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1 Institute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany
2 Institute for Chemistry and Biochemistry, Ernst-Moritz-Arndt-Universität, Greifswald, Germany
3 alicon AG, Wagistrasse 23, CH-8952 Schlieren, Switzerland
Correspondence
Martin H. Groschup
martin.groschup{at}fli.bund.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Two models explaining the conversion of PrPC into PrPSc have been proposed (reviewed by Aguzzi & Polymenidou, 2004
). The first is referred to as template-directed refolding in which the formation of a heterodimer between endogenous PrPC and exogenous PrPSc induces the transformation of PrPC into PrPSc. Conversion of PrPC itself into PrPSc is inhibited by a high-energy barrier. A second model, called seeded nucleation, suggests that both monomeric forms – PrPC and PrPSc – are in an equilibrium. After the formation of monomeric PrPSc into multimeric ‘infectious' seeds, PrPC is converted and incorporated into the growing fibrillar amyloid aggregates.
A major problem with the ‘protein-only’ hypothesis (Prusiner, 1984) of prion propagation is the explanation of the existence of multiple, phenotypically distinct prion isolates or strains. These different strains show distinct incubation times and specific patterns of neuropathological targeting in the brain. Prion strains may also differ in their biochemical properties such as glycoform ratios (Kascsak et al., 1986), aggregation state and PK digestion patterns of PrPSc (Kuczius & Groschup, 1999). As the amino acid sequence of the prion protein is identical in different prion strains within the same species, it is considered that the conformation and/or post-translational modifications of PrPSc carry the information that defines these strains. For instance, there are two strains of hamster-adapted transmissible mink encephalopathy (TME) called Hyper (HY) and Drowsy (DY) (Bessen & Marsh, 1992). Treatment of PrPSc of these two strains with PK results in fragments of different sizes. These differently sized fragments could also be transmitted to newly formed PrPSc/PrPres in vivo and in vitro (Bartz et al., 2000). However, the molecular mechanism of this strain specificity remains unclear.
Although most investigations have been carried out in vivo in the form of infection studies, suitable in vitro systems have recently been established that permit the biological characteristics of TSEs to be studied. Neuronal cells (ScN2A, SMB) that are persistently infected with mouse-passaged scrapie strains are commonly used to analyse prion conversion in vitro. These cells are able to convert endogenous PrPC into PrPSc and accumulate PrPSc over several passages (Race et al., 1987; Butler et al., 1988).
In contrast to infection studies in animals or with scrapie-infected cultured tissue cells, cell-free conversion assays utilize purified PrPC and PrPSc, where PrPC is converted into its protease-resistant form, PrPres, with characteristics similar to those of the progenitor PrPSc (Kocisko et al., 1994). In previously published cell-free conversion studies, radiolabelled PrPC was used as substrate and PrPres formation was visualized by phosphorimaging autoradiography (Caughey et al., 1999a). Cell-free conversion assays represent a precise system to analyse molecular factors as well as the kinetics of conversion reactions (Bossers et al., 2003). The system has been used for the analysis of species and strain specificities of the prion protein, e.g. the interaction of mouse and hamster PrPC with mouse- and hamster-derived PrPSc (Kocisko et al., 1995), of human PrPC with bovine spongiform encephalopathy (BSE) and scrapie (Raymond et al., 1997) and of several PrPC species with chronic wasting disease (Raymond et al., 2000). Additionally, the interaction of ovine PrPC variants with different scrapie strains (Bossers et al., 1996, 2000) and of hamster PrPC with TME strains (Bessen et al., 1995) has been examined.
Protein misfolding cyclic amplification is another cell-free conversion system, in which the seeding effect of PrPSc is enhanced by serial ultrasonification to repetitively generate conversion-active fibrils (Saborio et al., 2001). Recently, it has been reported that PrPres generated by this method is infectious (Castilla et al., 2005).
In another cell-free system, recombinant PrPC was turned into PK-resistant fibrillar aggregates, even without the addition of PrPSc, when suitable denaturants and buffer conditions were used (Bocharova et al., 2005; Baskakov & Bocharova, 2005).
