Current definitions.

Perhaps the best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid (28). Because a wealth of data supports the contention that scrapie prions are composed entirely of a protein that adopts an abnormal conformation, it is not unreasonable to define prions as infectious proteins (25, 27, 59, 60). But I hasten to add that we still cannot eliminate a small ligand bound to PrP^Sc as an essential component of the infectious prion particle. Learning how to renature PrP^Sc accompanied by restoration of prion infectivity or to generate prion infectivity de novo by using a synthetic polypeptide should help address this as-yet-unresolved issue (61). From a broader perspective, prions are elements that impart and propagate conformational variability.

Although PrP^Sc is the only known component of the infectious prion particles, these unique pathogens share several phenotypic traits with other infectious entities such as viruses. Because some features of the diseases caused by prions and viruses are similar, some scientists have difficulty accepting the existence of prions despite a wealth of scientific data supporting this concept (62–67).

Families of hypotheses.

Once the requirement for a protein was established, it was possible to revisit the long list of hypothetical structures that had been proposed for the scrapie agent and to eliminate carbohydrates, lipids, and nucleic acids as the infective elements within a scrapie agent devoid of protein (58). No longer could structures such as a viroid-like nucleic acid, a replicating polysaccharide, or a small polynucleotide surrounded by a carbohydrate be entertained as reasonable candidates to explain the puzzling properties of the scrapie agent (58, 68).

The family of hypotheses that remained after identifying a protein component was still large and required a continued consideration of all possibilities in which a protein was a critical element (49). The prion concept evolved from a family of hypotheses in which an infectious protein was only one of several possibilities. With the accumulation of experimental data on the molecular properties of the prion, it became possible to discard an increasing number of hypothetical structures. In prion research, as in many other areas of scientific investigation, a single hypothesis is all too often championed at the expense of a reasoned approach that requires entertaining a series of complex arguments until one or more can be discarded on the basis of experimental data (69).

Search for a scrapie-specific nucleic acid.

The inability to inactivate preparations highly enriched for scrapie infectivity by procedures that modify nucleic acids militates against the existence of a scrapie-specific nucleic acid (58, 110, 111). To explain the findings in terms of a virus, one must argue that PrP^Sc or an as-yet-undetected PrP-like protein of viral origin protects the viral genome from inactivation. The notion that the putative scrapie virus encodes a PrP-like protein was refuted by nucleic acid hybridization studies using a PrP cDNA probe. Less than 0.002 nucleic acid molecule encoding PrP per ID₅₀ unit was found in purified preparations of SHa prions (84). To circumvent this finding, it could be hypothesized that the genetic code used by the PrP gene differs so greatly from that found in the cell that a PrP cDNA probe failed to detect it in highly purified preparations.

If prions contained a genome with a unique genetic code, then it is likely that this genome would encode some specialized proteins required for replication as well as some unique tRNAs. But both UV and ionizing radiation inactivation studies as well as physical studies have eliminated the possibility of a large nucleic acid hiding within purified preparations of prions (110–112). Only oligonucleotides of fewer than 50 bases were found at a concentration of one molecule per ID₅₀ unit in prion preparations highly enriched for scrapie infectivity (113, 114). These small nucleic acids were of variable length and are thought to be degradation byproducts generated during purification of prions. Failure to find a bona fide genome was attributed to the unusual properties of the putative viral nucleic acid or technical incompetence on the part of the investigators who were unable to find it (63, 115).

PrP amyloid.

In preparations highly enriched for scrapie infectivity and containing only PrP 27–30 by silver staining of gels after SDS/PAGE, numerous rod-shaped particles were seen by electron microscopy after negative staining (Fig. ​(Fig.3)3) (107). Each of the rods was slightly different, in contrast to viruses, which exhibit extremely uniform structures (116). These irregular rods, composed largely, if not entirely, of PrP 27–30, were indistinguishable morphologically from many other purified amyloids (117). Studies of the prion rods with Congo red dye demonstrated that the rods also fulfilled the tinctorial criteria for amyloid (107), and immunostaining later showed that PrP is a major component of amyloid plaques in some animals and humans with prion disease (118–120). Subsequently, it was recognized that the prion rods were not required for scrapie infectivity (121); furthermore, the rods were shown to be an artifact of purification during which limited proteolysis of PrP^Sc generated PrP 27–30 that polymerized spontaneously in the presence of detergent (Fig. ​(Fig.3)3) (122).

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Figure 3

Electron micrographs of negatively stained and ImmunoGold-labeled prion proteins. (A) PrP^C. (B) PrP^Sc. Neither PrP^C nor PrP^Sc forms recognizable, ordered polymers. (C) Prion rods composed of PrP 27–30 were negatively stained. The prion rods are indistinguishable from many purified amyloids. (Bar = 100 nm.)

