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REVIEWS THE STATE OF THE PRION Charles Weissmann Abstract | There is little doubt that the main component of the transmissible agent of spongiform encephalopathies — the prion — is a conformational variant of the ubiquitous host protein PrPC, and that the differing properties of various prion strains are associated with different abnormal conformations of this protein. The precise structure of the prion is not yet known, nor are the mechanisms of infection, conformational conversion and pathogenesis understood. STRAINS Types of prions differing in regard to the clinical course of the disease and the neuropathology they elicit, their transmissibility and the physico-chemical properties of the PrP isoforms that they are associated with. PRION Protein-containing infectious agent causing transmissible spongiform encephalopathy (TSE), unusually resistant to agents known to inactivate nucleic acids. As used in this article the term does not imply any specific components or structure. VIRINO An infectious particle that is conjectured to consist of a TSE-specific nucleic acid enveloped by PrPSc. MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG, UK, and Department of Infectology, Scripps Research Institute Florida, Boca Raton, Florida 33431, USA. e-mail: charles.weissmann@ prion.ucl.ac.uk doi:10.1038/nrmicro1025 Transmissible spongiform encephalopathies (TSEs, or prion diseases), such as Creutzfeldt–Jakob disease (CJD) and kuru in man, bovine spongiform encephalopathy (BSE) in cattle or scrapie in sheep, still present major challenges to biomedical research. The first phase of investigation of these diseases was dominated by experiments at the classical biological level and led to the discovery of transmissibility of scrapie, kuru and CJD to appropriate recipients, the existence of different STRAINS and recognition of the peculiar properties of the transmissible agent, such as lack of immunogenicity and long incubation times. The unusual resistance of these agents to radiation led to the proposal that the agent, later named ‘PRION’, might be devoid of nucleic acid (in this review I will use the term prion to signify the infectious TSE agent, regardless of its composition or structure). The next phase of research involved the isolation and biochemical characterization of PrPSc, a protein that is found only in scrapie-infected animals1. This was followed by the cloning of PrP cDNA2,3 and Prnp (the cognate gene)4, the recognition that this gene encodes a normal host protein, PrPC, from which PrPSc is derived by conformational rearrangement4–6, and the establishment of a link between Prnp and familial prion disease7. In the third phase of research, transgenic experimentation strengthened the link between the PrP gene and susceptibility to prion disease by showing that the so-called species barrier could be overcome, at least in some cases, by introducing the PrP gene of a donor into a recipient8. The strongest evidence for the essential role of PrP in prion disease was the demonstration that PrP-knockout mice9 were resistant to NATURE REVIEWS | MICROBIOLOGY prion disease and were incapable of propagating the infectious agent10. The discovery of ‘yeast prions’ has provided profound insights into the mechanism of self-propagating conformational changes11. The precise nature of the transmissible agent has been debated since the mid-1960s. As a virus is understood to consist of a protein-encased nucleic acid encoding some or all of its constituent proteins, the concept of a ‘slow’ or ‘unconventional’ virus has lost support because intense efforts in many laboratories have failed to identify a TSE-specific nucleic acid, or even a nucleic acid that is long enough to encode a small protein12. Nonetheless, the idea that a virus — perhaps an endogenous virus, the nucleic acid of which would not score as extraneous — is responsible for TSEs persists in some quarters13,14. The VIRINO hypothesis, which asserts that the infectious agent consists of an agent-specific nucleic acid enveloped in a host-specified protein, was proposed to explain the lack of an immune response by the host as well as strain variation15 (FIG. 1c). However, the failure to identify a TSE-associated nucleic acid, the biochemical linkage of the mouse scrapie prions and PrPSc (which is a protease-resistant, aggregated conformational isoform of the normal host protein PrPC), and the link between the PrP gene and susceptibility to prions and familial prion diseases have provided support for an updated version of the ‘protein-only’ hypothesis16, namely that the infectious agent is PrPSc, which ‘multiplies’ by catalysing the conversion of PrPC into a likeness of itself17 (FIG. 1b). The finding that PrP knockout mice were resistant to scrapie fulfilled a central prediction of the protein-only hypothesis, and significantly promoted its acceptance, without however proving it. VOLUME 2 | NOVEMBER 2004 | 8 6 1 REVIEWS a Normal cell A further proposal18 attempted to mediate between the two camps (protein only and protein plus nucleic acid) by hypothesizing that the conversion of PrPC to an abnormal conformer that can cause disease is indeed the essential