The present study was performed to characterize both the conversion kinetics of bacterial prion protein and the interaction of BSE and scrapie strains in a modified, cell-free assay. De novo-formed PrPres was detected using a specific antibody that discriminated between PrPres and the PrPSc seed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This construct was cloned into the Escherichia coli expression vector pET19b (Novagen) using the XhoI and BamHI cleavage sites. Due to the vector composition, this construct contained an N-terminal histidine (His) tag and an additional enterokinase cleavage site. The construct was expressed in Origami (DE3) cells (Novagen). Purification was performed under denaturing conditions according to the manufacturer's instructions (Qiagen): cells of a 50 ml E. coli culture were harvested with lysis buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris, adjusted to pH 8.0 with NaOH) and gently shaken for 1 h. Samples were centrifuged at 14 000 r.p.m. (30 min, 4 °C) in an FA-45-30-11 rotor (Eppendorf) and the supernatant was incubated with Ni-NTA resin (Qiagen). After 1 h of shaking, the lysate resin mixture was loaded on to a column, which was subsequently washed with buffer containing 8 M urea, 0.1 M NaH2PO4 and 0.01 M Tris/HCl (pH 6.3). The protein was eluted in buffer D [8 M urea, 0.1 M NaH2PO4, 0.01 M Tris/HCl (pH 5.9)] followed by elution in buffer E [8 M urea, 0.1 M NaH2PO4, 0.01 M Tris/HCl (pH 4.5)]. Eluted fractions were dialysed and refolded against 20 mM HEPES/KOH (pH 7.4). The protein yield was approximately 20 µg ml–1.
In order to obtain the high yields of pure protein needed for circular dichroism (CD) spectroscopy, PrPC was purified according to a protocol described previously (Zahn et al., 1997). For cell-free conversion studies, this PrPC preparation was diluted to a final concentration of 20 µg ml–1 in 20 mM HEPES/KOH (pH 7.4). Full-length recombinant hamster PrPC was purchased from Prionics AG.
CD spectra were recorded on a Jasco J810 spectropolarimeter using a cuvette with a 2 mm path length. Measurements were carried out at 20 °C in H2O at a final protein concentration of 100 µg ml–1.
Propagation of BSE and scrapie strains.
C57BL/6 mice were inoculated intracerebrally with 20 µl 1 % mouse brain homogenate using mouse scrapie strains ME7, 22A and RML and with a BSE strain. The BSE strain was initially isolated from a German BSE case using C57BL/6 mice and was repeatedly subpassaged thereafter. The scrapie strain 87V was propagated in VM mice (kindly provided by Dr M. Bruce, Institute for Animal Health, Edinburgh, UK) and the hamster scrapie strain 263K was propagated in Syrian golden hamsters. After the onset of clinical symptoms, animals were euthanized and their brains were removed and stored at –20 °C.
Purification of PrPSc.
Purification was performed according to an established procedure (Caughey et al., 1999b) with some modifications. Mouse scrapie brain (1 g) was homogenized on ice in 15 ml TEND buffer [10 mM Tris/HCl (pH 8.3), 1 mM EDTA, 130 mM NaCl, 1 mM DTT] containing 10 % N-lauroyl sarcosine with protease inhibitors (0.1 mM Pefabloc, 0.5 µg leupeptin ml–1, 1.0 µg aprotinin ml–1, 0.7 µg pepstatin ml–1) and then centrifuged at 23 000 r.p.m. in a TLA 100.4 rotor (Beckman) for 30 min (4 °C). The supernatant was then recentrifuged for 2.5 h at 58 000 r.p.m. (TLA 100.4 rotor, 4 °C). The resulting pellet was resuspended in 15 ml TEND buffer containing 10 % NaCl and 1 % sulfobetaine 14 (SB14). This suspension was then centrifuged at 68 000 r.p.m. for 1.5 h (TLA 100.4 rotor, 20 °C). The remaining pellet was resuspended in TMS buffer (10 mM Tris/HCl, pH 7.4, 5 mM MgCl2, 0.1 M NaCl) containing 10 % (w/v) NaCl and 1 % (w/v) SB14 and carefully loaded on to a sucrose cushion consisting of 1.0 M sucrose, 0.1 M NaCl and 0.5 % SB14 for a final centrifugation at 68 000 r.p.m. for 1.5 h (TLA 100.4 rotor, 20 °C). The pellet was collected in PBS containing 0.5 % SB14, sonicated and homogenized in a Teflon glass dounce homogenizer. Aliquots were stored at –20 °C.