The idea that scrapie prions were composed of an amyloidogenic protein was truly heretical when it was introduced (107). Since the prevailing view at the time was that scrapie is caused by an atypical virus, many argued that amyloid proteins are mammalian polypeptides and not viral proteins!

Some investigators have argued that the prion rods are synonymous with scrapie-associated fibrils (123–125) even though morphologic and tinctorial features of these fibrils clearly differentiated them from amyloid and as such from the prion rods (126, 127). The scrapie-associated fibrils were identified by their unique ultrastructure in which two or four subfilaments were helically wound around each other (126) and were proposed to represent the first example of a filamentous animal virus (128). After the argument for a filamentous animal virus causing scrapie faded, it was hypothesized that a virus induces the formation of PrP amyloid to explain the accumulation of PrP^Sc in prion diseases (129). Besides the lack of evidence for a virus of any shape, no compelling data have been offered in support of the idea that prion diseases are caused by a filamentous bacterium called a spiroplasma (130).

Search for the ubiquitous “scrapie virus.”

When PrP gene mutations were discovered to cause familial prion diseases (4), it was postulated that PrP^C is a receptor for the ubiquitous scrapie virus that binds more tightly to mutant than to wt PrP^C (131). A similar hypothesis was proposed to explain why the length of the scrapie incubation time was found to be inversely proportional to the level of PrP expression in transgenic (Tg) mice and why Prnp^0/0 mice are resistant to scrapie (132). The higher the level of PrP expression, the faster the spread of the putative virus, which results in shorter incubation times; conversely, mice deficient for PrP lack the receptor required for spread of the virus (63). The inability to find virus-like particles in purified preparations of PrP^Sc was attributed to these particles being hidden (115) even though tobacco mosaic viruses could be detected when one virion was added per ID₅₀ unit of scrapie prions (121).

Recent studies on the transmission of mutant prions from FFI and fCJD(E200K) to Tg(MHu2M) mice, which results in the formation of two different PrP^Sc molecules (27), has forced a corollary to the ubiquitous virus postulate. To accommodate this result, at least two different viruses must reside worldwide, each of which binds to a different mutant HuPrP^C and each of which induces a different MHu2M PrP^Sc conformer when transferred to Tg mice. Even more difficult to imagine is how one ubiquitous virus might acquire different mutant PrP^Sc molecules corresponding to FFI or fCJD(E200K) and then induce different MHu2M PrP^Sc conformers upon transmission to Tg mice.

Artificial prions.

To explain the production of artificial prions from chimeric or mutant PrP transgenes in terms of a virus (133–135), mutated PrP^Sc molecules must be incorporated into the virus. In the case of mice expressing chimeric PrP transgenes, artificial prions are produced with host ranges not previously found in nature. Similarly, deleting specific regions of PrP resulted in the formation of “miniprions” with a unique host range and neuropathology as described below. The production of artificial prions that were generated by modifying the PrP gene sequence and exhibit unique biological properties is another compelling argument against the proposition that scrapie and CJD are caused by viruses.

Modeling PrP structures.

Molecular modeling studies predicted that PrP^C is a four-helix bundle protein containing four regions of secondary structure denoted H1, H2, H3, and H4 (Fig. ​(Fig.4)4) (146, 147). Subsequent NMR studies of a synthetic PrP peptide containing residues 90–145 provided good evidence for H1 (148). This peptide contains the residues 113–128, which are most highly conserved among all species studied (Fig. ​(Fig.44A) (147, 149, 150) and correspond to a transmembrane region of PrP that was delineated in cell-free translation studies (151, 152). Recent studies show that a transmembrane form of PrP accumulates in GSS caused by the A117V mutation and in Tg mice overexpressing either mutant or wild-type (wt)PrP (153). The paradoxical lack of evidence for an α-helix in this region from NMR studies of recombinant PrP in aqueous buffers (154–156) could be explained if the recombinant PrPs correspond to the secreted form of PrP that was also identified in cell-free translation studies. This contention is supported by studies with recombinant antibody fragments (Fabs) showing the GPI-anchored PrP^C on the surface of cells exhibits an immunoreactivity similar to that of recombinant PrP that was prepared with an α-helical conformation (157, 158). GPI-anchored PrP^C is synthesized within the secretory pathway and transported to the surface of the cell (139, 159).

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Figure 4

Species variations and mutations of the prion protein gene. (A) Species variations. The x-axis represents the human PrP sequence, with the five octarepeats and H1–H4 regions of putative secondary structure shown as well as the three α-helices A, B, and C and the two β-strands S1 and S2 as determined by NMR. The precise residues corresponding to each region of secondary structure are given in Fig. ​Fig.5.5. Vertical bars above the axis indicate the number of species that differ from the human sequence at each position. Below the axis, the length of the bars indicates the number of alternative amino acids at each position in the alignment. (B) Mutations causing inherited human prion disease and polymorphisms in human, mouse, and sheep. Above the line of the human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotype of disease. Data were compiled by Paul Bamborough and Fred E. Cohen.

