The colony-forming abilities of the cells were expressed as the surface of the well (either 25, 33, 50, 75 or 100%) covered by the colonies after 1, 2 and 3 weeks. On the other hand, the plating efficiency was also determined by plating 100–10000 GSM (control primary and hTERT-transfected) cells into 25 cm2 flasks. The assay was carried out using three flasks per dilution and per cell line. After 7 days of culture, the flasks were fixed with methanol, stained with 5% Giemsa and the colonies were counted. Punctuate outgrowth comprising a minimum of 40 cells was considered as a colony. The colony-forming efficiency (CFE) was expressed as: CFE (%) = (number of colonies per flask/number of cells plated per flask) × 100.

2.3.6. Virus infectivity assay The permissiveness to infection of GSM (primary and hTERT-transfected) cells was tested with two SRLV strains: the North American CAEV Cork (Cork et al., 1974) and the Icelandic MVV K1514 (Sonigo et al., 1985). Midconfluence proliferating cultures of primary GSM and hTERT-transfected GSM cells were infected with each strain by incubation in serum-free DMEM supplemented with 2 ␮g/ml polybrene. After 4 h at 37 ◦ C, the cells were rinsed with PBS and finally incubated with DMEM supplemented with 2% FCS. The infected cells were incubated at 37 ◦ C for 7–10 days, after which the supernatant was harvested. The supernatants were added to GSM and GSM T cells seeded in 48-well flat-bottom plates. After incubation for 7–10 days, cells were fixed and stained with 5% Giemsa. The cytopathic effect was scored by the presence of syncytia. The virus titers were estimated by serial limiting dilutions.

2.3.3. Statistical analysis The statistical significance of these growth assays was assessed with a Student’s t-test.

3. Results

2.3.4. Reverse transcription-PCR analysis of endogenous versus exogenous hTERT expression Total RNA was isolated from cells using RLB (RNA Lysis Buffer). Three micrograms of total RNA from each cell culture (GSM and goat synovial membrane telomerase (GSM T)) were reverse-transcribed in a 20 ␮l reaction using the first strand synthesis kit SuperScript (Invitrogen). A 2 ␮l aliquot of cDNA was used for PCR amplifications. hTERT was amplified using the oligonucleotide primer hTERT (5 -GACTCGACACCGTGTCACCTAC3 ) paired with either the primer endo (5 -ACGTAGAGCCCGGCGTGACAG-3 ) or the primer pLPC (5 -CCACTAGTTCTAGAGCGGCCGCGT-3 ), which selectively amplify endogenous and exogenous hTERT, respectively. For both primer pairs, thermocycling conditions were: 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 45 s. Amplified products (exogenous hTERT: 182 bp; endogenous hTERT: 219 bp) were resolved on a 2% agarose gel and visualized by ethidium bromide staining. 2.3.5. Telomere length Genomic DNA was isolated from cultured cells with the Isoquick kit (Bioprobe). Two micrograms of DNA were digested with the restriction enzymes MspI and RsaI. DNA fragments were separated by electrophoresis on a 0.7% agarose gel, transferred to a Hybond-N+ membrane and hybridized in 6X SSC at 37 ◦ C for 12 h with a [␥-32 P] ATP radiolabeled 6 bp telomeric repeat (TTAGGG)3 oligonucleotide probe. The membrane was washed in 3X SSC at 42 ◦ C before exposure to a preflashed KODAK XAR film for 3 days. Mean telomere restriction fragment (TRF) lengths were determined from densitometric analysis of autoradiograms as described (Harley et al., 1990).