Each conversion reaction was carried out with approximately 400–800 ng PrPSc material. As the exact concentration was impossible to measure due to the fibrillar properties of PrPSc, quantification was estimated on the basis of immunoblot signal intensities.
PrP conversion assay.
For the conversion reaction, 400 ng bacterial prion protein PrPC was incubated with 400–800 ng PrPSc in a conversion buffer consisting of 50 mM citrate/NaOH (pH 6.0), 200 mM KCl, 5 mM MgCl2 and 1.25 % Sarkosyl (Horiuchi et al., 2000). A standard incubation was carried out for 3 days at 37 °C. Three controls were used: (i) PrPC without PrPSc or PK, (ii) PrPC without PrPSc, incubated with PK, and (iii) PrPC incubated together with PrPSc. The third control was immediately frozen (t=0). After the end of the incubation, the samples were treated with PK at a final concentration of 30 µg ml–1 (in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl in 50 µl) and incubated for 1 h at 37 °C. The reaction was stopped with 10 mM PMSF and 20 µg of a carrier protein (thyroglobulin) was added. Samples were then incubated with 4 vols of methanol at –20 °C. The precipitated proteins were pelleted by centrifugation at 14 000 r.p.m. in an FA-45-30-11 rotor (Eppendorf) for 15 min.
SDS-PAGE and immunoblotting.
Pellets were resuspended in a loading buffer containing 1 % (w/v) SDS, 25 mM Tris/HCl (pH 7.4), 0.5 % mercaptoethanol and 0.001 % bromophenol blue, heat-denatured for 5 min at 95 °C and loaded on to SDS-polyacrylamide gels containing 16 % bisacrylamide acrylamide/bisacrylamide (37.5 : 1). After electrophoresis, proteins were transferred to a PVDF membrane in a semi-dry chamber. Membranes were incubated in blocking buffer (PBS containing 0.1 % Tween 20 and 5 % non-fat dried milk powder), followed by incubation for 60 min with monoclonal antibody (mAb) L42 (Harmeyer et al., 1998) or polyclonal antibody (pAb) Ra10 (Groschup et al., 1997). Ra10 was generated against a 16mer peptide based on the ovine sequence (aa 107–122) (Groschup et al., 1994). Membranes were washed three times for 10 min each with PBS containing 0.1 % Tween 20 and incubated for 60 min with a secondary antibody conjugated to alkaline phosphatase (AP) (goat anti-mouse–AP or goat anti-rabbit–AP). Membranes were washed again three times for 10 min each with PBS containing 0.1 % Tween 20. Subsequently, the chemiluminescence substrate CDP Star (Tropix) was applied and incubated on the membrane for 5 min before the light signals were detected directly using a camera. For stripping, the membranes were incubated twice for 15 min each with a buffer containing 0.2 M glycine/HCl (pH 2.0) and 1 % SDS.
Visualization was achieved using the Bio-Rad VersaDoc imaging system and quantification was measured using Quantity One quantification software (Bio-Rad). Conversion rate was estimated as the ratio of PrPres (PK-digested) to PrPC (undigested control). The relative conversion efficiency (%) was calculated as follows: (signal volume of PrPres digested with PK)/(signal volume of PrPC before PK digestion)x100. The conversion rate of each time point was calculated as a mean value from three independent reactions.
Molecular masses were determined using a molecular mass marker (FLI marker, developed at the Friedrich-Loeffler-Institut), which was a protein ladder with 1 kDa incremental steps (from 16 to 23 kDa). Each of these proteins harboured an N-terminal 6-His tag (Groschup et al., 2001), which was detected using an anti-His antibody (RGS-His; Qiagen).