Optical spectroscopic measurements of PrP^C provided the necessary background for more detailed structural studies (25). Unable to produce crystals of PrP, we and others utilized NMR to determine the structure of an α-helical form of a recombinant PrP. The NMR structure of a COOH-terminal fragment of MoPrP consisting of 111 residues showed three helices, two of which corresponded to H3 and H4 in the PrP^C model, and two small β-strands each consisting of three residues (154). How the structure of this MoPrP(121–231) fragment differs from PrP^C is of interest because this fragment is lethal when expressed in Tg mice (160). Subsequently, structural studies were performed on a longer fragment of PrP containing residues 90–231 and corresponding to SHaPrP 27–30 (155, 161, 162). Expression of PrP(90–231) in Tg mice did not produce spontaneous disease (163, 164). More recently, NMR structures of recombinant full-length PrP have been reported (156, 165).

Models of PrP^Sc suggested that formation of the disease-causing isoform involves refolding of residues within the region between residues 90 and 140 into β-sheets (166); the single disulfide bond joining COOH-terminal helices would remain intact because the disulfide is required for PrP^Sc formation (Fig. ​(Fig.55E) (167, 168). The high β-sheet content of PrP^Sc was predicted from the ability of PrP 27–30 to polymerize into amyloid fibrils (107). Subsequent optical spectroscopy confirmed the presence of β-sheet in both PrP^Sc and PrP 27–30 (25, 169–171). Deletion of each of the regions of putative secondary structure in PrP, except for the NH₂-terminal 66 amino acids (residues 23–88) (163, 172) and a 36-amino acid region (mouse residues 141–176) prevented formation of PrP^Sc as measured in scrapie–infected cultured neuroblastoma cells (168). With anti-PrP Fabs selected from phage display libraries (157) and two monoclonal antibodies (mAbs) derived from hybridomas (173–175), the major conformational change that occurs during conversion of PrP^C into PrP^Sc has been localized largely, but not entirely, to a region bounded by residues 90 and 112 (158). Similar conclusions were drawn from studies with an anti-PrP IgM mAb (176). While these results indicate that PrP^Sc formation involves primarily a conformational change in the domain composed of residues 90–112, mutations causing inherited prion diseases have been found throughout the protein (Fig. ​(Fig.44B). Interestingly, all of the known point mutations in PrP with biological significance occur either within or adjacent to regions of putative secondary structure in PrP and as such, appear to destabilize the structure of PrP (147, 148, 154).

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Figure 5

Structures of prion proteins. (A) NMR structure of SHa recombinant (r) PrP(90–231). Presumably, the structure of the α-helical form of rPrP(90–231) resembles that of PrP^C. rPrP(90–231) is viewed from the interface where PrP^Sc is thought to bind to PrP^C. The color scheme is as follows: α-helices A (residues 144–157), B (172–193), and C (200–227) in pink; disulfide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to β-sheet (155). (B) NMR structure of rPrP(90–231) is viewed from the interface where protein X is thought to bind to PrP^C. Protein X appears to bind to the side chains of residues that form a discontinuous epitope: some amino acids are in the loop composed of residues 165–171 and at the end of helix B (Gln-168 and Gln-172 with a low-density van der Waals rendering), whereas others are on the surface of helix C (Thr-215 and Gln-219 with a high-density van der Waals rendering) (178). (C) PrP residues governing the transmission of prions (180). NMR structure of recombinant SHaPrP region 121–231 (155) shown with the putative epitope formed by residues 184, 186, 203, and 205 highlighted in red. Residue numbers correspond to SHaPrP. Additional residues (138, 139, 143, 145, 148, and 155) that might participate in controlling the transmission of prions across species are depicted in green. Residues 168, 172, 215, and 219 that form the epitope for the binding of protein X are shown in blue. The three helices (A, B, and C) are highlighted in pink. (D) Schematic diagram showing the flexibility of the polypeptide chain for PrP(29–231) (156). The structure of the portion of the protein representing residues 90–231 was taken from the coordinates of PrP(90–231) (155). The remainder of the sequence was hand-built for illustration purposes only. The color scale corresponds to the heteronuclear {¹H}-¹⁵N nuclear Overhauser enhancement data: red for the lowest (most negative) values, where the polypeptide is most flexible, to blue for the highest (most positive) values in the most structured and rigid regions of the protein. (E) Plausible model for the tertiary structure of HuPrP^Sc (166). Color scheme is as follows: S1 β-strands are 108–113 and 116–122 in red; S2 β-strands are 128–135 and 138–144 in green; α-helices H3 (residues 178–191) and H4 (residues 202–218) in gray; loop (residues 142–177) in yellow. Four residues implicated in the species barrier are shown in ball-and-stick form (Asn-108, Met-112, Met-129, Ala-133).

