Monoclonal antibodies used for epitope mapping.

a<p>Epitope on human PrP.</p>b<p>Repeat region aa 59–65, 67–73, 75–81, 83–89. Epitope reference: SAF32, 8G8,12F10, SAF60 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-Feraudet1" target="_blank">[61]</a>; 12B2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-Jeffrey1" target="_blank">[62]</a>; 9A2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-Yull1" target="_blank">[63]</a>; 6D11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-Miller1" target="_blank">[64]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-Pankiewicz1" target="_blank">[65]</a>; F89 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-ORourke1" target="_blank">[66]</a>; L42 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405-Gretzschel1" target="_blank">[54]</a>.</p><p>−no signal; +/−weak signal; +strong signal.</p

Titration of proteinase K digestion.

<p>Representative western blots of brain homogenates from Nor98 (#2), VPSPr (#7), GSS F198S (#11), A117V (#13), P102L (#15) and sCJD treated with increasing concentrations of PK (0–1000 µg/ml). Given the various PK-cleavage sites of the PrP^Sc types under investigation which affects mAbs binding to PrP^res (see Fig. 3), membranes stained with different antibodies were selected in order to show the PK resistance of strong PrP^res in each sample. Membranes were probed with 12B2 for Nor98 and GSS P102L, 9A2 for GSS A117V, 1E4 for VPSPr, and 3F4 for GSS F198S and sCJD samples.</p

Epitope mapping of PrP^Sc treated with different PK concentrations.

<p>PK digestion curves of VPSPr (#7), GSS A117V (#13) and GSS F198S (#11) were probed with 12B2, 9A2 and L42.</p

Human and ovine cases used for comparative analyses.

a<p>Ovine polymorphism: amino acids at codon 136, 141, 154 and 171 of ovine PrP gene; human mutation at codon 102, 117 or 198 and polymorphism at codon 129 of human PrP gene.</p>b<p>Cer: cerebellum; Fr cx: frontal cortex.</p>c<p>Mean ± SEM.</p

Conformational stability of insoluble PrP^Sc.

<p><b>A:</b> Western blot analysis of VPSPr 129MV (#7) and 129VV (#3) and GSS P102L (#15 cerebellum) cases, showing separation of insoluble PrP^Sc by a solubility assay. Samples were centrifuged at 20000 g for 1 h in presence of 1% sarkosyl. Supernatant (S) and pellet (P) fractions were analysed with (+) or without (-) PK treatment (50 µg/ml), along with aliquots of samples before centrifugation (Tot). Note that PrP^res segregated into the insoluble fraction (compare lanes S+ and P+ in each blot). In each lane 0,7 mg TE were loaded. Membranes were probed with L42 mAb. <b>B:</b> Representative western blots of CSSA experiments in Nor98 (#1), VPSPr (#9), GSS F198S (#11), GSS A117V (#14) and GSS P102L (#16) cases. Lanes were loaded with insoluble PrP obtained as shown in Fig. 6A, with or without previous denaturation with increasing concentrations of GdnHCl, as shown on the top of each lane. Membranes were probed with L42. Molecular size markers are shown in kilodaltons on the right of each blot. <b>C:</b> Dose-response curves obtained by plotting the fraction of PrP^Sc remaining in the pellet as a function of GdnHCl concentration and best-fitted and using a four parameter logistic equation. Individual [GdnHCl]_1/2 values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone-0066405-t001" target="_blank">Table 1</a>.</p

Comparison of PrP^res fragments in Nor98, GSS and VPSPr.

<p>The aligned amino acid sequences at the N and C termini of all PrP^res fragments, derived from epitope mapping as described in SI 5, are depicted. Amino acid numbering refers to sheep PrP for Nor98 and to human PrP for all other fragments. Coloured letters highlight the relevant epitopes as follows: orange for SAF32; blue for 12B2; green for 9A2; pink for 8G8; red for 12F10 and violet for L42. Note that when epitopes of mAbs partially overlap, only the aa differentiating the epitopes were coloured. On the right of each PrP^res fragment is reported the predicted MW estimated by using the ProtParam (ExPASy) software.</p

Isolation of a Defective Prion Mutant from Natural Scrapie

<div><p>It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur <i>in vitro</i> during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the <i>in vitro</i> propagation of natural scrapie isolates. Although <i>in vitro</i> vole-adapted PrP^Sc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrP^Sc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrP^Sc conformers showed to be efficiently autocatalytic <i>in vitro</i> and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different <i>bona fide</i> PrP^Sc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for <i>in vivo</i> replication while the mutant was unable to infect voles, <i>de facto</i> behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least <i>in vitro</i>. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrP^C and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for <i>in vitro</i> replication. Finally, we propose a molecular mechanism able to account for the discordant <i>in vitro</i> and <i>in vivo</i> behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.</p></div

Additional file 1: Figure S1. of Atypical Creutzfeldt-Jakob disease with PrP-amyloid plaques in white matter: molecular characterization and transmission to bank voles show the M1 strain signature

Western blot analysis of np-CJDMM1 and p-CJDMM1 (case #1) subcortical white matter. FC: frontal cortex; PC: parietal cortex. (a) Electrophoretic mobility of PK-digested PrPSc (i.e. PrP27–30) after separation in a 7 cm long gel. Blot was probed with the primary antibody 3F4. (b) CTF13 analysis after PrP deglycosylation with PNGase F. Blot was probed with the primary antibody SAF60. Relative molecular masses are expressed in kDa. Percentages (mean ± standard deviation) of CTF13 are referred to the total PrPSc amount: np-CJDMM1 = 12.8 ± 5.0, p-CJDMM1 = 14.1 ± 2.9. (TIFF 824 kb

Identification of 14K from a natural scrapie sample.

<p><b>A</b>) Serial 10-fold dilutions of 2 Italian scrapie samples (ES47/10/3 and 198/9) were used as seeds in serial PMCA reactions using vole brain homogenate substrate. Products from rounds 4°, 6° and 8° (indicated in roman numbers) were digested with PK and analyzed by Western blot with antibody SAF84. After 8 PMCA rounds both samples were positive up to dilution 10⁻⁷. An atypical PrP^Sc with smaller PrP^res (indicated by the asterisk) emerged after the sixth round only from the last detectable dilution of sample 198/9, and was propagated until the end of the experiment. <b>B</b>) Three <i>in vitro</i> selected prion populations (18K, 14K/1 and 14K/2, as indicated on the top of each blot) were serially propagated for 4 successive PMCA rounds (represented in roman numbers). After each round, aliquots of the PMCA products were digested with PK and analyzed by Western blot with antibody SAF84. For 14K/1, PK-digested PrP^res is also shown after enzymatic removal of N-linked oligosaccarides, which allows to better appreciate the co-presence and evolution of 14K and 18K PrP^res (indicated by a single and a double asterisk respectively) during the experiment.</p