Prion Protein Molecules Research Articles

Accumulation of prion protein in muscle fibers of experimental chloroquine myopathy: in vivo model for deposition of prion protein in non-neuronal tissues

Prion protein (PrP) is known to accumulate in some non-neuronal tissues under conditions unrelated to prion diseases. The biochemical and biological nature of such accumulated PrP molecules, however, has not been fully evaluated. In this study, we established experimental myopathy in hamsters by long-term administration of chloroquine, and we examined the nature of the PrP molecules that accumulated. PrP accumulation was immunohistochemically demonstrated in autophagic vacuoles in degenerated muscle fibers, and this was accompanied by the accumulation of other molecules related to the neuropathogenesis of prion diseases such as clathrin, cathepsin B, heparan sulfate, and apolipoprotein J. Accumulated PrP molecules were partially insoluble in detergent solution and were slightly less sensitive to proteinase K digestion than normal cellular PrP. Muscle homogenates containing these PrP molecules did not cause disease in inoculated hamsters. The findings indicate that the PrP molecules that accumulated in muscle fibers have distinct biochemical and biological properties. Therefore, experimental chloroquine myopathy is a novel and useful model to investigate the mechanism of deposition of PrP in non-neuronal tissues and might provide new insights in the pathogenesis of prion diseases.

Purification of recombinant bovine normal prion protein PrP(104–242) by HPHIC

Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104–242, PrP(104–242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104–242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104–242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104–242) by HPHIC. This procedure yield PrP(104–242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min.

Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies

We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrP Sc) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56–90, 119–127, 137–143, 143–149, 147–151, 163–169, and 219–229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrP Sc. However, no mAbs reacted with protease-treated PrP Sc purified from scrapie-affected mice, even when PrP Sc was dispersed into a detergent–lipid protein complex, to reduce the size of PrP Sc aggregates. In contrast, denaturation of PrP Sc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrP Sc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrP Sc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrP Sc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases.

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein.

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.

Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process.

The infectious form of prion protein, PrP(Sc), self-propagates by its conversion of the normal, cellular prion protein molecule PrP(C) to another PrP(Sc) molecule. It has not yet been demonstrated that recombinant prion protein can convert prion protein molecules from PrP(C) to PrP(Sc). Here we show that recombinant hamster prion protein is converted to a second form, PrP(RDX), by a redox process in vitro and that this PrP(RDX) form seeds the conversion of other PrP(C) molecules to the PrP(RDX) form. The converted form shows properties of oligomerization and seeded conversion that are characteristic of PrP(Sc). We also find that the oligomerization can be reversed in vitro. X-ray fiber diffraction suggests an amyloid-like structure for the oligomerized prion protein. A domain-swapping model involving intermolecular disulfide bonds can account for the stability and coexistence of two molecular forms of prion protein and the capacity of the second form for self-propagation.

Effect of Glycosylphosphatidylinositol Anchor-dependent and -independent Prion Protein Association with Model Raft Membranes on Conversion to the Protease-resistant Isoform

Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI(+)) and GPI anchor-deficient (GPI(-)) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34-94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI(+) PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI(+) PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI(-) PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res.

Does transmissibility necessarily imply infectivity in spongiform encephalopathy?