NMR structure of recombinant PrP.

The NMR structure of recombinant (r) SHaPrP(90–231) derived from Escherichia coli was determined after the protein was purified and refolded (Fig. ​(Fig.55A). Residues 90–112 are not shown because marked conformational heterogeneity was found in this region, while residues 113–126 constitute the conserved hydrophobic region, which also displays some structural plasticity (162). Although some features of the structure of rPrP(90–231) are similar to those reported earlier for the smaller recombinant MoPrP(121–231) fragment (154, 177), substantial differences were found. For example, the loop at the NH₂ terminus of helix B is well defined in rPrP(90–231) but is disordered in MoPrP(121–231); in addition, helix C is composed of residues 200–227 in rPrP(90–231) but extends only from 200–217 in MoPrP(121–231). The loop and the COOH-terminal portion of helix C are particularly important because they form the site to which protein X binds as described below (Fig. ​(Fig.55B) (178). Whether the differences between the two recombinant PrP fragments are because of (i) their different lengths, (ii) species-specific differences in sequences, or (iii) the conditions used for solving the structures remains to be determined.

Studies of chimeric SHa/Mo and Hu/Mo PrP transgenes identified a domain composed of residues 95–170, where PrP^C binds to PrP^Sc (133, 179). When chimeric bovine (Bo)/Mo PrP transgenes failed to render mice sensitive to BSE prions, we examined the differences among the sequences in the chimeric and parent PrP genes (180). The findings identified a second domain in PrP composed of residues 180–205 that seems to modulate the interaction between PrP^C and PrP^Sc (Fig. ​(Fig.55C).

Recent NMR studies of full-length MoPrP(23–231) and SHaPrP(29–231) have shown that the NH₂ termini are highly flexible and lack identifiable secondary structure under the experimental conditions employed (Fig. ​(Fig.55D) (156, 165). Studies of SHaPrP(29–231) indicate transient interactions between the COOH terminus of helix B and the highly flexible, NH₂-terminal random-coil containing the octareapeats (residues 29–125) (156); such interactions were not reported for MoPrP(23–231) (165).

PrP gene dosage controls length of incubation time.

Scrapie incubation times in mice were used to distinguish prion strains and to identify a gene controlling its length (135, 215). This gene was initially called Sinc on the basis of genetic crosses between C57BL and VM mice that exhibited short and long incubation times, respectively (215). Because of the restricted distribution of VM mice, we searched for another mouse with long incubation times. I/Ln mice proved to be a suitable substitute for VM mice (216); eventually, I/Ln and VM mice were found to be derived from a common ancestor (217). With a PrP cDNA probe, we demonstrated genetic linkage between the PrP gene and a locus controlling the incubation time in crosses between NZW/LacJ and I/Ln mice (213). We provisionally designated these genes as components of the Prn complex but eventually found that the incubation time gene, Prn-i, is either congruent with or closely linked to the PrP gene, Prnp (195).

Although the amino acid substitutions in PrP that distinguish NZW (Prnp^a ) from I/Ln (Prnp^b) mice argued for congruency of Prnp and Prn-i, experiments with Prnp^a mice expressing Prnp^b transgenes demonstrated a “paradoxical” shortening of incubation times (196). We had predicted that these Tg mice would exhibit a prolongation of the incubation time after inoculation with RML prions on the basis of (Prnp^a × Prnp^b)F₁ mice, which do exhibit long incubation times. We described those findings as “paradoxical shortening” because we and others had believed for many years that long incubation times are dominant traits (213, 215). From studies of congenic and transgenic mice expressing different numbers of the a and b alleles of Prnp, we learned that these findings were not paradoxical; indeed, they resulted from increased PrP gene dosage (218). When the RML isolate was inoculated into congenic and transgenic mice, increasing the number of copies of the a allele was found to be the major determinant in reducing the incubation time; however, increasing the number of copies of the b allele also reduced the incubation time, but not to the same extent as that seen with the a allele. From the foregoing investigations, we concluded that both Sinc and Prn-i are congruent with PrP (218), and recent gene targeting studies have confirmed this view (219).

Overexpression of wtPrP transgenes.

Mice were constructed expressing different levels of the wt SHaPrP transgene (214). Inoculation of these Tg(SHaPrP) mice with SHa prions demonstrated abrogation of the species barrier, resulting in abbreviated incubation times (220). The length of the incubation time after inoculation with SHa prions was inversely proportional to the level of SHaPrP^C in the brains of Tg(SHaPrP) mice (220). Bioassays of brain extracts from clinically ill Tg(SHaPrP) mice inoculated with Mo prions revealed that only Mo prions but no SHa prions were produced. Conversely, inoculation of Tg(SHaPrP) mice with SHa prions led only to the synthesis of SHa prions. Although the rate of PrP^Sc synthesis appears to be a function of the level of PrP^C expression in Tg mice, the level to which PrP^Sc finally accumulates seems to be independent of PrP^C concentration (220).

