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1 Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France
2 Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Rome, Italy
3 Department of Biochemistry and Molecular Biology, Bio21, Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia
Correspondence
D. Vilette
d.vilette{at}envt.fr
H. Laude
hubert.laude{at}jouy.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Laboratoire de Biologie Cellulaire et Moléculaire, UFR des Sciences Exactes et Naturelles, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France. 
Present address: Prion Center, University of Alberta, Edmonton, AB T6G 2M8, Canada.
Present address: Ludwig Institute for Cancer Research, Post Office Royal Melbourne Hospital, Parkville, VIC 3050, Australia.
||Present address: UMR 1225, INRA/ENVT, 23 Chemin des Capelles, 31076 Toulouse, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and tissues (Bueler et al., 1993), several lines of evidence indicate that PrPC expression is not sufficient to confer permissiveness to prion propagation (Enari et al., 2001; Raeber et al., 1999). Although several new permissive cell lines were recently identified, the number of cell models remains quite limited (for a review, see Vilette, 2007). More importantly, the ex vivo multiplication of a number of strains of particular interest has not yet been achieved. This suggests that prion multiplication may require strain-specific, PrPC-independent factors, the conjunction of which would be restricted to a few cell lines. Previous work has identified epithelial RK13 cells as an efficient and robust cell system for the multiplication of a natural strain of sheep scrapie, provided that these non-ovine cells expressed sheep PrPC (Rov cells; Vilette et al., 2001). Here, we have shown that RK13 cells genetically engineered to express rodent PrPC, either from the mouse or the bank vole, a species highly permissive to various prion isolates or strains (Cartoni et al., 2005; Chandler & Turfrey, 1972; Nonno et al., 2006), were readily infected by prions adapted to and propagated in these two species, thus extending the range of prion strains that can be multiplied in this cell-culture model. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Generation of RK13 cells expressing mouse and vole PrPC.
The open reading frame (ORF) of vole PrPC was PCR amplified from bank vole (Myodes glareolus, formerly Clethrionomys glareolus) genomic DNA, cloned into pBluescript plasmid and verified by sequencing. The ORF encoding the vole PrPC with methionine at position 109 (Cartoni et al., 2005) was then subcloned into the pTRE plasmid (Clontech). The pTRE plasmid encoding the mouse PrPC (allele a) was provided by Dr S. Lehmann (Institut de Génétique Humaine, France). Each plasmid was introduced by transfection into RK13 cells as described previously (Vilette et al., 2001), and puromycin-resistant cell clones were selected and tested for doxycycline (dox)-inducible expression of PrPC. The data reported in this study with mouse (moRK13) and bank vole (voRK13) RK13 cells were obtained using clones #55 and #1/9, respectively, but similar findings were observed with other cell clones. The cultures were maintained at 37 °C in 6 % CO2 in Opti-MEM (Invitrogen/Gibco) supplemented with 10 % fetal bovine serum, 100 U penicillin ml–1 and 10 µg streptomycin ml–1. Cell lines were split by a 1 : 4 dilution every week. To induce expression of PrPC in the cell clones, 1 µg dox ml–1 was added to the culture medium.
Prion strains.
The murine strains Fukuoka-1 (Tateishi et al., 1979, originating from the laboratory of S. Katamine, Nagasaki, Japan), Chandler, 22L and ME7 (Bruce & Fraser, 1991, originating from the laboratory of R. Carp, Staten Island, NY, USA) were maintained in C57BL/6 mice. Transmission and stabilization of the natural sheep scrapie isolate Ss3 and of sheep bovine spongiform encephalopathy (BSE) into bank voles have been described previously (Nonno et al., 2006; Piening et al., 2006). The survival times of bank voles serially infected with cattle BSE were similar to those of bank voles infected with sheep BSE (U. Agrimi, unpublished data). Infected brains were homogenized at 10 % (w/v) in a sterile 5 % glucose solution with a Ribolyser (Hybaid) and sonicated for 1–2 min before incubation with the cells.
Infection of RK13 cultures.
Confluent cultures grown for 2 days in single wells of 12-well plates in the presence of 1 µg dox ml–1 were incubated in culture medium containing 2.5 % infected brain homogenates. After 2 days, the medium was removed and the cells rinsed with PBS and split into two 25 cm2 flasks. Each week, one flask was used for subcultivation, whilst the other was used to prepare a cell lysate for PrP analysis.
Isolation and Western blot analysis of PrPres.