3.1. Primary GSM cells are immortalized by the transfection of hTERT Primary cultures of GSM cells were transfected with a vector containing the catalytic subunit of the human telomerase and the puromycin resistance genes. Cells having integrated the vector were selected by resistance to puromycin. At confluency, successfully transfected cells were passed into larger flasks and subcultured in standard culture medium every 3–4 days at a 1:4 split ratio. These cells were named GSM T cells. As seen in Fig. 1A, the GSM T cells have the same morphology as normal primary GSM cells, although they are smaller and constitute a more homogeneous population. Thus far, the GSM T cell line has undergone over 150 passages, i.e. more than 300 population doublings, whereas the untransduced primary GSM cells underwent 15 passages at a constant proliferation rate before dividing less than once per week, which is typical of cells near senescence. The senescent GSM cells displayed an increased number of vacuoles, a flattened shape and an enlarged size, all phenotypical characteristics of replicative senescence. Cultures of untransduced primary GSM cells underwent irreversible growth arrest after 17 passages, after which the cells remained viable for 2 weeks with a senescent phenotype. These cultures progressively accumulated cells with aberrant morphologies consistent with the definition of the crisis phase of culture, leading to progressive cell death. In contrast, senescence was overcome for the GSM T cell line, and no subsequent crisis was observed over the 2-year-period of continuous culture that this cell line underwent. As a whole, the GSM T cell line is homogeneous and stationary, having been passaged continuously for over 2 years, without showing any morphological changes. During this time, the cells were submitted to numerous freeze-thaw cycles without any

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Fig. 1. Morphology of uninfected or CAEV-infected GSM and GSM T cells, observed by phase contrast microscopy after Giemsa staining. (A) primary GSM and immortalized GSM T cells. Left: GSM cells (40×). Right: GSM T cells (40×). (B) GSM and GSM T cells infected with CAEV Cork. Left: infected GSM cells (40×). Right: infected GSM T cells (40×).

loss of the growing performance of the cell line. The GSM T cell line has accumulated up to 170 passages after explantation at a constant proliferation rate and is thus functionally immortalized. 3.2. Expression of endogenous and exogenous hTERT mRNA We tested if the hTERT-transfected GSM T cells actually expressed the hTERT mRNA and if the GSM and GSM T cells exhibited any level of endogenous hTERT mRNA expression. We determined the expression of endogenous and exogenous hTERT mRNA in GSM and GSM T cells by RT-PCR with specific primers. As seen in Fig. 2, both primary presenescent passage 12 GSM cells and hTERT-transfected GSM T cells did not express endogenous hTERT mRNA. As anticipated, only GSM T cells expressed the exogenous hTERT mRNA. Thus, as previously reported for other cell types, ectopic expression of exogenous hTERT enables GSM T cells to proliferate well beyond their expected crisis point.

3.3. The GSM T cell line has a constant proliferation rate The growth patterns of the GSM and GSM T cell lines were compared with an MTT assay (Mosmann, 1983). Cells were plated in 96-well culture plates at a density of either 1000 or 8000 cells per well and the proliferation rate was measured every 2 days. The GSM and GSM T cells reached confluency at day 10 of culture, except for the primary GSM cells plated at a low density (1000 cells per well) which

Fig. 2. Expression of endogenous and exogenous hTERT mRNA. Reverse-transcription PCR for hTERT was done on whole RNA extracted from GSM (lanes 1 and 4) and GSM T cells (lanes 2 and 5) using primers specific for either endogenous hTERT (endo, 219 bp, lanes 1 and 2) or exogenous hTERT (exo, 182 bp, lanes 4 and 5). Lane 3: 100 bp DNA ladder.

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2,5

100

GSM

1,5

1 GSM 1000 cells

confluency (%)

2

Mean values (OD540)

GSM T

75

50

GSM 8000 cells

0,5

GSM T 1000 cells GSM T 8000 cells

25

0 450

0

growth index (%)

400 1000 cells

350

8000 cells

300 250 200 150

Day 7

Day 14

Day 21

Fig. 4. Colony-forming abilities of the GSM and GSM T cell lines. The colony-forming ability was assessed by plating 1 cell per well in 96-well plates, then the confluency was evaluated at days 7, 14 and 21 of culture. The colony-forming abilities are expressed as a percentage of confluency, i.e. the surface of the well covered by the colonies.