During transgenetic studies, we discovered that uninoculated older mice harboring numerous copies of wtPrP transgenes derived from Syrian hamsters, sheep, and Prnp^b mice spontaneously developed truncal ataxia, hind-limb paralysis, and tremors (198). These Tg mice exhibited a profound necrotizing myopathy involving skeletal muscle, a demyelinating polyneuropathy, and focal vacuolation of the CNS. Development of disease was dependent on transgene dosage. For example, Tg(SHaPrP^+/+)7 mice homozygous for the SHaPrP transgene array regularly developed disease between 400 and 600 days of age, whereas hemizygous Tg(SHaPrP^+/0)7 mice also developed disease, but only after >650 days.

Transmission of Hu prions.

Protein X was postulated to explain the results on the transmission of Hu prions to Tg mice (Table ​(Table4)4) (134, 179). Mice expressing both Mo and HuPrP were resistant to Hu prions, whereas those expressing only HuPrP were susceptible. These results argue that MoPrP^C inhibited transmission of Hu prions—i.e., the formation of nascent HuPrP^Sc. In contrast to the foregoing studies, mice expressing both MoPrP and chimeric MHu2MPrP were susceptible to Hu prions and mice expressing MHu2MPrP alone were only slightly more susceptible. These findings contend that MoPrP^C has only a minimal effect on the formation of chimeric MHu2MPrP^Sc.

Table 4

Evidence for protein X from transmission studies of humanprions

Inoculum	Host	MoPrP gene	Incubation time, days	(n/n0)
sCJD	Tg(HuPrP)	Prnp+/+	721	(1/10)
sCJD	Tg(HuPrP)	Prnp0/0	263 ± 2	(6/6)
sCJD	Tg(MHu2M)	Prnp+/+	238 ± 3	(8/8)
sCJD	Tg(MHu2M)	Prnp0/0	191 ± 3	(10/10)

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Data with inoculum RG (134); incubation times are given ±SEM. 

Genetic evidence for protein X.

When the data on Hu prion transmission to Tg mice were considered together, they suggested that MoPrP^C prevented the conversion of HuPrP^C into PrP^Sc but had little effect on the conversion of MHu2M into PrP^Sc by binding to another Mo protein. We interpreted these results in terms of MoPrP^C binding to this Mo protein with a higher affinity than does HuPrP^C. We postulated that MoPrP^C had little effect on the formation of PrP^Sc from MHu2M (Table ​(Table4)4) because MoPrP and MHu2M share the same amino acid sequence at the COOH terminus. We hypothesized that MoPrP^C only weakly inhibited transmission of SHa prions to Tg(SHaPrP) mice (Table ​(Table3)3) because SHaPrP is more closely related to MoPrP than is HuPrP.

Using scrapie-infected Mo neuroblastoma cells transfected with chimeric Hu/Mo PrP genes, we extended our studies of protein X. Substitution of a Hu residue at position 214 or 218 prevented PrP^Sc formation (Fig. ​(Fig.5B)5B) (178). The side chains of these residues protrude from the same surface of the COOH-terminal α-helix, forming a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at position 167, 171, or 218 prevented PrP^Sc formation; these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings within the context of protein X explain the protective effects of basic polymorphic residues in PrP of humans and sheep (199, 232, 233).

Transgene-specified susceptibility.

Tg(PrP106)Prnp^0/0 mice that expressed PrP106 developed neurological dysfunction ≈300 days after inoculation with RML prions previously passaged in CD-1 Swiss mice (S. Supattapone, T. Muramoto, D. Peretz, S. J. DeArmond, A. Wallace, F. E. Cohen, S.B.P., and M. R. Scott, unpublished results). The resulting prions containing PrP^Sc106 produced CNS disease in ≈66 days upon subsequent passage in Tg(PrP106)Prnp^0/0 mice (Table ​(Table5).5). Besides widespread spongiform degeneration and PrP deposits, the pyramidal cells of the hippocampus constituting the CA-1, CA-2, and CA-3 fields disappeared in Tg(PrP106)Prnp^0/0 mice inoculated with prions containing PrP^Sc106 (Fig. ​(Fig.7).7). In no previous study of Tg mice have we seen similar neuropathological lesions. The Tg(MoPrP-A) mice overexpressing MoPrP are resistant to RML106 miniprions but are highly susceptible to RML prions. These mice require more than 250 days to produce illness after inoculation with miniprions but develop disease in ≈50 days when inoculated with RML prions containing full-length MoPrP^Sc.

