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1 Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France
2 MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK
Correspondence
Didier Vilette
d.vilette{at}envt.fr
Hubert Laude
hubert.laude{at}jouy.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: UMR INRA/ENVT 1225 Interactions Hôte–Agent Pathogène, 23 chemin des Capelles, 31076 Toulouse Cedex 03, France. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Weissmann, 2004), and a wealth of experimental data indicates that an abnormal form of PrP (PrPSc) is the infectious agent (Prusiner, 1998). PrPSc apparently triggers conformational conversion of PrPC into abnormal PrP; the newly produced conformers can further propagate misfolding of the protein, resulting in multiplication of the infectious agent (Castilla et al., 2005). In vivo, prions can multiply and spread along particular neuroanatomical pathways to reach the central nervous system (Beekes et al., 1998, and references therein), consistent with step-by-step infection of closely apposed cells. Extracellular forms of PrPSc can also be detected in infected tissues (Jeffrey et al., 2001), and diffusion of these extracellular forms (Brandner et al., 1996) could contribute to the spread of prion infection among more distant cells. These observations suggest that prion dissemination from non-circulating cells may occur through different modes. Infected cultures, in which the possible spread of prions may contribute to maintenance of infection, have been used as models to investigate mechanisms of prion cell-to-cell spreading. Experiments using paraformaldehyde-fixed cells as donors of prion infectivity have shown that target cells can be infected through cell membrane contact (Kanu et al., 2002). Additionally, the presence of infectivity in the culture medium of several infected cell lines (Archer et al., 2004; Baron et al., 2006; Schatzl et al., 1997) raises the possibility that cell-free infectivity participates significantly in the spread of prion infection among cells. In the present study, the issue of prion spreading was investigated in two cell lines identified as permissive for sheep prions. MovS is a cell line with Schwann-like features established from ovine transgenic mice (Archer et al., 2004). The epithelial RK13 cell line, which does not show detectable levels of rabbit endogenous PrPC, has been transfected to allow stable expression of ovine PrPC (Vilette et al., 2001), resulting in the Rov cell line. We showed that intercellular transfer of prions was much more efficient in MovS cells than in Rov cultures. Analysis of the spatial distribution of the newly infected MovS cells revealed that, whilst distant cells could also become infected, cells proximal to the infected donor cells consistently accumulated more abnormal PrP. Our findings indicate that, despite the presence of extracellular, cell-free infectivity, the progression of infection in this experimental model occurred mainly through infection of neighbouring cells. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Staining of abnormal PrP in infected cultures was performed using mAb ICSM33. This antibody was obtained through immunization of PrP knockout mice with a beta form of recombinant human PrP (Jackson et al., 1999). This antibody recognizes the abnormal form of ovine PrP in various experimental settings, including staining of infected cells (this study) and immunoprecipitation. Full characterization of this antibody will be published elsewhere. Immunoblot analysis of 
-tubulin was performed with mAb B-5-1-2 (Sigma).
Cell culture.
MovS cells (clone MovS6; Archer et al., 2004), MS0/0 (clone F10; Archer et al., 2004), Rov cells (clone Rov9; Vilette et al., 2001) and RK13 cells (Christofinis & Beale, 1968) were maintained at 37 °C in 6 % CO2 in a mixture of three parts Dulbecco's modified Eagle's medium to one part F12 medium (MovS cells) or in -minimal essential medium (-MEM) (RK13 and Rov cells) supplemented with 10 % fetal bovine serum, 100 U penicillin ml–1 and 10 µg streptomycin ml–1. The cell lines were split 1 : 4 (RK13 and Rov cells) or 1 : 10 (MovS cells) every week. To induce the expression of ovine PrPC in Rov cells, 1 µg doxycycline ml–1 was added to the culture medium.
Sheep prion strain.
The PG127 sheep isolate (Vilotte et al., 2001) was serially propagated and biologically cloned into Prnp0/0 transgenic mice expressing the VRQ allele of ovine PrPC (Vilotte et al., 2001) to obtain the 127S strain used in this study.
Co-cultures.
