Sensitivity of 4E1-HP and 5C1-P21 PCR assays.

<p>Panel A: sensitivity test on serial dilutions of reference DNAs (WB for assemblage A and Ad28 for assemblage B). Left images show results obtained with assemblage A; right images show results obtained with assemblage B. The amount of DNA template, reported as the number of cysts equivalent, is shown at the top of the images. Panel B: results of assays tested on different proportions of the same reference DNAs. The following A to B ratios were tested: 6∶4 (1); 7.5∶2.5 (2,); 9∶1 (3); 1∶9 (4); 2.5∶7.5 (5); and 4∶6 (6). N indicates the negative control.</p

Primer design for the 4E1-HP assay.

<p>Alignment of the sequence of the 4E1 clone (assemblage B) with the homologous sequence of HP 13988 (assemblage A). Dots correspond to identical nucleotides. The primers designed for assemblage A amplification are underlined with single lines, whereas the primers designed for assemblage B amplification are underlined with double lines.</p

List of primers for the assemblage-specific PCRs and sizes of the amplicons.

<p>List of primers for the assemblage-specific PCRs and sizes of the amplicons.</p

PCR assays at six independent genetic loci.

<p>Locus names are shown at the upper left corner of each panel. Capital letters in the upper lane indicate the reference DNA in each sample: A for assemblage A, B for assemblage B. Neg indicates the negative control (no DNA). Lower cases in the lower lane indicate the specific primer pair used for each reaction: a for the assemblage A specific pair; b for the assemblage B specific pair; a+b for the two primer pairs combined. Sizes of the assemblage-specific bands are indicated on the right. Size markers are indicated on the left.</p

Selected clones from the library of <i>Giardia duodenalis</i> Ad-28 strain and their genome localizations on both assemblages B (GS) and A (WB).

*<p>Orthologous gene identified on the basis of the similarity of the encoded peptide.</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

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

Vole bioassay.

<p><b>A</b>, <b>B</b> and <b>C</b> show the neuropathological and PrP^res phenotypes observed in voles after primary transmission (left panels, and indicated as I, roman number, in the blot on the right) and second passage (central panels and indicated as II, roman numbers, in the blot) of sheep 198/9 (<b>A</b>), PMCA-derived 18K (<b>B</b>) and PMCA-derived 14K/1 (<b>C</b>). Brain-scoring areas in lesion profiles are: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), cingulate and adjacent motor cortex (9). For each blot a vole adapted scrapie was added (BvScr, last lane). PrP^res was detected by antibody SAF84. <b>D</b>) Graph depicting the denaturation profiles obtained by CSA from PrP^Sc in sheep 198/9 (Sh198/9) or from voles infected with 18K (Bv18K) and vole-adapted 198/9 (Bv198/9). <b>E</b>) Graph depicting the comparison of denaturation profiles of 18K before (18K) and after (Bv18K) transmission in voles. [GdnHCl]_1/2 values are reported in the graph. <b>F</b>) Graph depicting the fate of 18K and 14K/2 after intracerebral inoculation in voles. Two groups of 8 voles were inoculated with 14K/2 or 18K and 2 voles for each group were sacrificed at different time points (0, 3, 14 and 52 dpi). Their brains were homogenized and used as seed for PMCA reactions in 3 independent experiments. The values on y axis represent the overall percentage of positive samples per time point after 3 PMCA rounds.</p

Hypothetical mechanisms underpinning the defective nature of 14K PrP^Sc.

<p>The cartoon depicts the hypothetical interaction between physiological proteases responsible for α-cleavage, here supposed to belong to the family of ADAM proteases [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006016#ppat.1006016.ref035" target="_blank">35</a>] and 18K or 14K PrP^Sc (<b>A</b>), and how it changes in the <i>in vitro</i> (<b>B</b>) and the <i>in vivo</i> (<b>C</b>) replication environments. Distinct symbols indicate PrP^C monomers, 18K or 14K PrP^Sc aggregates, as well as the α-cleavage site and the location of the polybasic domains in PrP^Sc, as shown in the graphical legend below the cartoon. (<b>A</b>) In 18K PrP^Sc aggregates the physiological α-cleavage site (residues 110–111, vole PrP numbering) and the central polybasic domain (in green, aa ~ 101–110), are tightly packed in the PK-resistant core of PrP^Sc. On the contrary, in mutant 14K PrP^Sc the α-cleavage site is available for hydrolysis. (<b>B</b>) During <i>in vitro</i> propagation by PMCA, the activity of ADAM proteases is purposely prevented by protease inhibitors, a factor which allows to keep full length PrP in solution. Under these conditions, both 18K and 14K PrP^Sc are full length and preserve intact polybasic domains, which would allow them to interact with PrP^C and replicate. (<b>C</b>) <i>In vivo</i>, 18K PrP^Sc is still protected from ADAM proteolysis as the cleavage site is buried within the PK-resistant core, and thus it is still fully competent for replication as it retains the N-terminus. In contrast, 14K PrP^Sc would be cleaved at 110–111 and would lose the central polybasic domain, supposed to be a key mediator of the interaction with partners indispensable for prion replication (PrP^C or cofactors).</p

Transmission of 198/9, 18K, 14K/1 and 14K/2 in bank voles.

<p>Transmission of 198/9, 18K, 14K/1 and 14K/2 in bank voles.</p