Table 5

Susceptibility and resistance of Tg mice to artificialminiprions

Host	Incubation time, days (n/n0)
	RML106 miniprions	RML prions
Tg(PrP106)Prnp0/0 mice	66±3(10/10)	300 ± 2(9/10)
Tg(MoPrP-A) mice	>250(0/11)	50 ± 2(16/16)

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Data are from ref. 218 and S. Supattapone, T. Muramoto, D. Peretz, S. J. DeArmond, A. Wallace, F. E. Cohen, S.B.P., and M. R. Scott, unpublished work. Incubation times are given ±SEM. 

Sporadic CJD.

Sporadic forms of prion disease constitute most cases of CJD and possibly a few cases of Gerstmann–Sträussler–Scheinker disease (GSS) (Table ​(Table1)1) (4, 240, 241). In these patients, mutations of the PrP gene are not found. How prions causing disease arise in patients with sporadic forms is unknown; hypotheses include horizontal transmission of prions from humans or animals (242), somatic mutation of the PrP gene, and spontaneous conversion of PrP^C into PrP^Sc (5, 15). Because numerous attempts to establish an infectious link between sporadic CJD and a preexisting prion disease in animals or humans have been unrewarding, it seems unlikely that transmission features in the pathogenesis of sporadic prion disease (9–12, 243).

Inherited prion diseases.

To date, 20 different mutations in the human PrP gene resulting in nonconservative substitutions have been found that segregate with the inherited prion diseases (Fig. ​(Fig.44B). Familial CJD cases suggested that genetic factors might influence pathogenesis (1, 2, 244), but this was difficult to reconcile with the transmissibility of fCJD and GSS (3). The discovery of genetic linkage between the PrP gene and scrapie incubation times in mice (213) raised the possibility that mutation might feature in the hereditary human prion diseases. The P102L mutation was the first PrP mutation to be genetically linked to CNS dysfunction in GSS (Fig. ​(Fig.44B) (4) and has since been found in many GSS families throughout the world (245–247). Indeed, a mutation in the protein coding region of the PrP gene has been found in all reported kindred with familial human prion disease; besides the P102L mutation, genetic linkage has been established for four other mutations (16, 29–31).

Tg mouse studies confirmed that mutations of the PrP gene can cause neurodegeneration. The P102L mutation of GSS was introduced into the MoPrP transgene, and five lines of Tg(MoPrP-P101L) mice expressing high levels of mutant PrP developed spontaneous CNS degeneration consisting of widespread vacuolation of the neuropil, astrocytic gliosis, and numerous PrP amyloid plaques similar to those seen in the brains of humans who die from GSS(P102L) (248–250). Brain extracts prepared from spontaneously ill Tg(MoPrP-P101L) mice transmitted CNS degeneration to Tg196 mice but contained no protease-resistant PrP (249, 250). The Tg196 mice do not develop spontaneous disease but express low levels of the mutant transgene MoPrP-P101L and are deficient for mouse PrP (Prnp^0/0) (221). These studies, combined with the transmission of prions from patients who died of GSS to apes and monkeys (3) or to Tg(MHu2M-P101L) mice (134), demonstrate that prions are generated de novo by mutations in PrP. Additionally, brain extracts from patients with some other inherited prion diseases, fCJD(E200K) or FFI, transmit disease to Tg(MHu2M) mice (27). An artificial set of mutations in a PrP transgene consisting of A113V, A115V, and A118V produced neurodegeneration in neonatal mice; these Val substitutions were selected for their propensity to form β-sheets (153, 251). In preliminary studies, brain extracts from two of these mice transmitted disease to hamsters and to Tg mice expressing a chimeric SHa/Mo PrP.

Genetic disease that is transmissible.

Had the PrP gene been identified in families with prion disease by positional cloning or through the purification and sequencing of PrP in amyloid plaques before brain extracts were shown to be transmissible, the prion concept might have been more readily accepted. Within that scenario, it seems likely that we would have explored the possibility that the mutant protein, upon inoculation into a susceptible host, stimulated production of more of a similar protein. Postulating an infectious pathogen with a foreign genome would have been the least likely candidate to explain how a genetic disease could be experimentally transmissible.

PrP^Sc conformation enciphers diversity.

Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrP^Sc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene (27). In FFI, the protease-resistant fragment of PrP^Sc after deglycosylation has a mass of 19 kDa, whereas in fCJD(E200K) and most sporadic prion diseases it is 21 kDa (Table ​(Table6)6) (273, 274). This difference in molecular size was shown to be due to different sites of proteolytic cleavage at the NH₂ termini of the two human PrP^Sc molecules reflecting different tertiary structures (273). These distinct conformations were understandable because the amino acid sequences of the PrPs differ.