The essential biologic properties inherently acquired subsequent to conformational transformation of the α-helical molecular structure of the normal cellular PrPc isoform to the β-sheet molecular tertiary structure of the abnormal PrPsc associated with a rapidly spreading form of neuronal cell death of spongiform encephalopathy are unknown. However, the vacuolization that chiefly characterizes the morphology of neurons in spongiform encephalopathy might constitute a physical disruption with subsequent rapidly progressive impairment of maintenance of homeostatic viability of neurons due to precisely loss of membrane integrity of the plasmalemma and cell organelles. As far as transmission of the prion particle is concerned, it would appear that active incorporation of this agent under the direction of the affected neuronal cell itself would implicate host attributes as paramount factors determining not only susceptibility to the pathologic effects of the prion particle but also to the mode of such infliction as arising in and constituting spongiform encephalopathy beyond its acquisition and progression. As a single set of acquired circumstances determining both transmissibility and also pathologic lesion creation, the spongiform neuronal change might arise directly from a membrane abnormality of water ingress and egress in and out of the neuron. An excess of water ingress intra-neuronally might actually constitute a phenomenon of active lesion induction even in terms simply of biophysical stress intra-neuronally. In simple terms, an understanding of pathogenesis in spongiform encephalopathy might actually implicate aspects of transmissibility as direct attributes of processes of template replication within a system of utilization and elimination of the prion particle. Indeed, susceptibility to spongiform change might constitute one aspect of a biologic process that arises from conformational change of the prion protein molecule that would in turn result from variable polymorphisms in modes of reactive handling of PrPc and PrPsc by the neurons and other constituent cell elements in the central nervous system.

Fundamentals of prion biology and diseases

One of the most remarkable changes in medicine during the last 20 years of the 20th century was the shift from the clinical-neuropathological classification of Creutzfeldt–Jakob disease (CJD) and related disorders as ‘transmissible spongiform encephalopathies’ to a molecular-etiologic classification as ‘prion diseases’. We now know that these diseases are caused by abnormalities of the prion protein (PrP). Specifically, CJD is caused by the conversion of the normal, protease-sensitive PrP isoform, designated PrP C, to a protease resistant isoform, designated PrP Sc. PrP Sc forms into an infectious particle, named a ‘prion’, that can transmit the disease. Accumulation of PrP Sc in the brain causes neurodegeneration. The main goals of this review are to summarize our understanding of the attributes of the PrP molecule that give it the properties of an infectious agent and to describe how different alterations of the PrP molecule cause the multiple known prion disease variants. Finally, the emergence of a new variant of CJD in Great Britain and to a lesser extent in Europe and its relationship to the emergence of a particularly virulent form of bovine spongiform encephalopathy will be discussed.

Glycosylation influences cross-species formation of protease-resistant prion protein.

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease-resistant prion protein, PrP-res, from a normally soluble, protease-sensitive and glycosylated precursor, PrP-sen. While amino acid sequence similarity between PrP-sen and PrP-res influences both PrP-res formation and cross-species transmission of infectivity, the influence of co- or post-translational modifications to PrP-sen is unknown. Here we report that, if PrP-sen and PrP-res are derived from different species, PrP-sen glycosylation can significantly affect PrP-res formation. Glycosylation affected PrP-res formation by influencing the amount of PrP-sen bound to PrP-res, while the amino acid sequence of PrP-sen influenced the amount of PrP-res generated in the post-binding conversion step. Our results show that in addition to amino acid sequence, co- or post-translational modifications to PrP-sen influence PrP-res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.

Mutant prion proteins are partially retained in the endoplasmic reticulum.

Familial prion diseases are linked to point and insertional mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP to the infectious isoform. In this report, we have investigated the subcellular localization of PrP molecules carrying pathogenic mutations using immunofluorescence staining, immunogold labeling, and PrP-green fluorescent protein chimeras. To facilitate visualization of the mutant proteins, we have utilized a novel Sindbis viral replicon engineered to produce high protein levels without cytopathology. We demonstrate that several different pathogenic mutations have a common effect on the trafficking of PrP, impairing delivery of the molecules to the cell surface and causing a portion of them to accumulate in the endoplasmic reticulum. These observations suggest that protein quality control in the endoplasmic reticulum may play an important role in prion diseases, as it does in some other inherited human disorders. Our experiments also show that chimeric PrP molecules with the sequence of green fluorescent protein inserted adjacent to the glycolipidation site are post-translationally modified and localized normally, thus documenting the utility of these constructs in cell biological studies of PrP.

Efficient conversion of normal prion protein (PrP) by abnormal hamster PrP is determined by homology at amino acid residue 155.