pathogenic event, but that a small hostspecified nucleic acid associated with PrP Sc — the ‘co-prion’ — is a crucial component that is required to modulate strain specificity. These different ideas are reviewed here in the light of recent developments. PrPc PrP mRNA Abnormal forms of PrP Nucleus PrP gene b Scrapie-infected cell: Protein-only model PrPSc PrPSc PrPc PrP mRNA Nucleus PrP gene c Scrapie-infected cell: Virino model Virino Replication TSE-specific nucleic acid Virino PrPc PrP mRNA Nucleus PrP gene Figure 1 | Models for the propagation of the TSE agent (prion). a | In a normal cell, PrPC (yellow square) is synthesized, transported to the cell surface and eventually internalized. b | The protein-only model postulates that the infectious entity, the prion, is congruent with an isoform of PrP, here designated as PrPSc (blue circle). Exogenous PrPSc causes catalytic conversion of PrPC to PrPSc, either at the cell surface or after internalization. c | The virino model postulates that the infectious agent consists of a TSE-specific nucleic acid associated with or packaged in PrPSc. The hypothetical nucleic acid is replicated in the cell and associates with PrPC, which is thereby converted to PrPSc. Reproduced with permission from REF. 123 © (1994) Elsevier. 862 | NOVEMBER 2004 | VOLUME 2 PrP C, which is the normal form of the protein encoded by Prnp, is attached to the outer surface of the plasma membrane by a glycosylphosphatidyl inositol (GPI) anchor (see BOX 1 for prion nomenclature). It is readily released from the cell surface by cleavage with phosphatidyl inositol-specific phospholipase C (PIPLC) and is highly susceptible to proteinase K (PK) digestion. PrPSc was originally defined as a form of PrP that was largely resistant to PK digestion under conditions in which PrPC and most other proteins were readily degraded19, but is somewhat confusingly now also used as the designation for the infectious isoform of PrP, whatever its properties might be 20,21. Importantly, digestion of PrP Sc by PK (but not by other proteinases such as trypsin) causes cleavage at residues 87 to 91 (the exact position depends on the prion strain) of the mature PrP sequence, leading to a characteristic electrophoretic mobility shift of the three bands that correspond to di-, mono- and unglycosylated species (designated PrP27-30). It should be noted that ‘protease resistance’ is a relative concept; the ‘resistant’ moiety is also susceptible to degradation, at a rate that depends critically not only on the concentration of PK22,23 and the PK to protein ratio, but also on the prion strain with which the PrPSc is associated24. Some forms of PrP are more resistant to PK than PrPC but are nonetheless non-infectious25,26. Purified preparations of prions contain aggregates of PrPSc (or PrP27-30 if PK-treated) as the main protein component, but also contain several non-protein components such as glycosaminoglycans and polysaccharides27. Strikingly, hamster prion infectivity shows a similar resistance to PK as PrPSc and, under stringent digestion conditions, PrPSc concentration and infectivity decrease at similar rates23. Complete dissociation of the aggregates by alkali, strong acids, detergents or chaotropic agents leads to a loss of infectivity that so far has remained irreversible; so, attribution of infectivity to a monomeric component of the aggregates has not been achieved. Usually, the number of PrP molecules in a scrapieinfected brain homogenate or in purified preparations is several orders of magnitude greater than the number of infectious units28, which raises the question as to whether the infectious process is very inefficient, the infectious entity is an aggregate of a large number of PrPSc molecules or whether it is a minority component of the preparation, perhaps a different isoform of PrP, generically designated PrP* (REF. 29). Several studies have reported very low30 or undetectable levels of www.nature.com/reviews/micro REVIEWS Box 1 | Prion nomenclature α-PrP The α-helix-rich form of PrP, represented by PrPC. β-PrP The β-sheet-rich forms of PrP can be generated from the oxidized and from the reduced form of PrP by exposure to various chemical treatments. They can form fibrillary structures, particularly when amino-terminally truncated. PrPC The physiologically occurring, mainly GPI-linked form of PrP, or prion protein, that can be glycosylated on one or both of two asparagine residues with a variety of glycans. As shown by NMR and X-ray crystallography, it is rich in α-helical structure and contains only a little β-sheet structure. PrPiso A designation I propose, for any stable form of PrP that differs from PrPC only by virtue of its conformation but not primary structure. Such differences may currently be detected by a variety of methods, such as reactivity to certain monoclonal antibodies, conformation-dependent immunoassay, susceptibility to proteinases, including the location of cleavage site(s), and optical measurements such as infra-red or circular dichroism. PrPiso comprises, among others, PrP-res, PrPSc or sPrPSc, as defined below. PrPSc An isoform of PrPC that is almost invariably detected in TSE-infected tissues and cells. It comprises a