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RESULTS |
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In the first experiments, PrPC was incubated for 3 days with PrPSc from scrapie strain ME7. After PK digestion and Western blotting, a PK-resistant PrPres fragment with a molecular mass of 17 kDa could be detected by mAb L42 (Fig. 1, lanes 4–5). Two controls were carried out: PrPC without PrPSc (Fig. 1, lane 2) and PrPC incubated with PrPSc that was stopped immediately after incubation (Fig. 1, lane 3). The L42-tagged PrPres fragment was partially PK resistant, i.e. had a 9 kDa lower molecular mass than the undigested precursor, which resulted from the removal of the N terminus (Fig. 1, lane 1). The conversion rate (% PrPres compared with the undigested PrPC control) was approximately 1 % (as determined by photoimaging).
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, exhibited CD spectra similar to unmodified recombinant hamster PrPC (Fig. 2a) and human PrPC (Zahn et al., 1997), which were indicative of an authentic secondary structure and protein folding. Proteins obtained from both purification methods were converted with the same conversion rate and PrPres fragments showed biochemical characteristics similar to the PK-resistant PrPres fragments (Fig. 2b). All subsequent reactions were carried out with the recombinant prion protein purified according to the Qiagen protocol (see Methods).
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) (Kuczius & Groschup, 1999; Hayashi et al., 2005). This molecular size difference observed in eukaryotic PrPSc was also apparent in the cell-free-converted prokaryotic PrPres fragments (Fig. 4). PrPres fragments derived from the BSE strain (Fig. 4a, lanes 7–8) exhibited a molecular mass that was 1 kDa lower than the ME7-derived PrPres fragments (Fig. 4a, lanes 5–6). The cell-free conversion of PrPC with BSE PrPSc also produced two additional fragments at approximately 14 and 15 kDa (Fig. 4a, lanes 7–8). These additional fragments corresponded to the C terminus and may have resulted from incompletely converted and hence further truncated PrPres. Similar low-molecular-mass fragments have been reported for cell-free-converted eukaryotic PrPres (Horiuchi et al., 2000). To visualize the PrPSc seed, the same blot was stripped and reincubated with pAb Ra10, which detects wild-type mouse PrPSc in strain ME7 and in BSE as unglycosylated, monoglycosylated and diglycosylated bands (Fig. 4b, lanes 3–8). Again, the differences in molecular size between the two strains were observed (Fig. 4b, compare lanes 3 and 4). In contrast to mAb L42 (Fig. 4a, lane 1), pAb Ra10 had a significantly lower affinity to L42-tagged PrPC (Fig. 4b, lane 1). For this reason, no PrPres fragments were visualized by pAb Ra10.
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). For unknown reasons, the mouse scrapie strain RML converted PrPC very inefficiently, if at all. The hamster scrapie strain 263K was used as another control and also did not convert murine PrPC, due to the existing species barrier (data not shown).
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) and are depicted in Fig. 5(a)
. For each graph, three independent conversion reactions were carried out. After an initial lag phase, which lasted up to 48 h, there was an elevated conversion activity, so that the PrPres signals peaked at 72 h. No further increase was seen and a steady state was reached, which lasted until approximately 168 h. The peak values for the newly converted prion proteins ranged from around 1 % in strains ME7 and BSE to 2 % in the 22A strain. Intermediate values were seen in strain 87V.
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). This co-incubation produced a PrPres doublet band, having two distinct fragments with a size difference of 1 kDa (Fig. 6, lanes 5–6). This result indicated that both conversion reactions were equally successful and without any intrinsic inhibitory effects. Due to the different molecular masses of the PrPres fragments (17 kDa derived from ME7 compared with 16 kDa derived from BSE), the quantification of the corresponding fragments was possible, even in co-incubation studies. Interestingly, co-incubation of ME7 and BSE PrPSc led to significantly higher amounts of ME7-derived and BSE-derived PrPres fragments compared with the amounts derived by each in single conversion reactions (Fig. 6, lanes 1–4 compared with lanes 5 and 6). Quantification and the temporal kinetics of the co-incubation are summarized in Table 2 and illustrated in Fig. 5(b).