The isolation of cell-derived PrPres has been described previously (Paquet et al., 2004; Vilette et al., 2001). Briefly, cell cultures were solubilized in lysis buffer [50 mM Tris/HCl (pH 7.4), 0.5 % Triton X-100, 0.5 % sodium deoxycholate]. Lysates were clarified (2000 r.p.m. for 1 min in a microcentrifuge) and cellular proteins were quantified by bicinchoninic acid using a BCA Protein Assay kit (Pierce). Identical amounts of cellular protein (500 µg) were digested with 2 µg proteinase K (PK) for 2 h at 37 °C in the presence of 0.02 % bromophenol blue for increased visualization of the pellets obtained after centrifugation. Pefabloc (4 mM) was added to stop the reaction and blue pellets containing aggregated PK-resistant PrP (PrPres) were collected by centrifugation at 13 000 r.p.m. in a microcentrifuge for 30 min at room temperature and separated by 12 % SDS-PAGE before transfer to nitrocellulose filters. In some experiments, pellets were treated with the endoglycosidase PNGaseF (New England Biolabs) prior to immunoblotting. For brain-derived PrPres analysis, 150–250 µg brain tissue equivalent was solubilized in lysis buffer. Clarification, PK digestion and PrPres recovery was as described for cell-derived PrPres.
Monoclonal antibody (mAb) 4F2 (Krasemann et al., 1996) recognizing the N-terminal region of PrPC was used to detect expression of full-length PrPC. As this antibody cannot react with N-terminally truncated PrPres, immunoblot analysis of abnormal PrP in PK-digested cell lysates and brain homogenates was performed with mAb Sha31 (Feraudet et al., 2005). Blots were developed using an ECL+ reagent kit (Amersham).
Pharmacological treatment of infected voRK13 cultures.
Infected voRK13 cultures were seeded in six-well plates in the presence of DS 500 (1 µg ml–1), MS-8209 (50 µg ml–1) or with vehicle only and incubated for 5 days with one medium change. The different treatments did not induce any obvious phenotypic effect, and after solubilization of the cultures, the protein concentration was similar in treated and untreated cultures. The same amount of cellular protein (250 µg) was digested with PK and analysed for PrPres levels by immunoblotting.
Bioassay.
Cultures were rinsed three times with PBS, scrapped into PBS and recovered by centrifugation. The cells were resuspended in a sterile 5 % glucose solution, frozen (–80 °C) and thawed four times, sonicated and stored at –80 °C until inoculation. MoRK13 and voRK13 cultures (20 µl) were inoculated intracerebrally into ovine transgenic tga20 mice (Fischer et al., 1996) and bank voles, respectively. Animals showing neurological signs were monitored almost daily and euthanized in extremis.
Histoblotting.
The procedure for histoblot analysis (Taraboulos et al., 1992) of tga20 mice was as described previously (Beringue et al., 2007).
Histopathology and paraffin-embedded tissue (PET) blots.
Each bank vole brain was divided into two parts by a sagittal paramedian cut. One was frozen for immunoblot analysis and further passages into vole, and the remaining part was embedded in paraffin. Lesion profiles were established using the first and the second passage of bank voles inoculated with vole-adapted sheep BSE agent serially propagated in voRK13 cells. PET blots were performed with the second vole passage. The procedures for the construction of lesion profiles and PET blot analysis have been described previously (Nonno et al., 2006).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. These data confirmed that RK13 cells did not express detectable levels of endogenous PrPC and established that PrPC from various species could be expressed efficiently in these cells.
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; Piening et al., 2006), whilst the other two were the BSE agent (either from cattle or from sheep, see Methods) serially transmitted to bank voles (Piening et al., 2006). Prior to exposure of the cell cultures, abnormal PrP (PrPres) in brain homogenates from the infected animals was analysed by immunoblotting. Fig. 2 confirmed that PrPres can show strain-specific features. Brain-derived PrPres from strain Fukuoka-1 migrated more slowly than PrPres from strains Chandler and ME7 (Fig. 2a), as reported previously (Arima et al., 2005). PrPres from the BSE agent in bank vole had a higher electrophoretic mobility compared with that from Ss3 sheep scrapie (Fig. 2b), consistent with the unique pattern of BSE PrPres observed in different species (Beringue et al., 2006; Castilla et al., 2003; Eloit et al., 2005; Hill et al., 1997; Houston et al., 2000; Scott et al., 1997).
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, lanes 1 and 3). Additional experiments indicated that PrPres was also readily detected in moRK13 cells exposed to the 22L strain of mouse prions (data not shown), whilst no PrPres was detectable in cultures exposed to ME7 (Fig. 2c, lane 2). Cell-derived PrPres was produced in voRK13 cells infected with the bank vole BSE agent originating from either cattle or sheep (Fig. 2d, lanes 2, 3, 6), whilst no PrPres was observed in voRK13 inoculated with the vole-adapted sheep scrapie Ss3 isolate (Fig. 2d, lane 1). In each case of successful transmission, no PrPres was observed when infections were carried out in the absence of dox. This demonstrated that no residual abnormal PrP from the inoculum was detected under these experimental conditions and that PrPres was produced de novo by the cells.
