Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki 540 06, Greece1
Laboratory of Microbiology and Infectious Diseases, Department of Veterinary Sciences, Aristotle University of Thessaloniki, Thessaloniki 540 06, Greece2
Central Veterinary Laboratory, Addlestone, Surrey KT15 3NB, UK3
Author for correspondence: Theodoros Sklaviadis. Fax +3 031 997645. e-mail sklaviadis{at}auth.gr
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Abstract |
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Introduction |
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It has been shown that denaturation of PrPSc aggregates in vitro leads to the final loss of infectivity (Manuelidis et al., 1995 ; Riesner et al., 1996
). The same does not necessarily apply in the case of a report by Safar et al. (1993)
, where conformational transitions did not affect infectivity. However, a model for the transformation of PrPC into infectious PrPSc aggregates would be the ultimate proof of prion propagation. Several approaches have been reported in the literature, but so far none has achieved such a transformation conclusively with measurable infectivity (Gasset et al., 1993
; Kocisko et al., 1994
; Kaneko et al., 1995
; Riesner et al., 1996
). Moreover, qualitative and conformational studies of the infectious material are complicated by the limited solubility of prions. So far, attempts to solubilize scrapie infectivity have not been successful (Prusiner et al., 1980
; Millson & Manning, 1979
). Interestingly, the very property of insolubility of the TSE infectious agents is now used for the enrichment of infectivity, together with the introduction of proteinase K digestion in the purification protocols, thus giving rise to the PrP2730 fraction (Prusiner et al., 1982
). Even though this fraction is infectious, its infectivity when separated on SDSPAGE (Brown et al., 1990
) has not been confirmed (Prusiner et al., 1993
).
Attempts to renature scrapie infectivity from guanidine (Gdn)thiocyanide- or urea-treated samples were unsuccessful (Prusiner et al., 1993 ). There is considerable loss of infectivity ( 99·5%) in the soluble fraction of hamster CJD infectious agent upon denaturation with 2·5 M GdnHCl (Manuelidis et al., 1995
). It has also been reported that, on renaturation, PrPSc from the 263K strain of hamster scrapie that had been treated with 3 M GdnHCl regained resistance to proteinase K (Kocisko et al., 1994
; Kaneko et al., 1995
); however, as shown by subsequent investigators, this may not be the only factor governing infectivity (Hill et al., 1999
).
In the light of these findings, we have looked at the possibility of reconstructing the mouse-passaged BSE infectious agent after denaturation with GdnHCl and investigated the properties of the newly acquired product, including infectivity.
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Methods |
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Denaturation, renaturation and isopycnic fractionation.
For denaturation, 1 g wet tissue equivalent of p215-MN was resuspended in 340 µl of a solution containing 2·5 M GdnHCl in 25 mM TrisHCl, pH 8·9, 0·05% Sarkosyl and 0·5 mM PMSF and bath-sonicated five times (30 min each) with alternate vortexing (45 min each). The samples were then centrifuged at 13000 g for 30 min. The pellet, designated as pG, was kept for further use, while the supernatant, sG, was dialysed exhaustively at room temperature against 25 mM TrisHCl, pH 7·6, 1 mM PMSF and 0·05% Sarkosyl in a Gibco BRL Microdialysis system with a membrane cut-off of 1214 kDa. A linear, 3086% (w/v) sucrose gradient (3 ml) was poured over a cushion of 500 µl 86% (w/v) sucrose and the dialysed material (dsG) in 800 µl dialysis buffer, equivalent to 2 g tissue, was loaded on the sucrose gradient for isopycnic fractionation. Denaturation, dialysis and isopycnic fractionation took place at room temperature in the presence of 1 mM PMSF. The samples were spun at 220000 g for 41 h at 25 °C. At the end of the run, eight fractions of 500 µl each were collected carefully from the gradient and kept frozen. Blank sucrose gradients were also run in order to estimate the density of sucrose in each fraction by refractive index measurements (Index Instruments).
All fractions, including those from the sucrose gradient, were divided into three aliquots for the estimation of PrPSc, nucleic acid analysis and bioassays.
Estimation of PrPSc.