In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.

A protease-resistant 61-residue prion peptide causes neurodegeneration in transgenic mice.

An abridged prion protein (PrP) molecule of 106 amino acids, designated PrP106, is capable of forming infectious miniprions in transgenic mice (S. Supattapone, P. Bosque, T. Muramoto, H. Wille, C. Aagaard, D. Peretz, H.-O. B. Nguyen, C. Heinrich, M. Torchia, J. Safar, F. E. Cohen, S. J. DeArmond, S. B. Prusiner, and M. Scott, Cell 96:869-878, 1999). We removed additional sequences from PrP106 and identified a 61-residue peptide, designated PrP61, that spontaneously adopted a protease-resistant conformation in neuroblastoma cells. Synthetic PrP61 bearing a carboxy-terminal lipid moiety polymerized into protease-resistant, beta-sheet-enriched amyloid fibrils at a physiological salt concentration. Transgenic mice expressing low levels of PrP61 died spontaneously with ataxia. Neuropathological examination revealed accumulation of protease-resistant PrP61 within neuronal dendrites and cell bodies, apparently causing apoptosis. PrP61 may be a useful model for deciphering the mechanism by which PrP molecules acquire protease resistance and become neurotoxic.

Primary Myopathy and Accumulation of PrPSc-Like Molecules in Peripheral Tissues of Transgenic Mice Expressing a Prion Protein Insertional Mutation

A nine-octapeptide insertional mutation in the prion protein (PrP) gene is associated with an inherited variant of Creutzfeldt–Jakob disease in humans. Transgenic mice that express the mouse PrP homologue of this mutation (designated PG14) under control of a PrP promoter display a progressive neurological disorder characterized by ataxia, apoptosis of cerebellar granule cells, and accumulation in the brain of mutant PrP molecules that display the biochemical hallmarks of PrPSc, the pathogenic isoform of PrP. In this report, we have investigated the expression of PG14 PrP in the peripheral tissues of these mice. We found highest levels of mutant PrP in the brain and spinal cord, intermediate levels in skeletal muscle, heart, and testis and low levels in kidney, lung, spleen, intestine, and stomach. Up to 70% of the PG14 PrP expressed in peripheral tissues was detergent-insoluble, and digestion with low concentrations of proteinase K yielded a PrP 27–30 fragment. These results suggest that the mutant protein was converted to a physical state reminiscent of PrPSc, although its infectivity remains to be determined. Histological analysis of skeletal muscle, one of the peripheral tissues with the highest level of PG14 PrP, revealed features indicative of a progressive, primary myopathy, including central nuclei, necrotic and regenerating fibers, and variable fiber size. These results indicate that the PG14 mutation structurally alters the protein in a way that promotes conversion to a PrPSc-like state, regardless of the tissue context, and suggest that accumulation of PrPSc can have deleterious effects on skeletal muscle cells as well as on neurons.

Identification of two prion protein regions that modify scrapie incubation time.

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.

A 7-kDa Prion Protein (PrP) Fragment, an Integral Component of the PrP Region Required for Infectivity, Is the Major Amyloid Protein in Gerstmann-Sträussler-Scheinker Disease A117V

Gerstmann-Sträussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Sträussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.

Quantitative Trait Loci Affecting Prion Incubation Time in Mice

Although the gene encoding prion protein (PrP) is the major determinant of susceptibility to prion disease, other genes also affect prion incubation time in mice and may be involved in prion replication. Scrapie incubation time was analyzed as a quantitative trait using crosses between SJL/J and CAST/Ei mice; these mouse strains encode identical PrP molecules but have different incubation periods. Our analysis revealed loci on Chromosomes 9 and 11 that affect prion susceptibility.

Nerve growth factor-induced differentiation does not alter the biochemical properties of a mutant prion protein expressed in PC12 cells.