Persistently infected Rov and MovS cells were obtained by exposing uninfected cultures to 2.5 % infectious brain homogenate from ovine transgenic mice infected with the 127S strain, as described previously (Vilette et al., 2001). The resulting infected cultures were then grown for at least 1 month to ensure that cultures were infected at a high and stable level before being used in co-culture experiments.
Co-cultures between infected MovS or Rov cells and recipient target cells (either MovS, Rov, MS0/0 or RK13 cells) were seeded at a high density (1.2x106 cells per well of a six-well plate). The cell culture medium was changed after 1 week. Co-cultures were healthy, with no or very few free-floating cells in the medium. In MovS and Rov cultures, the amount of total cellular protein increased by only 16±7 and 39±7 %, respectively, after 2 weeks of culture, indicating that the cells were far from having doubled during the experiments. A ratio of one infected cell per 10 or 100 target cells was used, as indicated. After 1 or 2 weeks, co-cultures were analysed for the presence of abnormal PrP by immunofluorescence or by immunoblotting. In some experiments, target recipient cells were co-cultivated with infected cells that had been killed previously by one of the following methods: 6 h after seeding, infected donor cells were killed by (i) four cycles of freezing/thawing; (ii) drying for 2 days; (iii) irradiation for 30 min at a distance of 10 cm under a UV lamp (Philips ULTRA-VIOLET, 5.39 J cm–2 and 1.2 mV cm–2); or (iv) fixation for 30 min at room temperature in PBS containing 4 % paraformaldehyde and 4 % sucrose, followed by five washes in PBS, after which the cells were kept for 2 days at 4 °C in PBS. The efficiency of killing of all of these treatments was verified by trypan blue staining. In addition, no evidence of cell growth was observed when dead cells were further incubated in cell culture medium. Recipient target cells were then added to the dead cells. In some other experiments, co-cultures were incubated in complete -MEM containing 0.6 % agarose.
Isolation and Western blot analysis of abnormal PrP.
The procedures have been described in detail elsewhere (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]. Cellular proteins were quantified by bicinchoninic acid and identical amounts of cellular proteins (usually 500 µg) were digested with 2 µg proteinase K (PK) for 2 h at 37 °C. Pellets of aggregated, PK-resistant PrP (PrPres) were collected by centrifugation and electrophoresed on 12 % SDS-polyacrylamide gels before transfer to nitrocellulose filters.
Detection of abnormal PrP by immunofluorescence microscopy.
Co-cultures (5x105 cells with a ratio of one infected cell per 100 uninfected cells) were grown on coverslips in 12-well plates. Cells were fixed with 4 % paraformaldehyde/4 % sucrose in PBS for 10 min, permeabilized for 5 min with 0.1 % Triton X-100 in PBS and treated for 5 min with 3 M guanidine thiocyanate in PBS (Taraboulos et al., 1990) at room temperature. Cells were then incubated with ICSM33 anti-PrP mAb (1.5 µg ml–1) for 1 h. Bound mAbs were visualized with Alexa-conjugated secondary antibodies and coverslips were mounted on slides using Vectashield anti-fading medium (Vector Laboratories). Nuclei were stained with DAPI. Immunofluorescence images were acquired with a Leica DMR microscope equipped with a Leica DC 330F camera.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Rov cells are RK13 kidney epithelial cells stably transfected with the ovine PrPC (Vilette et al., 2001). As controls, we used MS0/0 Schwann-like cells derived from Prnp0/0 mice (Archer et al., 2004) and RK13 cells. Neither RK13 nor MS0/0 cells express PrPC and they are not permissive for prion replication. Infected MovS and Rov cells used as a source of infectivity were from cultures in which 127S sheep prion replicated at similar, steady-state levels. Infected donor and uninfected target cells were seeded in six-well plates at a high density to minimize cellular proliferation as far as possible and we verified that the number of cells did not increase significantly during the experiments. Under these experimental conditions, an increase in PK-resistant PrP (PrPres), as assessed by immunoblotting or immunofluorescence analysis, could therefore be considered to reflect the infection of more cells in the co-culture.