Proteins were separated on 13% SDSPAGE and blotted onto a PVDF membrane and stained with AuroDye Forte (Amersham Life Science) for the estimation of total protein. For PrPSc-specific staining, the membranes were blocked (1 h, room temperature) in 3% BSA, 1% Ficoll and 1% polyvinyl pyrrolidone and incubated with a polyclonal anti-PrP primary antibody (78295; Kascsak et al., 1997 ), a generous gift of R. Kascsak, diluted 1:5000 in the blocking solution, for 1 h at room temperature, washed three times for 10 min in 100 ml PBS, 0·1% Tween and incubated further with a goat anti-rabbit secondary antibody conjugated with alkaline phosphatase (1:3000). NBT and BCIP were used as substrates to visualize the proteins. Quantification of PrPSc on PVDF membranes (3419 kDa bands) was performed by densitometry (ScanPack version 3.0, Biometra Biomedizinische Analytik). Proteins from the sucrose-density fractions were concentrated by precipitating with 10 vols ethanol before immunoblotting.
Analysis of nucleic acids.
Nucleic acids were isolated from the second portion of each fraction. The procedure that was employed has been reported previously in detail (Sklaviadis et al., 1990 ; Akowitz et al., 1994
). In brief, nucleic acids were extracted with phenolchloroform, precipitated with ethanol and end-labelled with 32P by standard methods. After further precipitation with ethanol and separation on 1% agarose gels, the nucleic acids were analysed by densitometry (ScanPack version 3.0) on autoradiographs.
Bioassays.
The third portion from each fraction was suspended in normal saline and used for bioassays in 3-week-old IM (Sincp7) mice, in groups of 20 per fraction. Each mouse received the inoculum through two routes, 20 µl intra-cranially and 100 µl intra-peritoneally (Bruce et al., 1994 ). Mice in the control group were inoculated intra-cranially with normal saline. Mice were scored from day 90 post-inoculation for early clinical and terminal signs of disease over a period of up to 400 days. Infectious units were calculated from mean incubation time for each group, based on a titration curve obtained from mice of the same genotype infected with the mouse BSE strain 301V (D. Taylor, Institute of Animal Health, Neuropathogenesis Unit, Edinburgh, UK, personal communication).
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Results |
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Composition of the sucrose gradient fractions
The sucrose-density gradient provided eight fractions, with densities ranging from 1·30 to 1·03 g/cm3. PrPSc was found to be concentrated mainly in fractions 6, 7 and 8, with densities of 1·11, 1·09 and 1·03 g/cm3, respectively. Most of it moved into fractions 6 and 7 and only traces of the protein were seen in fraction 8 (Fig. 2a, lanes 68).
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Bioassays
Data on infectivity in the various fractions (Table 1) showed that p215 itself was highly infectious (5·8 log10 LD50 units), with no considerable loss of infectivity in the p215-MN fraction (5·4 log10 LD50 units). Following exposure of p215-MN to GdnHCl, the pG fraction exhibited low but measurable infectivity (1·9 log10 LD50 units), while the corresponding soluble sG fraction was not infectious. Partial infectivity (3·5 log10 LD50 units, two log units less than that of the pre-denatured material) was recovered in dsG, however, the dialysed supernatant.
Only fractions 6 (2·6 log10 LD50 units), 7 (2·8 log10 LD50 units) and 8 (2·3 log10 LD50 units) from the sucrose-density gradient were infectious (Tables 1 and 2
, Fig. 2c
). Infectivity that was detected in fraction 5 (Fig. 2 c
) had to be from residual material from the fraction(s) above because, in addition to the negligible infectivity (0·8 log10 LD50 units) that this fraction showed, only six of 20 mice were infected, with an incubation period of 329±48 days, way beyond those presented by other infectious fractions. For these reasons, it was decided to not consider this fraction for further analysis. Comparison of the infectious fractions in terms of percentages (Table 2
) showed that fraction 6, where nearly all of the nucleic acids were found, contained 0·6% of the total PrPSc and 31% of the total infectivity. Fraction 7, with no detectable nucleic acids, contained 99·2% of PrPSc and 52% of total infectivity, while fraction 8, with no detectable nucleic acids and only 0·2% of PrPSc, showed 17% of infectivity. Obviously, the majority of the nucleic acids did not contribute towards infectivity, because both fractions 7 and 8 were infectious without detectable nucleic acids. The important observation is that fractions 6 and 8 were infective but had only meagre amounts of PrPSc (0·6 and 0·2%, respectively), thus making it clear that infectivity was not associated directly with abundance of PrPSc. The absence of infectivity in fractions 14 precludes any cross-contamination among the fractions due to handling (Table 1
). These fractions also served as internal negative controls.