carboxy-proximal segment of about 140 residues that is resistant to defined conditions of PK treatment. The term PrPSc is used by some interchangeably with prion, a usage that should be avoided. PrPSc designates a structure, prion is a functional concept. The implication that a particular form of PrP is the only essential constituent of the prion remains to be proven. PrP27-30 The PrP fragment remaining after controlled PK digestion of PrPSc. PrP-res Alternative designation for PrPSc, that has been proposed to generalize the term for all types of TSEs and not only scrapie. PrP-sen The designation for PrPC and forms of PrP that are equally susceptible to PK digestion. PrP* A hypothetical isoform of PrP that is the essential component of the TSE agent or prion. Prnp The gene encoding PrP. Prnpo/o Genotype in which both copies of the PrP gene are inactivated or ablated. rPrP Denotes recombinant PrP. When produced in Escherichia coli it lacks the GPI anchor and the glycan residues. sPrPSc A term used by Prusiner to designate a protease-sensitive isoform of PrP that is detected in prion-infected tissue. This terminology is contradictory because PrPSc was originally defined as a protease-resistant entity. PrPSc in homogenates31, or partially purified preparations, of prion-infected hamster brains14 and brains of fatal familial insomnia patients32, which suggests that PrPSc is not required for infectivity33. However, failure to detect PrPSc in infectious brain homogenates, or fractions thereof, does not disprove that PrPSc is all or part of the infectious agent unless it can be shown that the detection limits for PrPSc are commensurate with those for infectivity, a hurdle that is difficult to overcome. Within the framework of the protein-only hypothesis it could be argued that at least some infectivity might be associated with an abnormal isoform of PrP that is sensitive to PK digestion (designated sensitive PrPSc or sPrPSc)20,34,35. It would be useful to revise the PrP nomenclature in the near future. As PrPSc implies ‘scrapie-specific PrP’ and therefore relates to the sheep disease, some NATURE REVIEWS | MICROBIOLOGY authors use the term ‘PrP-res’ for protease-resistant PrP. However, as mentioned above, protease resistance is a relative term, and the main issue is whether one is dealing with the naturally occurring, well-characterized α-helix-rich PrPC, or with one of the many isoforms that are found in association with different host species and/or prion strains, or generated in vitro, for which one might propose the generic designation ‘PrPiso’ (BOX 1). Spontaneous forms of prion disease The most common form of human prion disease is the so-called sporadic Creutzfeldt–Jakob disease (sCJD), which occurs with an incidence of about 1–2 per million individuals per year. It is considered to be spontaneous because no epidemiological evidence for association with any exogenous factors, in particular animal or VOLUME 2 | NOVEMBER 2004 | 8 6 3 REVIEWS The proponents of the virus or virino hypothesis predicate that these agents are ubiquitous and that, in the case of sCJD, the outbreak of disease is promoted by unknown exogenous factors, in analogy perhaps to herpesvirus, which is almost ubiquitous in the population but only rarely gives rise to disease symptoms. However, there is no evidence to support this suggestion. a Refolding model Prpc PrPSc b Seeding model Prpc Very slow Replication of prions in organisms PrPSc Rapid Rapid Figure 2 | Models for the conversion of PrPC to PrPSc. a | The refolding model. The conformational change is kinetically controlled, a high activation energy barrier preventing spontaneous conversion at detectable rates. Interaction with exogenously introduced PrPSc (blue circle) causes PrPC (yellow square) to undergo an induced conformational change to yield PrPSc. This reaction could be facilitated by an enzyme or chaperone. In the case of certain mutations in PrPC, spontaneous conversion to PrPSc can occur as a rare event, explaining why familial Creutzfeldt– Jacob disease (CJD) or Gerstmann–Sträussler–Sheinker syndrome (GSS) arise spontaneously, albeit late in life. Sporadic CJD (sCJD) might arise when an extremely rare event (occurring in about one in a million individuals per year) leads to spontaneous conversion of PrPC to PrPSc. b | The seeding model. PrPC (yellow square) and PrPSc (or a PrPSc-like molecule; shown as a blue circle) are in equilibrium, with PrPC strongly favoured. PrPSc is only stabilized when it adds onto a crystal-like seed or aggregate of PrPSc. Seed formation is rare; however, once a seed is present, monomer addition ensues rapidly. To explain exponential conversion rates, aggregates must be continuously fragmented, generating increasing surfaces for accretion. Reproduced with permission from REF. 124 © (2002) National Academies of Sciences, USA. human sources of infection, has been uncovered. The protein-only hypothesis proposes that either normal PrPC rarely converts spontaneously to PrPSc, or that a somatic mutation in PrP renders it susceptible to conversion to PrPSc. Once PrPSc has been formed it is hypothesized to elicit an autocatalytic conversion process. All familial forms of prion disease are associated with one of 20 or more mutations of the PrP gene, of which most are amino acid substitutions but some are insertions of supernumerary repeats in the octarepeat region36. It is not understood how PrP mutations favour the generation of prions; within the framework of the protein-only hypothesis it is assumed that the pathogenic mutations increase the probability of spontaneous conversion of the mutated PrPC into the cognate PrPSc form. There is however, no evidence that any of the mutations significantly destabilize the PrPC conformation, at least in studies with the recombinant, unglycosylated protein37. 