Table 6

Distinct prion strains generated in humans with inherited prion diseases and transmitted to Tgmice

Inoculum	Host species	Host PrP genotype	Incubation time, days	(n/n0)	PrPSc, kDa
None	Human	FFI(D178N, M129)			19
FFI	Mouse	Tg(MHu2M)	206 ± 7	(7/7)	19
FFI → Tg(MHu2M)	Mouse	Tg(MHu2M)	136 ± 1	(6/6)	19
None	Human	fCJD(E200K)			21
fCJD	Mouse	Tg(MHu2M)	170 ± 2	(10/10)	21
fCJD → Tg(MHu2M)	Mouse	Tg(MHu2M)	167 ± 3	(15/15)	21

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Data are from ref. 27 and G. Telling and S.B.P., unpublished work. Incubation times are given ±SEM. 

Extracts from the brains of FFI patients transmitted disease to mice expressing a chimeric MHu2M PrP gene about 200 days after inoculation and induced formation of the 19-kDa PrP^Sc, whereas fCJD(E200K) and sCJD produced the 21-kDa PrP^Sc in mice expressing the same transgene (27). On second passage, Tg(MHu2M) mice inoculated with FFI prions showed an incubation time of ≈130 days and a 19-kDa PrP^Sc, whereas those inoculated with fCJD(E200K) prions exhibited an incubation time of ≈170 days and a 21-kDa PrP^Sc (28). The experimental data demonstrate that MHu2MPrP^Sc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet the amino acid sequence of MHu2MPrP^Sc is invariant.

The results of our studies argue that PrP^Sc acts as a template for the conversion of PrP^C into nascent PrP^Sc. Imparting the size of the protease-resistant fragment of PrP^Sc through conformational templating provides a mechanism for both the generation and propagation of prion strains.

Interestingly, the protease-resistant fragment of PrP^Sc after deglycosylation with a mass of 19 kDa has been found in a patient who developed a sporadic case of prion disease similar to FFI but with no family history. Because both PrP alleles encoded the wt sequence and a Met at position 129, we labeled this case fatal sporadic insomnia (FSI). At autopsy, the spongiform degeneration, reactive astrogliosis, and PrP^Sc deposition were confined to the thalamus (275). These findings argue that the clinicopathologic phenotype is determined by the conformation of PrP^Sc in accord with the results of the transmission of human prions from patients with FFI to Tg mice (27).

Origin of BSE prions?

The origin of the bovine prions causing BSE cannot be determined by examining the amino acid sequence of PrP^Sc in cattle with BSE because the PrP^Sc in these animals has the bovine sequence whether the initial prions in MBM came from cattle or sheep. The bovine PrP sequence differs from that of sheep at 7 or 8 positions (287–289). In contrast to the many PrP polymorphisms found in sheep, only one PrP polymorphism has been found in cattle. Though most bovine PrP alleles encode five octarepeats, some encode six. PrP alleles encoding six octarepeats do not seem to be overrepresented in BSE (Fig. ​(Fig.44B) (290).

Brain extracts from BSE cattle cause disease in cattle, sheep, mice, pigs, and mink after intracerebral inoculation (291–295), but prions in brain extracts from sheep with scrapie fed to cattle produced illness substantially different from BSE (296). However, no exhaustive effort has been made to test different strains of sheep prions or to examine the disease after bovine-to-bovine passage. The annual incidence of sheep with scrapie in Britain over the past two decades has remained relatively low (J. Wilesmith, personal communication). In July 1988, the practice of feeding MBM to sheep and cattle was banned. Recent statistics argue that the epidemic is now disappearing as a result of this ruminant feed ban (Fig. ​(Fig.88A) (281), reminiscent of the disappearance of kuru in the Fore people of New Guinea (242, 253) (Fig. ​(Fig.88B).

Monitoring cattle for BSE prions.

Although many plans have been offered for the culling of older cattle to minimize the spread of BSE (281), it seems more important to monitor the frequency of prion disease in cattle as they are slaughtered for human consumption. No reliable, specific test for prion disease in live animals is available, but immunoassays for PrP^Sc in the brainstems of cattle might provide a reasonable approach to establishing the incidence of subclinical BSE in cattle entering the human food chain (176, 209, 289, 297–299, 345). Determining how early in the incubation period PrP^Sc can be detected by immunological methods is now possible, since a reliable bioassay has been created by expressing the BoPrP gene in Tg mice (180). Prior to development of Tg(BoPrP)Prnp^0/0 mice, non-Tg mice inoculated intracerebrally with BSE brain extracts required more than 300 days to develop disease (67, 295, 300, 301). Depending on the titer of the inoculum, the structures of PrP^C and PrP^Sc, and the structure of protein X, the number of inoculated animals developing disease can vary over a wide range (Table ​(Table3).3). Some investigators have stated that transmission of BSE to mice is quite variable with incubation periods exceeding 1 year (67), whereas others report low prion titers in BSE brain homogenates (300, 301) compared with rodent brain scrapie (54, 56, 302, 303).

Have bovine prions been transmitted to humans?