Our findings indicate that permissiveness of RK13 appears to be strain-specific. No PrPres was detected in voRK13 cells inoculated with Ss3 or in moRK13 cells exposed to the ME7 strain. This latter observation is reminiscent of previous observations with N2a and GT1, two cell lines permissive to the Chandler/RML strain but resistant to ME7 (Bosque & Prusiner, 2000; Klohn et al., 2003). However, successful multiplication of ME7 in other cell lines [e.g. SN56 (Baron et al., 2006), MG20 (Iwamaru et al., 2007) and L929 (Vorberg et al., 2004)] indicate that the cell tropism of prion multiplication observed in vivo can manifest in cultured cells.
To compare more accurately the electrophoretic mobility of abnormal PrP of these strains after propagation in culture, PrPres in moRK13 cells infected with strains Chandler and Fukuoka-1 and in voRK13 cells infected with vole-adapted BSE agent were deglycosylated by PNGaseF treatment and analysed by Western blotting. Fukuoka-1 PrPres retained a lower electrophoretic mobility compared with Chandler PrPres (Fig. 3, lanes 1 and 2), whilst PrPres generated in voRK13 cells infected with the vole-adapted BSE agent migrated faster (Fig. 3, lanes 3 and 4).
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). The survival time of tga20 mice inoculated with moRK13 cultures (3x105 cells per mouse) was 127±3 days, and all had PrPres in their brain, the pattern of which was indistinguishable from that in the brain material used as inoculum (data not shown). Bank voles inoculated with infected voRK13 cultures (3x106 cells) had a survival time of 90±5 days, an incubation period similar to that obtained when the strain was grown in the animal (Piening et al., 2006). No detectable infectivity was associated with inoculated cells expressing no bank vole PrPC (parental RK13 cells, Table 1). All of the diseased bank voles showed PrPres in the brain with an electrophoretic pattern indistinguishable from that in the original inoculum (data not shown). These results demonstrated that mouse and bank vole infectious prions are propagated efficiently in RK13 cells.
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; Taraboulos et al., 1992) and the extent of vacuolation in selected brain areas as originally described by Fraser & Dickinson (1968) were used to investigate strain-specific features. The PrPres regional distribution in tga20 mice inoculated with cell-passaged Fukuoka-1 and 22L strains was compared by histoblotting with that of Fukuoka-1 and 22L serially passaged in the same mouse line. As illustrated in Fig. 4(a), PrPres distribution was essentially similar in both groups, but was clearly distinguishable between the Fukuoka-1 and 22L strains. After infection by RK13- or mouse-passaged Fukuoka-1 strain, PrPres deposits were diffuse and present in numerous areas of the brain such as the fimbria, corpus callosum, anteromedial thalamus nuclei and, to a lesser extent, the lateral hypothalamus (Fig. 4a and data not shown). 22L staining was more punctate and for both groups predominantly involved the septum, basal nuclei, anteromedial and ventral thalamic nuclei, lateral hypothalamus, habenular nuclei, inferior colliculus, raphe nuclei and aqueduct region (Fig. 4a and data not shown). voRK13- and bank vole-passaged sheep BSE caused indistinguishable PrPres distribution, as assessed by PET blot analysis of bank vole brains (Fig. 4b). Accumulation of PrPres was evident throughout the brain and in particular in the medulla, cerebellar nuclei, lateral geniculate nuclei, superior colliculus, red nucleus, thalamic nuclei, septum and cingulate, and parietal cortices. The lesion profile in the brains of bank voles inoculated with infected voRK13 cells was also determined (Fig. 4c). Bank vole- and voRK13-passaged sheep BSE produced identical patterns of vacuolar degeneration in bank voles, characterized by abundant grey-matter spongiform degeneration in all brain areas examined with the exception of the cerebellum. Collectively, these findings supported the view that the three strains studied were not modified through multiplication in RK13 cells.
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). To determine whether the multiplication of bank vole-adapted BSE agent was sensitive to pharmacological treatment, infected cultures were incubated for 1 week in the presence of DS 500 and the amphotericin B derivative MS-8209, two molecules known to delay the onset of disease in experimentally infected rodents (Adjou et al., 1995; Ehlers & Diringer, 1984). Fig. 5 shows that DS 500 and MS-8209 decreased PrPres levels in infected, treated voRK13 cultures. As no cell system enabling replication of cattle BSE or human vCJD agents is available to date, voRK13 cells, together with a recently identified cell line permissive to mouse-adapted BSE (Iwamaru et al., 2007), may provide improved tools for the screening of drugs potentially effective against this human-affecting agent.
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), confirming that RK13 represents an interesting biological cell system to study how distinct abnormal PrP from various prion strains and species can be generated in a single cell type. This cell model may provide new opportunities for investigating the basis of strain identity.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 31 July 2007;
accepted 29 September 2007.
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, first passage; 
, second passage) or with the vole-passaged sheep BSE agent (
). Brain-scoring positions are medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8) and cingulate and adjacent motor cortex (9).