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In additional experiments, co-incubation of ME7 or BSE with RML scrapie prions was carried out (Table 2 and Fig. 5b). PrPSc derived from the scrapie strain RML displayed no detectable cell-free conversion activity (Table 1), and this was also seen during simultaneous incubation (Table 2 and Fig. 5b): maximum conversion values were 0.53 (for ME7) and 0.98 (for BSE). In accordance with these results, co-incubation with RML prions did not display the synergistic effects that were observed in the co-incubation of scrapie ME7 and BSE prions.
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DISCUSSION |
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) and does not interfere with its conversion into PrPSc (Telling et al., 1997). Moreover, prion protein fused at its N terminus to green fluorescent protein supports prion replication in transgenic mice (Bian et al., 2006). We also showed by CD spectroscopy that the additional and/or mutated residues (L42 epitope, His tag and enterokinase cleavage site) of L42-tagged PrPC had no significant effect on the secondary structure (Fig. 2a).
Based on the immunoblotting data, approximately 1–3 % of the recombinant PrPC was converted into PrPres using this cell-free conversion assay when PrPSc was added at equimolar concentrations. This efficiency was ten times lower than in previous reports using cell-free conversion with radiolabelled PrPC (Bossers et al., 1997; Caughey et al., 1999b; Kirby et al., 2003). Hence, it cannot be excluded that the L42 epitope is disproportionally less accessible in the denatured PrPres fragment than in PrPC. However, the signal intensities were in the linear range of the Versadoc quantification system and permitted an exact and specific data analysis.
The cell-free conversion assay was carried out with six different scrapie strains/isolates. Four mouse strains (ME7, 87V, 22A and BSE) converted the recombinant mouse prion protein, mimicking the situation in vivo. A clear species specificity was observed, as the hamster scrapie strain 263K did not convert mouse PrPC in the cell-free assay. This is in accordance with a previously published report (Kirby et al., 2003) and reflects the inefficient experimental transmissibility of scrapie strain 263K to mice. However, a rather low efficiency, if any, was also found for the mouse scrapie strain RML, although most in vivo transmission characteristics of RML are similar to those of the other four mouse scrapie strains. Unusual behaviour with the RML strain during conversion in vitro has been described previously (Vorberg & Priola (2002). Indeed, biochemical differences are seen in PrPSc glycosylation: RML-derived PrPSc consists of a higher proportion of monoglycosylated protein compared with that derived from strains ME7, 87V, 22A and from BSE (Groschup & Kuczius, 2002), which may contribute to the failure to convert the bacterial prion protein by strain RML.
According to the data presented here, the kinetic stages involving the conversion of PrPC into PrPres using the three scrapie (ME7, 87V and 22A) strains and the BSE strain can be subdivided into three phases: an initial lag phase, followed by an efficient amplification phase and finally a plateau phase. These three stages can be explained by modified seeded nucleation reactions. Initially, PrPSc fibrils are broken into aggregates by the sonic treatment. However, they seem to require further maturation before efficient conversion can take place. It remains to be established whether conversion-active seeds are generated by further dissociation or by regeneration of the existing fragments. This is in accordance with a recent investigation (Silveira et al., 2005) in which particles consisting of 14–28 PrPSc molecules were identified to be the most efficient initiators of TSE disease. A second explanation for this initial delay may be the low binding efficiency of recombinant PrPC to PrPSc, which retards the conversion rate considerably in the first 48 h. However, the conversion of recombinant PrPC increases when sufficient amounts of de novo-formed PrPres are generated, which may function more efficiently as the progenitor seed. An equilibrium may eventually be reached between inactive PrPSc multimers and conversion-active seeds, resulting in the PrPres plateau phase.
These observations differ from previously reported conversion kinetics (Chabry et al., 1999). These authors observed no initial delay and the de novo PrPres levels had already peaked after 36 h and remained stable for up to 80 h. However, in previous studies, the PrPSc used was pretreated with 2.5 M guanidine hydrochloride. In another study (Mulcahy & Bessen, 2004), the authors applied milder denaturation conditions (1.0 M guanidine hydrochloride) for PrPSc and reported an elongation phase that continued up to 72–96 h. A significant increase in the conversion rate was observed after 48 h, which is similar to the data presented in this study. A steady state was reached after 120 h (compared with 72 h in this study) and was preceded by a depolymerization phase. The lack of a depolymerization phase in our study is in accordance with data from a previous study (Callahan et al., 2001) in which PrP aggregation was not reversible in the absence of guanidine hydrochloride.