Efficient spread of prion infection in MovS cultures
Cultures of chronically infected MovS cells co-seeded with uninfected MovS cells or with control MS0/0 cells were lysed after 1 or 2 weeks and the amount of PrPres was estimated by Western blotting. When infected MovS cells were co-cultivated with non-permissive recipient MS0/0 cells (10 % infected cells), a weak and constant PrPres signal was detected (Fig. 1, upper panel, lanes 3 and 4), corresponding to PrPres of the donor infected cells. When permissive MovS cells were used as recipient cells, PrPres levels were much higher (Fig. 1, upper panel, lanes 1 and 2) and typically reached those seen in the undiluted, infected culture (Fig. 1, upper panel, lanes 5 and 6). Similar levels of -tubulin were observed when the same lysates were analysed prior to PK digestion (Fig. 1, lower panel). These results suggested that infected MovS cells could infect additional cells efficiently, implying effective spread of prions within the culture. To confirm this point directly, abnormal-PrP-containing cells in the co-cultures were visualized by immunofluorescence microscopy using mAb ICSM33, which preferentially stains abnormal PrP-containing cells (G. S. Jackson, unpublished data, and Fig. 2a). When co-cultures of infected and uninfected (1 % infected cells) MovS cells were analysed 10 days after seeding, most of the cells were positive for abnormal PrP (Fig. 2b, left panel). This was not observed in co-cultures with MS0/0 cells, where staining was restricted to isolated cells (Fig. 2b, right panel), presumably corresponding to the 1 % of infected MovS cells seeded in the culture. When co-cultures with permissive MovS cells were analysed earlier (5 days after seeding), clearly delineated areas stained strongly for abnormal PrP (Fig. 2d, upper left panel). In addition, a low-level, widespread immunoreactivity was detected in the remaining part of the cell monolayer (Fig. 2d, lower left panel). We have shown previously that infected MovS cultures secrete prion infectivity into the cell culture medium (Cronier et al., 2004; Fevrier et al., 2004). Thus, to investigate the extent to which extracellular infectivity participated in the spread of prions, co-cultures were overlaid with semi-solid medium (liquid medium containing 0.6 % agarose) to hinder diffusion. PrPres levels, as assessed by immunoblotting, were similar in 5 day co-cultures grown in the presence or absence of semi-solid medium (Fig. 2c). When analysed by immunofluorescence, co-cultures in semi-solid medium showed areas of abnormal-PrP-positive cells (Fig. 2d, upper right panel), but the widespread, low-level immunoreactivity was no longer observed (Fig. 2d, lower right panel). However, after 10 days, most of the cells in the co-cultures were positive for abnormal PrP (not shown), as observed in co-cultures in liquid medium (Fig. 2b, left panel). These results indicated that infection progressed mainly through neighbouring cells (visualized as foci of strongly stained cells), although it also involved more distant cells to some extent.
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, compare lanes 3 and 5). It should be noted that the low levels of PrPres in MS0/0 co-cultures (Fig. 3, lane 4), corresponding to abnormal PrP in the 10 % of infected donor cells, were also observed in cultures infected with killed, donor MovS cells (Fig. 3, lane 5), indicating that input PrPSc in the dead, infecting cells was present throughout the entire period of the co-culture. Similar results (not shown) were obtained with infected cells killed by various other means (fixation with paraformaldehyde, UV irradiation and drying), confirming that prion infection was much more efficient when initiated with living cells than with killed cells.