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Discussion |
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It was observed that, on treatment with 2·5 M GdnHCl, there was complete loss of infectivity in the supernatant, and only residual infectivity was detected in the pellet. These results are similar to those of Manuelidis et al. (1995) , where a loss of 99·5% of the starting infectivity, under similar conditions, was reported with hamster CJD preparations. In other studies, where the 263K strain of hamster scrapie has been denatured with 2·5 M GdnHCl, partial disaggregation of PrPSc has been noticed (Kocisko et al., 1996
) and, in a more recent work (Caughey et al., 1997
), a >1000-fold reduction in infectivity has been observed without complete loss in the supernatant. None of the above workers, however, attempted to recover infectivity from the denatured preparations. Interestingly, in our hands, we have regained a portion of the infectivity after the removal of GdnHCl by dialysis (Fig. 2c
; dsG). The differences between the above findings and those of our own study might be attributable to the different strains used in the studies. On the other hand, similarities that the results of this work present to those of Manuelidis et al. (1995)
, where hamster CJD was used, may be explained by a common and an increased sensitivity of the two agents to chaotropic agents. The implications of such a similarity on the passing of the BSE agent to humans to cause variant CJD remains to be seen.
On further analysis with an isopycnic sucrose-density gradient, it was discovered that, in the renatured fraction, only a portion of the total PrPSc was responsible for infectivity (Table 2). It has been reported earlier that overexpression of PrPSc was not a criterion for the onset of prion diseases in PrP+/- mice (Bueler et al., 1994
). It has also been shown that the BSE infectious agent can be transmitted to mice in the absence of detectable prion protein (Somerville & Dunn, 1996
; Lasmezas et al., 1997
). Therefore, in accordance with the findings of other workers and using experiments in vitro, the present work confirms that, during infection, pathogenicity of the infectious agent (murine BSE in this study) is dependent on the correct conformation in vivo and not on the bulk of PrPSc in the inoculum. A role of other macromolecules, not detected under the present conditions, as part of the infectious agent cannot be excluded. However, as shown by gold staining (Fig. 1
, lane 1), the relative purity of the starting preparation in terms of contamination with other proteins precludes such a possibility. In order to use a more defined starting material, we repeated our experiments with p215-MN after digestion with proteinase K (Fig. 1
, lane 3), but the preparation was not easily denatured with 2·5 M GdnHCl. Denaturation with 5 M GdnHCl to overcome this difficulty led to unrecoverable loss of infectivity (data not shown). It was also observed that only a part of the total infectivity in the starting material was recovered at the end of the denaturationrenaturation steps (Table 1
). This could be due to only partial recovery of the correct conformation that may contribute structurally towards infectivity.
Although the nature of the prion itself is proteinaceous (Prusiner, 1982 ), its ability to associate with nucleic acids (Akowitz et al., 1994
) and also with RNA aptamers (Weiss et al., 1997
) has been demonstrated earlier. Our own experiments have demonstrated the capability of PrPSc-enriched preparations to bind nucleic acids in vitro (unpublished data). In this study, dsG was found to be associated with nucleic acids that were structurally protected, since dsG was derived from the p215-MN (micrococcal nuclease-treated) preparation. Even though their presence has been demonstrated for the first time in mouse BSE preparations, these endogenous nucleic acids did not seem to contribute towards infectivity and were bound non-specifically to the infectious complexes.
It is likely that novel states of the murine BSE agent were generated under the denaturingrenaturing conditions used in this study. Such unfolded/refolded stages of the agent may be extremely useful as templates for studying in vitro proteinprotein interactions of PrPSc with its own cellular isoform or even other macromolecules from the same or different species, since these steps are known to precede/accompany transmission of prion diseases.
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Acknowledgments |
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References |
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Received 11 January 2000;
accepted 22 February 2000.