Our study shows that PrPSc derived from two strains (ME7 and BSE) can convert the same unglycosylated PrPC precursor into two different PrPres fragments. Moreover, the newly formed fragments retained the same difference in molecular size of approximately 1 kDa in comparison with the PK-digested and unglycosylated PrPSc fragments. These results show that scrapie and BSE strains can transmit their own biochemical properties, even to unglycosylated bacterial PrPC. Both strains converted the prion protein at equal efficiencies of about 1 %. The formation of strains is attributed to different conformations of PrPSc, which lead to different PK cleavage sites. Similar results were achieved in a previous study where two distinct hamster strains, HY and DY, were able to transmit their conformation to eukaryotic PrPC (Bessen et al., 1995). In these studies, however, PrPSc was partly denatured by chaotropic salts (guanidine hydrochloride).
In a co-incubation study, where scrapie strain ME7 and BSE were incubated simultaneously with PrPC, PrPres fragments generated from ME7 as well as from BSE were detectable after 72 h. Due to their size difference of 1 kDa, both PrPres fragments could be quantified independently. Newly formed PrPres levels in the co-incubation experiment were significantly higher than in separate ME7 or BSE seed-driven reactions.
This phenomenon indicates that these prion strains not only retain their ability to convert PrPC under such conditions, but may even cooperate in the generation of PrPres. One explanation according to the modified seeded nucleation model is that after sonication the dissolved PrPSc monomers reassociate into oligomeric or multimeric seeds that are composed of both ME7 and BSE PrPSc molecules. These aggregates may then function as accessory initiation sites for the conversion of bacterial PrPC.
Co-incubation of the scrapie ME7 or the BSE strain with the RML strain showed no cooperative effects on the conversion reaction. As before, RML PrPSc lacked an intrinsic conversion activity, which was demonstrated during the co-incubation. In fact, it appeared to show a moderate inhibitory effect on PrPres generation by ME7 or BSE PrPSc.
This is the first time that a cooperative interaction between two prion strains has been shown in a cell-free conversion assay. Further experiments will focus on longer incubation times (>168 h) to reveal possible selection and competition effects between strains. Such effects have been described in experiments in vivo. For example, a blocking effect of the long-incubation-period scrapie strain 22A on its short-incubation-period counterpart 22C has already been reported (Kimberlin & Walker, 1985). Indeed, when strain 22A was inoculated 100 days prior to strain 22C, both incubation time and neuropathology were characteristic of the 22A strain. This strain selection, however, did not depend only on the incubation times, i.e. the kinetics of the PrP replication, but also on the challenge doses. Bartz et al. (2000) used a long-incubation-period (DY) and a short-incubation-period (HY) TME strain to examine the selection process. Using comparable infectious doses, the HY strain dominated the DY strain after several passages in a new host species. However, high doses of the DY strain also inhibited formation of the HY phenotype. Similarly, Baron & Bicabe (2001) showed that simultaneous inoculation of BSE and scrapie prions into mice resulted in the selection mainly of the scrapie isolate, which induced a characteristic PrPSc formation and neuropathology.
Our results were observed when using purified PrPSc seeds and recombinant PrPC as a substrate. The results suggest that the strain specificities and synergisms/antagonisms observed in vivo may at least partly be encoded by the intrinsic properties of PrPSc. Our data therefore underline the importance of cellular and abnormal prion proteins on the efficiency and quality of PrP conversion.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 October 2005;
accepted 31 July 2006.
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), 87V (
), BSE (
) and ME7 (
). (b) Co-incubation of ME7 and BSE PrPSc with PrPC showing the conversion rates of ME7-derived PrPres (
). Detection of PrPres following co-incubation of ME7 and RML PrPSc (
) and BSE and RML PrPSc (