864 | NOVEMBER 2004 | VOLUME 2 The protein-only hypothesis proposes that prions consist of an isoform of PrPC (generically designated PrP* and commonly assumed to be PrPSc) and that their replication comes about by a self-propagating conversion of PrPC to the pathogenic isoform (FIG. 2). According to the refolding model, PrPC unfolds to some extent and refolds under the influence of a PrPSc molecule17. The nucleation or seeding model however, proposes that PrPC is in equilibrium with PrPSc (or a precursor thereof), with the equilibrium strongly favouring PrPC, and that PrPSc is only stabilized when it adds to a crystal-like seed of PrPSc. Once a seed is present, monomer addition ensues rapidly38. Occasional cleavage of aggregates must be postulated to explain the exponential increase of PrPSc during infection39. The nucleation or seeding model has found convincing experimental support in the case of yeast prions, as discussed below, and more recently also with mammalian prions (REF. 40), where in vitro incubation of the normal form of a protein with a seed of its misfolded, fibrillary counterpart leads to rapid conversion, whereas spontaneous misfolding is a slow process. In yeast, propagation of yeast prions depends on a crucial concentration of the chaperone Hsp104 (REFS 41,42); there is currently no evidence for a similar requirement in mammals. In the case of mammalian prions, PrPC expression (or even overexpression) by a cell culture or tissue, is not sufficient to allow prion replication43,44, which might be due to the absence of a receptor or of an auxiliary host protein (such as the conjectured protein X; REF 45), or to a rate of degradation of PrPSc (REFS 43,46) that exceeds the rate of its formation, as detailed below. A high degree of identity between the sequence of the incoming PrPSc and the resident PrPC is often8,47, but not always, crucial for efficient prion replication. A few, or even a single, amino acid change in the PrP that is expressed by a normally susceptible host might confer protection against prion disease. For example, Cheviot sheep with two 171Q alleles (171Q/Q) are susceptible to scrapie, whereas the configurations 171Q/R and 171R/R confer protection48. Prnpo/o transgenic mice that expressed murine PrPQ167R at about the same level as wild-type animals remained healthy and did not accumulate PrPSc after inoculation with mouse scrapie prions. Moreover, expression of PrPQ167R affords partial protection to mice containing wild-type PrP; it has been suggested that this is due to sequestration of the conjectural protein X (REF.49). This protective effect should not be confused with the partial protection against sCJD that is afforded by the M/V heterozygosity www.nature.com/reviews/micro REVIEWS 2.3 2.2 2.1 2.0 1.9 1.8 Lethal limit 1.7 1.6 k = ln3/tD PrPsc per cell (arbitrary units) 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 k = ln2/tD 0.7 0.6 0.5 0.4 0.3 Detection limit 0.2 0.1 k = ln1.4/tD 0.0 0 1 2 3 4 5 6 7 Cell generations Figure 3 | The dynamic susceptibility model for prion propagation. Even if the genetic and biochemical prerequisites for prion replication in a cell population are given, propagation of prions seems to be subject to epigenetic determinants. The dynamic susceptibility model proposes that the determinants are the relative rates of synthesis (ks) and degradation (kd) of prions in individual cells, and that stable propagation of prions is only possible within narrow limits of k = ks/kd. The amount of infectivity per cell can be calculated by P = P0 × e kt D, where P0 is the infectivity content of the cell at the beginning of the cell cycle and tD is the doubling time of the host cell. After each cell division, infectivity per cell is halved. To successfully initiate infection, k must be equal to, or larger than, ln2/tD, otherwise the amount of infectivity per cell would not increase, or continuously diminish, respectively, as the cell divides. However, if k > tD, there will be a steady accumulation of infectious agent, which, if not arrested by an eventual decrease in k, must lead to the death of the cell. In the figure, we assume that at the time of infection k = ln3/tD, resulting in an overall logarithmic increase in the cellular content of infectivity. Starting after the third division, we plot the outcome if k continues at ln3/tD (blue line), resulting in continuous accumulation of infectivity until the lethal limit is reached, if k diminishes to ln2/tD (red line), leading to a stabilization of the infectivity level averaged over time, and if k decreases to ln1.4/tD (green line), leading