In 1994, the first cases of CJD in teenagers and young adults that were eventually labeled new variant (v) CJD occurred in Britain (304). Besides these patients being young (305, 306), their brains showed numerous PrP amyloid plaques surrounded by a halo of intense spongiform degeneration (Fig. ​(Fig.10)10) (307). Later, one French case meeting these criteria followed (308). These unusual neuropathologic changes have not been seen in CJD cases in the United States, Australia, or Japan (307, 309). Both macaque monkeys and marmosets developed neurologic disease several years after inoculation with bovine prions (310), but only the macaques exhibited numerous PrP plaques similar to those found in vCJD (311) (R. Ridley and H. Baker, personal communication).

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Figure 10

Histopathology of vCJD in Great Britain. (a) Section from frontal cortex stained by the periodic acid–Schiff method, showing a field with aggregates of plaques surrounded by spongiform degeneration. (×93.) (b) Multiple plaques and amorphous deposits are PrP immunopositive. (×460.) Specimens were provided by James Ironside, Jeanne Bell, and Robert Will; photomicrographs were prepared by Stephen J. DeArmond.

The restricted geographical occurrence and chronology of vCJD have raised the possibility that BSE prions have been transmitted to humans. That only ≈25 vCJD cases have been recorded and the incidence has remained relatively constant make establishing the origin of vCJD difficult. No set of dietary habits distinguishes vCJD patients from apparently healthy people. Moreover, there is no explanation for the predilection of vCJD for teenagers and young adults. Why have older individuals not developed vCJD-based neuropathologic criteria? It is noteworthy that epidemiological studies over the past three decades have failed to find evidence for transmission of sheep prions to humans (9–12). Attempts to predict the future number of cases of vCJD, assuming exposure to bovine prions prior to the offal ban, have been uninformative because so few cases of vCJD have occurred (312–314). Are we at the beginning of a human prion disease epidemic in Britain like those seen for BSE and kuru (Fig. ​(Fig.8),8), or will the number of vCJD cases remain small as seen with iCJD caused by cadaveric HGH (255, 256)?

Hallmark of prion diseases.

The hallmark of all prion diseases—whether sporadic, dominantly inherited, or acquired by infection—is that they involve the aberrant metabolism and resulting accumulation of the prion protein (Table ​(Table1)1) (23). The conversion of PrP^C into PrP^Sc involves a conformation change whereby the α-helical content diminishes and the amount of β-sheet increases (25). These findings provide a reasonable mechanism to explain the conundrum presented by the three different manifestations of prion disease.

Understanding how PrP^C unfolds and refolds into PrP^Sc will be of paramount importance in transferring advances in the prion diseases to studies of other degenerative illnesses. The mechanism by which PrP^Sc is formed must involve a templating process whereby existing PrP^Sc directs the refolding of PrP^C into a nascent PrP^Sc with the same conformation. A knowledge of PrP^Sc formation not only will help in the rational design of drugs that interrupt the pathogenesis of prion diseases but it may also open new approaches to deciphering the causes of and to developing effective therapies for the more common neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). Indeed, the expanding list of prion diseases and their novel modes of transmission and pathogenesis (Table ​(Table1),1), as well as the unprecedented mechanisms of prion propagation and information transfer (Tables ​(Tables55 and ​and6),6), indicate that much more attention to these fatal disorders of protein conformation is urgently needed.

But prions may have even wider implications than those noted for the common neurodegenerative diseases. If we think of prion diseases as disorders of protein conformation and do not require the diseases to be transmissible, then what we have learned from the study of prions may reach far beyond these common illnesses.

Multiple conformers.

The discovery that proteins may have multiple biologically active conformations may prove no less important than the implications of prions for diseases. How many different tertiary structures can PrP^Sc adopt? This query not only addresses the issue of the limits of prion diversity (345) but also applies to proteins as they normally function within the cell or act to affect homeostasis in multicellular organisms. The expanding list of chaperones that assist the folding and unfolding of proteins promises much new knowledge about this process. For example, it is now clear that proproteases can carry their own chaperone activity where the pro portion of the protein functions as a chaperone in cis to guide the folding of the proteolytically active portion before it is cleaved (347). Such a mechanism might well feature in the maturation of polypeptide hormones. Interestingly, mutation of the chaperone portion of prosubtilisin resulted in the folding of a subtilisin protease with different properties than the one folded by the wild-type chaperone. Such chaperones have also been shown to work in trans (347). Besides transient metabolic regulation within the cell and hormonal regulation of multicellular organisms, it is not unreasonable to suggest that polymerization of proteins into multimeric structures such as intermediate filaments might be controlled at least in part by alternative conformations of proteins. Such regulation of multimeric protein assemblies might occur either in the proteins that form the polymers or in the proteins that function to facilitate the polymerization process. Additionally, apoptosis during development and throughout adult life might also be regulated, at least in part, by alternative tertiary structures of proteins.