100 50 0 0

2

4

6

8

10

12

14

3.4. The GSM T cell line has a better plating efficiency than the GSM cells

Days

Fig. 3. Proliferation of GSM and GSM T cells. Proliferation was assessed over 14 days with a MTT-based assay by plating different numbers of GSM and GSM T cells. Each OD is the mean value of 5 experiments. The upper graph represents the proliferation rates of the GSM and GSM T cell lines. The bottom graph represents the growth index of the GSM T cell line. The growth index is the percentage ratio of the OD540 value of the GSM T cell line to that of the GSM cell line.

needed 2 more days to reach confluency (Fig. 3, top graph). Five MTT assays were performed with GSM T cells at passage 25, 41, 47, 61 and 73. These experiments gave similar results, confirming the stability of the GSM T cell line over time. The GSM T cells had a higher proliferation rate than the primary GSM cells, especially in the early phase of culture. This was particularly evident when the cells were seeded at a low density. The Fig. 3 (bottom graph) also shows the growth indices of the GSM T cells, i.e. the value of the OD540 of the GSM T cells over that of the GSM cells. When plated at the low density of 1000 cells per well, the GSM T cells had a strikingly higher growth index than the GSM cells from days 4 to 8 of culture, with the growth index of the GSM T cells being four times that of the GSM cells by day 4 of culture. Thus, GSM T cells are characterized by a higher proliferation rate when cells are seeded at a low density but, globally, GSM T cells exhibit constant proliferation rate and cell doubling time.

The plating efficiencies of GSM and GSM T cells were compared in two ways. Firstly, the colony-forming ability was assessed for individual cells by plating a maximum of one cell per well in a 96-well plate (in fact, 0.1 cells per well, corresponding to 1 cell in 10 wells). Fig. 4 represents the colony-forming abilities of GSM and GSM T cells expressed as a percentage of confluency, i.e. the surface of the well covered by the colonies. In a representative experiment, 12 individual GSM and 15 individual GSM T cells were plated. At day 21 of culture for the GSM T cells, we observed: eigtht wells which were at confluency, five punctuate outgrowths of cells, which covered at least 25% of the surface of the well, and two wells with less than 20 cells. On the other hand, no colony was seen in any of the 12 wells plated with 1 GSM cell. For those GSM cells, there were either only a couple of dead cells (in 2 wells) or less than 20 cells (in 10 wells), a number, however, too small to be considered as a colony. Secondly, plating efficiencies were determined for different numbers of cells seeded in flasks in order to take into account the influence of growth factors secreted by the cells in the culture medium. The plating efficiency was expressed as the percentage of colonies formed in comparison with the number of cells plated. The plating efficiencies were assessed after 7 days, a short period compared to the 3 weeks of the assay presented above. In contrast to the primary GSM cells, the GSM T cell line was able to grow rapidly when seeded at the low density of 10,000 cells per 25 cm2 flask, reaching confluency after 7 days of culture. When 100 cells are seeded, the colony-forming efficiency values were 6.7%

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(±1.25) for GSM cultures and 13% (±2.16) for GSM T cultures. The increase in the number of colonies for the GSM T compared to the GSM cells was significant (P < 0.05). These experiments were reproduced and gave similar results. When several cells were seeded, they had a better growth rate than when they were seeded individually. The better growth rate of multiple cells could be attributed to the release of growth factors in the culture medium. Moreover, the GSM T cells had a higher growth rate than the GSM. Overall, these results show that most GSM T cells were clonogenic and the GSM T cells exhibited better growth properties, such as colony-forming efficiency and growth rate than primary GSM cells. 3.5. Ectopic hTERT expression without telomere lengthening in the GSM T cells The introduction of hTERT into GSM cells resulted in the continuous expression of exogenous hTERT in GSM T cells, while no expression of endogenous hTERT was reported in either GSM or GSM T cells (Fig. 2). To assess the effect of hTERT expression on telomere length, we examined the mean terminal restriction fragment lengths. TRF were generated by cutting genomic DNA with restriction enzymes, which cut sites in subtelomeric regions but do not cleave telomeric repeats. The mean TRF length of the GSM T cells was measured at different passages and compared to those of primary GSM cells at various stages, including near crisis and crisis stage control. Genomic DNA from GSM T cells was harvested at various time points after the transfection of hTERT: passages 7, 26, 53 and 77. DNA was also harvested from primary GSM cells prior to the onset of senescence and twice thereafter. As expected for the GSM cell line, the average telomere length decreased slightly with increasing passages (from 5.3 to 5.0 Kb). Surprisingly, the GSM T cell line did not exhibit any marked lengthening, instead the average telomere length decreased slightly from 5.2 Kb at passage 7 to 4.8 Kb at passage 53, and then appeared to remain stable (4.8 Kb at passage 77). Besides, the telomeres in the late passage GSM T cells were shorter than in the GSM cells, even when GSM cells that had entered crisis are considered. In summary, hTERT-transfected GSM T cells showed that the telomere lengths, after an initial decrease, stabilized as the cells proliferated well beyond the expected crisis point. 3.6. The GSM T cells are susceptible to and propagate SRLV To assess the ability of GSM T cells to allow infection, cultures were infected with two different SRLV strains: the ovine maedi visna MVV K1514 from Iceland and the caprine CAEV Cork from the USA. Ovine and caprine lentiviruses led to different cytopathic phenotypes, which have already been described for GSM cells. By days 7–10 post-infection, we observed the characteristic formation of syncytia. In con-