to a decrease of infectivity below the limits of detection (dashed line). An infected cell population is a mixture of cells with a wide variety of k values. Infected cells secrete infectivity into the medium, infecting as-yet-uninfected cells59. Infected cells may die or lose their infectivity, but the infection is maintained by cells with appropriate k values. The population is thus in a dynamic equilibrium. at position 129 of human PrP because neither M/M nor V/V is protective 50 and therefore this is not due to dominance of an allele. As all clinical cases of variant CJD (vCJD) have occurred in humans with the 129M/M polymorphism51 it is possible that 129V exerts a dominant-negative effect. This effect is however not absolute because a subclinical case of vCJD was detected post-mortem in a 129V/M heterozygote52. NATURE REVIEWS | MICROBIOLOGY Replication of prions in cell lines Only a few cell lines, including the murine neuroblastoma line N2a (REF. 53), the rat adrenal phaeochromocytoma line PC12 (REF. 54) and the hypothalamic neuronal line GT1 (REF. 55), can propagate scrapie prions — they accumulate and maintain PrPSc and infectivity, as shown by the mouse bioassay — over many passages. Although most cell lines that are used for prion propagation are of VOLUME 2 | NOVEMBER 2004 | 8 6 5 REVIEWS UNIFIED THEORY This theory proposes that a PrP isoform, PrP*, is indeed the essential infectious component, but that its properties can be modified by a physically associated small RNA, the co-prion, such as a siRNA. The co-prion would have to be amplified in the host cell and remain bound to the newly formed PrP* to explain the stability of strains, and different siRNAs would be responsible for the phenotypic differences between prion strains. 866 neuronal origin, it is remarkable that the mouse fibroblast lines NIH/3T3 and L929 support prion propagation56 and that a rabbit kidney epithelial line expressing ovine PrP transgenes can propagate sheep scrapie prions57. Interestingly, N2a lines and sublines are susceptible to certain murine prion strains, such as RML, but not to other strains, such as Me7 (REFS 58,59), 87V or 22A (REF. 60), whereas L929 cells propagate RML, ME7 and 22L but not 87V prions56. A sensitive and rapid quantitative assay for murine RML scrapie prions that is based on highly susceptible N2a cells has recently been described and effectively replaces the slow and expensive mouse bioassay59. Overexpression of PrP is not a required parameter for rendering cells highly susceptible to prions43,56. Interestingly, overexpression of PrP in transgenic mice accelerates the course of scrapie disease but does not increase susceptibility to prions or the levels of infectivity or concentrations of PrP Sc in the affected brain 61. So which property of a cell line enables it to propagate prions and discriminate between strains? Similarity between the sequence of the PrPC that is expressed in the cell and that of the prion ‘donor’ is crucial — as small differences not only prevent conversion of the mismatched PrPC (REF. 47) but also have a dominant-negative effect on the conversion of matching PrPC in the same cell62. N2a cell populations are heterogeneous in regard to their susceptibility to infection43,58,59 — cloned sublines can exhibit a thousand-fold difference in susceptibility — however, this property is not stable and changes during prolonged propagation59, which might therefore reflect an epigenetic mechanism. The dynamic susceptibility model, which I propose herewith, suggests that the capacity of a cell to propagate prions, all genetic features being equal, depends on the ratio of prion synthesis to degradation (FIG. 3). Contrary to the earlier belief that PrPSc is inherently very stable, it has become clear that, at least in N2a cells, it has a half-life of less than 24 hours43,46. If this property is shared by prions, then clearly, if the rate of prion formation does not equal or exceed twice that of its degradation (assuming both rates to be constant), the infected state will be eliminated at some time after exposure of cells to exogenous prions. Conversely, if the rate of formation is more than twice that of degradation, continuous accumulation of PrPSc may eventually lead to the death of the cell. So, persistent infection of a cell might depend on the maintenance of a delicate balance of synthesis versus degradation that is subject to both internal and external factors. The unexpected finding that many murine prion strains that are readily propagated in mice do not infect N2a cells that are susceptible to the RML strain58–60 could be explained if, on average, the degradation of these strains is more rapid, or the synthesis slower, than that of RML prions. As mentioned above, PrPSc species that are associated with different prion strains have very different susceptibility to PK digestion63. In the brain, different cells may have subtly different prion synthesis and degradation rates for the various prion strains. | NOVEMBER 2004 | VOLUME 2 Conversion of PrPC to PrPSc in cell-free systems In vitro conversion of PrPC into a PrPSc-like conformation by incubation of PrP C or PrP C-containing brain homogenates with PrP Sc has been achieved. Using 