Insertional and point mutations in the gene encoding the prion protein (PrP) are responsible for familial prion diseases. We have previously generated lines of Chinese hamster ovary cells that express PrP molecules carrying pathogenic mutations, and found that the mutant proteins display several biochemical properties reminiscent of PrP(Sc), the infectious isoform of PrP. To analyze the properties and effects of mutant PrP molecules expressed in cells with a neuronal phenotype, we have constructed stably transfected lines of PC12 cells that synthesize a PrP molecule carrying a nine-octapeptide insertion. We report here that this mutant PrP acquires scrapie-like properties, including detergent insolubility, protease resistance, and resistance to phospholipase cleavage of its glycolipid anchor. A detergent-insoluble and phospholipase-resistant form of the mutant protein is also released spontaneously into conditioned medium. These scrapie-like biochemical properties are quantitatively similar to those seen in Chinese hamster ovary cells and are not affected by differentiation of the PC12 cells into sympathetic neurons by nerve growth factor. Moreover, there is no detectable effect of mutant PrP expression on the morphology or viability of the cells in either the differentiated or undifferentiated state. These results indicate that conversion of mutant PrP into a PrP(Sc)-like form does not depend critically on the cellular context, and they suggest that mutant PrP expressed in cultured cells, even those having the phenotype of differentiated neurons, is not neurotoxic.

Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation.

We report that lysosomotropic agents and cysteine protease inhibitors inhibited protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells. The inhibition occurred without either apparent effects on normal prion protein biosynthesis or turnover or direct interactions with prion protein molecules. The findings introduce two new classes of inhibitors of the formation of protease-resistant prion protein.

Dominant-negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein.

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrP(C) act as dominant-negative, inhibitors of PrP(Sc) formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069-10074, 1997). Trafficking of substituted PrP(C) to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrP(C) are responsible for dominant-negative inhibition of PrP(Sc) formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrP(C) to PrP(Sc). We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrP(C) molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrP(C) to an auxiliary molecule that participates in PrP(Sc) formation remains to be established.

Cerebral amyloidosis in prion diseases

rion diseases in animals and humans are included among the cerebral amyloidoses. They are Unique P neurodegenerative disorders because, while 85 to 90% of the human forms occur sporadically throughout the world and only 10 to 15% are dominantly inherited as is the case withAlzheimer’s disease, all forms ofprion disease are also transmissible, unlike other neurodegenerative diseases. Prion diseases are caused by abnormalities of the prion protein (PrP)’. The unique properties of the wild-type PrP molecule and the abnormal conformations and plasma membrane topographies of mutated (mu) PrP molecules account for disease and for amyloidosis. Full-length wild-type PrP can exist in two stable conformations. There is a normal, protease-sensitive conformer, designated PrF, which is expressed constitutively throughout life at particularly high levels by nerve cells2. It is glycolipid (GPI) anchored and almost exclusively localized to caveolae-like domains of the plasma membrane3. It appears to function as a Cu” binding protein4. The C-terminal half of the PrF is highly ordered consisting of two a-helices bound together by a disulfide bridge, two N-linked oligosaccharides and the GPI anchor; whereas, the N-terminal half of the molecule is relatively unstructured and contains the Cu” binding domain5. These features account for the 40% a-helix and 3% P-sheet conformation of the PrPC molecule6.’. The full-length PrP molecule can also exist in an abnormal, protease-resistant conformer that accumulates in Creutzfeldt-Jakob disease (CJD) and scrapie (Sc), designated FYP”. It consists of about 45% p-sheet and about 30% a-helix. It is predicted that the p-sheet results from intramolecular andor intermolecular interactions among residues 80 to 140 in the unstructured N-terminal domain*, 9, lo. The propensity of the PrP molecule to form p-structures and to associate with glycosaminoglycans”. l2 accounts for amyloidogenesis in prion diseases. Transmissibility in prion diseases is accounted for by the tendency of the PrF molecule to adopt the same P-sheet conformation as PrPsc when the former comes into contact with PrPsc comprising an infecting prion.