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Cell-to-cell spreading is much less efficient in epithelial Rov cultures
Spreading of prion infection was studied in epithelial Rov cultures. These cells express ovine PrPC and are permissive for multiplication of the ovine prion strain 127S. Although the abnormal PrP conformers produced by MovS and Rov cells have distinct ratios of glycoforms, these cells accumulated as much PrPres as infected MovS cells (Fig. 4a). The weak and constant PrPres signal observed when infected Rov cells were co-cultivated with non-permissive parental RK13 cells (Fig. 4b, lanes 3 and 4) was only marginally increased when permissive Rov cells were used as recipient cells (Fig. 4b, lanes 1 and 2) and no significant change was observed in 3 week co-cultures (data not shown). Similar co-culture experiments were conducted with another clone of Rov cells (clone RovF9). Co-cultures of 10 % infected RovF9 and 90 % uninfected RovF9 cells showed minimal infection of the target uninfected cells (data not shown).Therefore, unlike the observations in MovS co-cultures (Fig. 1
), PrPres levels in Rov co-cultures seeded with 10 % infected cells were far from being restored to the levels of the undiluted infected culture (Fig. 4b, lanes 5 and 6). Further evidence that dissemination of prion infection is less efficient in epithelial Rov cultures than in neuroglial MovS cultures was obtained by exposing both cell lines to serial 10-fold dilutions of the same infectious brain homogenate and periodically monitoring the accumulation of PrPres. Under these experimental conditions (i.e. using diluted inoculum), it has been shown that PrPres from the inoculum is not detected and signals correspond to PrPres with an electrophoretic mobility typical of cell-produced PrPres (Archer et al., 2004; Vilette et al., 2001). In early passages (3 weeks post-inoculation), levels of PrPres in infected MovS cultures varied according to the initial concentration of the inoculum, with low levels of PrPres being detected in cultures exposed to highly diluted infectious material (Fig. 5). In contrast, after 5 weeks, MovS cultures challenged with high or low concentrations of inoculum had similar levels of PrPres, consistent with the spread of infection in these cultures (Fig. 5). The situation was strikingly different in Rov cultures, where PrPres levels were dependent solely on the amount of inoculum, even at higher passage numbers (Fig. 5).
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). As observed previously, efficient cell-to-cell transmission of prion infection occurred in MovS cells exposed to infected MovS cells, but not in Rov cells exposed to infected Rov cells. Interestingly, the levels of PrPres observed in MovS cultures infected with Rov cells were low, indicating that MovS cultures were infected inefficiently by Rov cells. As discussed below, the simplest explanation for this result is that infected Rov cells allow the replication of high levels of infectivity but with inefficiently transmission of infection to recipient target cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), whilst no spread of prion infection was observed in fibroblast cultures infected with the 22L strain of murine prions (Vorberg et al., 2004). Transfer of infection from SMB cells infected with the Chandler strain to target cells has also been reported (Kanu et al., 2002). In the latter experimental approach, however, the infected donor cells were eliminated by drug selection during co-culture, and the possible infection of target cells by the products of dying, infected cells rather than by cell-to-cell transmission was not addressed. With the aim of further investigating prion spread in cell culture, we used short-term, confluent co-cultures of prion-infected cells and naïve target cells. Our main findings were that: (i) cell-to-cell spread of infection may be an efficient phenomenon, as a whole culture of MovS target cells could be infected within 10 days using 10 % infected cells; (ii) infection proceeded mainly through transmission to neighbouring cells, although transmission to more distant cells is also proposed to occur; (iii) the efficiency of cell-to-cell spreading varied greatly between the two cell models studied, arguing that the ability to replicate and to spread the agent are distinct phenomena.
Using infected MovS and Rov cultures replicating the same prion strain at similar titres (about 2 LD50 per cell) (Archer et al., 2004; Sabuncu et al., 2003), the present work provides evidence that the efficiency of dissemination in cell culture may vary depending on the cell model. As 127S prions disseminated poorly among Rov cells, we assumed that the increase in PrPres observed after infection of Rov cultures (Vilette et al., 2001) reflected an increase in abnormal PrP in cells infected ab initio by prions present in the inoculum. Once infected, these cells accumulated high levels of infectivity but were relatively inefficient in transmission of the infection to other cells in the culture. By contrast, we showed that dissemination of prion infection to additional cells clearly participated in the increase in levels of abnormal PrP in infected MovS cultures. The reasons why dissemination of prion infection is more efficient in MovS cultures than in Rov cells merit further study. As both infected cultures had a high (>50 %) proportion of infected cells (Archer et al., 2004; Vilette et al., 2001) and accumulated similar amounts of abnormal PrP (Fig. 4a), poor dissemination among Rov cells may have resulted from differences in the cell biology of PrPSc, possibly linked to the species of these cells. Possible differences in the subcellular distribution of abnormal PrP in the two cell types (e.g. cell surface vs intracellular compartments) might contribute to better transmission in MovS cells. The number of infectious microvesicles released by each cell type may also be involved (see below). In any case, our findings emphasize cell-to-cell spread as a potential limiting factor during prion propagation in vivo.