35S-labelled PrPC, Caughey and colleagues showed that the conversion product resembles the PrPSc template in regard to its electrophoretic mobility pattern after PK digestion. As the amount of newly formed PrPSc remained sub-stoichiometric relative to the amount of template64 it is not surprising that a possible increase in infectivity has not been reported. Modified protocols have also been described for PrPSc-elicited in vitro conversion of PrPC, such as the cyclic amplification procedure, in which samples are repeatedly sonicated and incubated to give an overall amplification of 30-fold or sometimes more65. Supattapone and co-workers used a similar protocol, but without sonication, to amplify PrPSc, and reported that conversion was stimulated by an RNA fraction from mammalian tissue and abolished by RNases66. This indicates that other components might be required, either in a structural or catalytic role, to enable or promote in vitro conversion. So far it has not been reported that an increase in the concentration of PrPSc results in an increase in infectivity. The discovery of siRNAs and microRNAs, which would have escaped notice in earlier analyses of prion preparations, owing to both their size and their host origin, provides candidates for the hypothetical co-prion that has been proposed by the UNIFIED THEORY18. It was recently reported that recombinant truncated PrPC (residues 89 to 230) could be converted in vitro into a β-sheet-rich fibrillary form. Injection of this material into wild-type mice did not cause disease; however, inoculation into transgenic mice overexpressing the PrP 89–230 fragment at a level 15-fold greater than that of PrPc in wild-type mice elicited scrapie-like disease after 380–660 days. Brain homogenate from these sick transgenic mice caused scrapie in wild-type mice after 150 days, indicating that a more efficient form of infectious agent was generated in the transgenic mice67. Although non-injected transgenic controls failed to exhibit clinical signs up to ~500 days, it was not reported whether these controls succumbed to spontaneous disease at a later time, whether they had histopathological changes in their brains or whether homogenates from their brains could elicit disease when injected into transgenic or wild-type mice. The possibility that a spontaneous, perhaps subclinical, form of scrapie can develop in transgenic mice overexpressing PrP, and that this process can be accelerated by the administration of PrP isoforms, has still to be considered. Prion strains One of the many remarkable features of prion diseases is the existence of distinct prion strains, which were originally characterized by incubation time and the neuropathology that they elicit in a particular host68. The finding that several different strains can be propagated indefinitely in hosts that are homozygous for the PrP gene (Prnp) has been used to support the existence of a nucleic-acid-containing agent; in the protein-only www.nature.com/reviews/micro REVIEWS hypothesis, the strain-specific properties would have to be encoded in a feature of the pathogenic PrP other than its amino acid sequence, such as its glycosylation pattern or its conformation. In fact, in many instances different strains are associated with PrPSc species that differ in physicochemical properties such as susceptibility to PK digestion24, electrophoretic mobility after PK treatment (which indicates different cleavage sites in the amino-proximal region69–71), stability towards denaturation agents21 or the ratio of di-, mono- and unglycosylated forms71. The conformation-dependent immunoassay (CDI) provides a sensitive tool for differentiating between different conformations of PrP that are associated with distinct prion strains20,72,73. Within the framework of the protein-only hypothesis each strain is assumed to be associated with a different isoform of PrP that can convert PrPC to a likeness of itself. This assumption predicates that there are as many stable conformations of PrP as there are stable prion strains that can be propagated in mice of a particular genotype, perhaps a dozen or more. The notion of dozens of stable conformations of a protein seems bizarre to classical protein biochemists thinking in terms of functional enzymes, but is less odd if one considers stable non-functional multimers or polymers. The concept of ‘conformation templating’ at the protein level is supported by the cell-free conversion experiments of Caughey and colleagues74, the seeding experiments with PrPC (REF. 40) and particularly by recent experiments with yeast prions (see below; REFS 75,76). Nonetheless, the finding that a particular prion strain can be transmitted between two species without changing strain-specific properties, even though the PrP sequences of the two species are very different77, is unexpected because it implies that the same conformation can be imposed on PrP molecules with different amino acid sequences (primacy of strain). A further question that is not readily explained by the protein-only hypothesis in its simplest form is why different prion strains cause lesions and PrPSc deposition at different locations in the brain; a role for the nature of the