Table 1 Comparison of the permissiveness of GSM and GSM T cells for CAEV infection Culture medium containing CAEV Corka

Infectious titerb GSM T cells

GSM cells

Fresh Fresh Fresh Frozen

0.5 × 105 105 0.4 × 104 1.1 × 105

ND ND 0.4 × 104 1.1 × 105

a The culture medium was used either fresh or after one freezing-thawing cycle. b Infectious titer was determined by the method of limiting dilutions.

trol primary GSM cells, CAEV Cork induced a persistent infection with the expected formation of syncytia with many nuclei (Fig. 1B, left), while the Icelandic MVV K1514 induced the formation of small syncytia but with only few nuclei (around 5) followed by the lysis of the infected tissue culture monolayer. The permissivity of the GSM T cells for CAEV Cork and MVV K1514 was observed for GSM T cells ranging from the 13th to the 79th passages. The GSM T cell type was susceptible to viral infection by both lentiviruses and, as expected, exhibited the same cytopathic effects as with the GSM cells. Hence, for the GSM T cells, CAEV Cork induced a persistent infection of the tissue culture monolayer with syncytia, however there were slightly less syncytia and those syncytia presented less nuclei in the GSM T cell line than in the CAEV-infected GSM cell line (Fig. 1B, right). The same held for the GSM T cells infected by the Icelandic MVV K1514, which exhibited syncytia and lysis of the infected tissue culture monolayer. On further examination, it appeared that the syncytia observed for the GSM T cell line were more regular and consequently easier to count than those of the GSM cell line. To determine whether the GSM T cells were as susceptible as the primary GSM cells to SRLV infection, we titrated different batches of CAEV Cork on both cell lines. As shown on Table 1 GSM T cells are identical to primary GSM cells for this criterium. Moreover, the GSM and GSM T cell lines ensured a complete virus cycle and infectious particles were produced in their culture media as indicated by their abilities to infect new batches of GSM T cells. Finally, the usefulness of GSM T cells for isolating new field viral isolates was tested. In a first series of experiments, three independent isolation assays were conducted. The GSM T cells were co-cultured with cells purified from the milk of three SRLV-infected goats. Within 1 month, the co-cultured GSM T cells exhibited syncytia and SRLV genomic DNA was successfully isolated from chromosomal DNA using the PCR technique. In a second series of experiment, chromosomal DNA originating from peripheral mononuclear cells isolated from SRLV-infected sheep was used to transfect 293T cells. These cells were then co-cultured with GSM T cells. Three weeks

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later syncytia appeared and the culture medium was used to infect a new batch of GSM T cells. These latter cells became syncytia positive and SRLV genomic DNA was successfully isolated from chromosomal DNA using the PCR technique. Thus, the GSM T cells allowed us to isolate the genetic information of new SRLVs originated from naturally infected sheep and goats.