Infectivity has been found in the culture medium using different cell models of prion infection, such as GT1 and SN56 cells infected with murine strains of prions (Baron et al., 2006; Schatzl et al., 1997) and MovS cells infected with ovine prions (Archer et al., 2004; Cronier et al., 2004), suggestive of a natural release mechanism. We have previously provided evidence that extracellular prion infectivity released from infected MovS cells is, at least in part, associated with small vesicles called exosomes (Fevrier et al., 2004), and the finding that cultured neurons release exosomes (Fauré et al., 2006) supports the proposal that microvesicles may be involved in the spread of prion infection (Fevrier et al., 2004; Porto-Carreiro et al., 2005). We think that the low-level, widespread PrPSc staining observed in Fig. 2(d) most likely reflects infection of distant cells by released microvesicles. Consistent with this interpretation, this staining was not observed in co-cultures in semi-solid medium.
A striking finding of this study was that, despite the presence of extracellular infectivity, infection proceeded mainly through transmission to neighbouring cells. The foci of strongly stained cells observed in this study were reminiscent of those observed in virus-infected cell cultures maintained either in semi-solid culture medium or in liquid medium for viruses that essentially remain cell-associated (Fields & Knipe, 1990). Different mechanisms, which are not mutually exclusive, could participate in the cell-to-cell transfer of prion infection. Glycosylphosphatidylinositol-linked glycoproteins can be released and reincorporated into the recipient target membrane, a process known as ‘painting’ (Medof et al., 1984). More specifically, intercellular transfer of PrPC after cellular activation with phorbol 12-myristate 13-acetate (PMA) has been reported (Liu et al., 2002). However, it is uncertain whether the abnormal form of PrP located at the cell surface could also be subjected to painting and hence could initiate conversion events in the recipient cell. Unlike PrPC, PrPSc is not released after phosphatidylinositol phospholipase C treatment, suggesting that this isoform has a distinct mode of association with the cell surface (Caughey et al., 1990; Stahl et al., 1990). In this regard, PMA treatment of MovS and Rov co-cultures failed to enhance the spread of prion infection (data not shown), possibly reflecting the inability of abnormal PrP to be subjected to painting.
Could abnormal PrP on the cell surface of an infected cell interact in trans with normal PrPC on the plasma membrane of an adjacent target cell and promote further conversion in the recipient cell? Some experiments suggest that abnormal PrP may act in trans to promote infection of target cells. Aldehyde-fixed, infected SMB cells are able to infect co-cultivated target cells, albeit at a relative low efficiency (Kanu et al., 2002). Also, neuroblastoma N2a cells can be infected with prions thought to be irreversibly bound to physical supports, such as stainless steel wires (Weissmann et al., 2002). However, trans interactions may not be the most efficient way to promote conversion of normal PrPC on a target cell. Indeed, cell-free reactions of detergent-resistant, membrane-bound PrPC with microsome-associated abnormal PrP show that conversion appears to be much more efficient if PrPC and PrPSc are in the same membrane (Baron et al., 2002). The fact that we (this study) and others (Kanu et al., 2002) found that prion infection is much more efficient when infectivity is administered in the form of living cells suggests that dissemination of prions involves active biological processes in addition to simple contacts between apposed PrP isoforms. We therefore propose that, at least in some situations, the release of infected microvesicles, probably involved in long-range dissemination, might primarily infect recipient cells adjacent to the secreting infected cells. Progression of infection is visualized as ‘prion plaques’, reminiscent of the plaques resulting from the step-by-step progression of virus infection. Finally, it would be of interest to apply the approach used in this study to assess the spatial progression of infection in other cell system/prion strain combinations, including those in which cell-free infectivity is released (Baron et al., 2006; Schatzl et al., 1997).
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ACKNOWLEDGEMENTS |
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Received 28 June 2006;
accepted 18 October 2006.
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