glycosylation of the misfolded PrP has been proposed to explain this78. The unanswered questions in connection with the strain issue have been used to support the proposal that the infectious agent has a nucleic-acid component in addition to misfolded PrP, as, for example, in the unified theory, or the proposal that a virus-like agent is involved. The finding that selected murine neuroblastoma (N2a) cell lines are susceptible to some murine prion strains (such as RML) but not to others (such as Me7)58,59, even though mice are susceptible to both, might provide an approach to analysing the basis of susceptibility to prions. Transmissibility of prions Under ‘natural’ circumstances — for example, in sheep scrapie, chronic wasting disease of mule deer or BSE — the transmissible agent is likely to be transmitted by ingestion of contaminated foodstuff 79. Experimental transmission is usually several orders of magnitude more efficient when administered intracerebrally than peripherally or orally, as judged by the amount of NATURE REVIEWS | MICROBIOLOGY infectious agent that is required to elicit clinical disease and by the shorter incubation time. By the same criteria, transmission of a TSE from one species to another is far less efficient than transmission within the same species and is sometimes impossible, giving rise to the concept of a species barrier80. However, the more recent finding that trans-species inoculation can lead to neuropathological changes, deposition of PrPSc and/or propagation of infectivity in the absence of clinical disease, albeit after long incubation times, undermines the concept of a species barrier, at least of an absolute one81,82. As it is not possible to determine whether clinical symptoms would or would not appear if the life of the affected animal could be prolonged sufficiently, the use of the neutral term ‘asymptomatic disease’ is better than subclinical or preclinical disease. The concept of species barrier is further complicated by the fact that some prion strains but not others can cause clinical or asymptomatic disease even though they are derived from the same donor species. A striking example is the greater susceptibility of wild-type mice to human vCJD than to sCJD prions83. Prusiner and colleagues showed that the barrier to transmission of Syrian hamster prions to mice, as assessed by the development of clinical disease, could be abrogated by introducing Syrian hamster PrP genes into wild-type mice; the incubation time was reduced with increasing concentrations of the hamster PrPC (REF. 8) and even further reduced in the absence of the resident murine PrP genes10. The interpretation of these results was that lack of similarity between Syrian hamster PrP and recipient murine PrP amino acid sequences hindered the postulated conversion process and that even if the recipient mouse was transgenic for Syrian hamster PrPC, the conversion was still inefficient owing to some form of competition by the mouse PrPC. Such a competitive effect was even more marked when human CJD prions were inoculated into transgenic mice expressing human (Tg[HuPrP]) and mouse–human chimeric PrP genes (Tg[MHu2M]), respectively. Although Tg[HuPrP] mice expressed high levels of human PrPC (HuPrPC), they were resistant to human prions but became susceptible after loss of the mouse PrP gene. By contrast, mice expressing low levels of the chimeric transgene mouse–human prion protein (MHu2M) (which contained the carboxyterminal portion of the mouse gene) were susceptible to human prions and disruption of the mouse PrP gene led to only a small reduction in incubation times. These findings were explained by postulating the existence of a species-specific protein X that binds near the C-terminus and is essential for the conversion process45. However, in the case of the BSE prions, mice expressing the chimeric PrP construct mouse–bovine prion protein (MBo2M) were resistant to infection, whereas mice carrying the complete bovine PrP transgene were susceptible84. Equally difficult to explain is the finding mentioned above, that wild-type mice are more susceptible to vCJD than to sCJD prions, even though the donors have the same PrP sequence83. VOLUME 2 | NOVEMBER 2004 | 8 6 7 REVIEWS Although PrP is essential for the propagation of prions and progression of disease, other genes in various loci of the mouse genome influence features such as the incubation time and, perhaps, susceptibility to infection; none of these genes has yet been identified85–88. Yeast prions Ten years ago Reed Wickner proposed that unexplained instances of ‘cytoplasmic inheritance’ in yeast and other fungi were due to the same phenomenon that had been proposed to underlie prion diseases — a self-perpetuating conformational change of a protein89,90. Although a rare event, a particular protein in a yeast cell can flip into a conformationally altered state, causing conversion of its counterparts to the same state and giving rise to functionally inactive aggregates. The resulting phenotype and the conformationally altered protein are transmitted to the offspring of the ‘mutated’ cell. In the case of the translation termination factor Sup35, for example, the conformational conversion results