4. Discussion The present study demonstrates that GSM cells are immortalized by the introduction of the catalytic subunit of the human telomerase. Thus, we can circumvent the life-span limitations and individual variation problems of the primary GSM cells. The GSM T cells can serve as an indefinite source of reliable GSM cells to propagate and study CAEV and MVV. Our result confirms previous observations by several groups indicating that the life-span of human fibroblasts cells was easily extended by the introduction of the hTERT gene (Bodnar et al., 1998; Vaziri and Benchimol, 1998; Zhu et al., 1999). Our hTERT-transfected GSM T cells could be passaged for an extended period of time: they continuously grew for more than 150 passages beyond the senescence of control cells. This result showed that hTERT allowed GSM cells to bypass senescence, and suggested that hTERT is sufficient to enable the cells to overcome the senescence and the subsequent crisis. It was concluded previously that telomere shortening in the absence of telomerase may be one signal that triggers senescence (Harley, 1991). As a corollary, the lengthening of telomeres, caused by the ectopic expression of hTERT, was thought to enable cultured cells to bypass senescence and become immortalized. Telomere elongation following the introduction of hTERT was demonstrated first by Bodnar et al. (1998); similar results were reported by Vaziri and Benchimol (1998). Surprisingly, telomeres in proliferating hTERT-transfected GSM T cells were, on average, no longer than telomeres in primary GSM cells in crisis, indeed the telomeres were slightly shorter in later passage GSM T cells, which were, nonetheless, fully viable and continued to proliferate. Nevertheless, it has already been reported that telomere lengthening and immortalization can be uncoupled. Zhu et al. (1999) showed that the ectopic expression of hTERT extended the life-span of virus transformed cells without net telomere lengthening. Moreover, many immortal tumor cell lines have stable telomeres that are significantly shorter than those found in young primary cells (de Lange et al., 1990). The results from the GSM T cells conform to the model, recently proposed by Blackburn (2001), which suggests that entry into senescence would depend on changes in the protected status of the telomere rather than on telomere shortening. The telomeric DNA/protein complex interconverts rapidly between two states: one which is competent for telomerase action, while the other is protected from telomerase action. For a telom-

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ere, the probability of being in a protected state decreases with increasing cell divisions. Several studies have demonstrated that different telomere-regulating factors, such as TR, TRF1, TRF2, or tankyrase, are also involved in regulating the telomere lengths (Blasco et al., 1997; Smogorzewska et al., 2000; Smith et al., 1998; Karlseder et al., 2002). We determined the characteristics of our hTERTtransfected GSM T cells, which showed that the GSM T cells are immortal, yet retaining their morphology. The GSM T cells kept a constant proliferation rate over their 2 years of continuous culture. The GSM T cells are able to grow when seeded at a low density, thanks to their higher colony-forming ability compared to the primary GSM cells. It is noteworthy that the GSM T cells were as susceptible as the primary GSM cells to both CAEV and MVV infections and supported complete virus replication cycles. Alike the original primary GSM cells, the GSM T cells exhibited different cytopathic phenotypes when infected by the Icelandic MVV strain K1514 or the North American CAEV strain Cork. Those typical cytopathic effects had previously been described for GSM cells, the Icelandic MVV K1514 strain being lytic but not the North American CAEV Cork strain, while both induced syncytia. The GSM T cells allowed us to isolate new field isolates of SRLVs originating from either infected goats or sheep. It must be noticed that the GSM T cells are the only tool allowing to rise infectious SRLV from proviral genome integrated in chromosomal DNA. Collectively, these data showed that GSM T cells are a useful tool to detect and study CAEV and MVV infection. Thus, the GSM T cell line could be used as the first reference material for both veterinary purposes as well as molecular and cellular biology studies. Acknowledgements We thank Cécile Martin for careful reading of the manuscript. References Blackburn, E.H., 2001. Switching and signaling at the telomere. Cell 106, 661–673. Blasco, M.A., Lee, H.W., Hance, M.P., Samper, E., Lanscorp, P.M., DePinho, R.A., Greider, C.W., 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., Wright, W.E., 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352. Cork, L.C., Hadlow, W.J., Crawford, T.B., Gorham, J.R., Piper, R.C., 1974. Infectious leukoencephalomyelitis of young goats. J. Infect. Dis. 129, 134–141. Da Silva Teixeira, M.F., Lambert, V., Mselli-Lakahl, L., Chettab, A., Chebloune, Y., Mornex, J.F., 1997. Immortalization of caprine fibroblasts permissive for replication of small ruminant lentiviruses. Am. J. Vet. Res. 58, 579–584.

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