in fibril formation and can be reproduced in a cell-free system91. Interestingly, depending on the conditions, different forms of fibrils can be generated in vitro, and these forms can be propagated indefinitely by seeding solutions of the recombinant protein with the fibrils. When introduced into normal yeast cells the polymerized forms of Sup35 cause them to adopt the ‘mutant’ or [PSI+] phenotype. It is of particular interest that the different forms of aggregates generated in vitro under specific conditions give rise to different phenotypes in the transformed yeast75,76, demonstrating the link between protein conformation and phenotype. It should be noted that yeast proteins showing the ‘prion phenomenon’ are not homologous to vertebrate PrPs at the level of amino acid sequence identity. Although mammalian prions have the ability to spread laterally under natural conditions, as is the case in scrapie and chronic wasting disease, penetrating their host and using remarkable mechanisms to propagate within it (see below), there seems to be no natural mechanism for prion spread in yeast, and introduction of yeast prions requires sophisticated experimental intervention. Nonetheless, the findings with yeast prions support the proposal that mammalian prion strains are encoded in protein conformation. Spread of prions in the organism SYMPATHECTOMY A chemical or surgical procedure that destroys innervation by the sympathetic nervous system. AMYLOID Fibrillary, mostly β-sheet-rich deposits of protein. PrP in amyloid form is found in some but not all forms of prion disease in humans and animals. 868 Under natural conditions, prion disease is mainly acquired through oral infection. Prions then spread from the periphery to the central nervous system (CNS; neuroinvasion) and once there cause neurodegeneration. PrPC expression is required not only for prion replication and pathogenesis but also for transporting the infectious agent both from the peripheral sites to the CNS (as shown by PrPC-expressing neurografts in PrP-knockout mice)92 and within the CNS93. However, reconstitution of Prnpo/o mice with wild-type bone marrow is insufficient to restore neuroinvasion in engrafted Prnpo/o mice, although the capacity of the spleen to accumulate prions of the RML strain is reconstituted92. This indicates that PrPC expression in an additional | NOVEMBER 2004 | VOLUME 2 compartment, presumably the peripheral nervous system, is required. Transport of prions from the peripheral entry site, particularly the digestive tract, to the lymphoreticular system is attributed to myeloid dendritic cells94. In the lymphoreticular system, prions are replicated and accumulated. B lymphocytes (not necessarily expressing PrPC) are crucial for efficient peripheral prion spread and neuroinvasion95. The dependence on lymphotoxin (LT)-mediated signalling by B cells might explain the requirement for B cells in peripheral pathogenesis: it is the follicular dendritic cells (FDCs) that accumulate PrPSc after scrapie infection96, and maturation of FDCs requires signalling by B cells expressing LTα/LTβ trimers on their surface97. Indeed, block of LTβ signalling by administration of soluble LTβR-immunoglobulin causes depletion of mature FDCs and markedly impairs neuroinvasion and accumulation of peripheral PrPSc and infectivity98,99. FDCs are crucial for efficient disease progression after oral scrapie challenge, but only within a short period of time100. After amplification in the spleen, prions are transferred to the CNS, presumably via the sympathetic nervous system, which provides the main innervation of lymphoid organs. Chemical SYMPATHECTOMY delays the onset of scrapie, whereas sympathetic hyperinnervation enhances splenic prion replication and neuroinvasion101. Massive peripheral inoculation of prions can however, bypass the lymphoreticular system altogether and colonize the CNS, as shown by the susceptibility of mice expressing PrP only in neuronal tissue102. Pathogenesis The major pathological changes resulting from prion disease are found in the CNS, with vacuolation, neuronal cell death and astrocytosis being the most common. As abrogation of PrP expression is not deleterious, whether it is inborn9 or elicited post-natally103, there is no reason to believe that depletion of PrPC owing to its conversion to PrPSc, if indeed such depletion occurs, is pathogenic. This raises the obvious question of whether PrPSc is toxic. Several findings cast doubt on this possibility. A first observation was that, although wild-type animals succumbed to scrapie about 22 weeks after inoculation, mice that were heterozygous for the PrP-knockout allele accumulated levels of PrPSc and infectivity as high as wild-type mice by 20 weeks after inoculation and yet survived without symptoms for more than 41 weeks104. When inoculated with prions, PrP-knockout mice with a PrP-expressing graft in their brains showed scrapie pathology, PrPSc and infectivity in the graft, whereas the surrounding tissue that did not contain PrP showed no changes, even when close to deposits of PrPSc that had been exported from the graft105. Even more impressive is the finding that mice in which neuronal expression of PrP was prevented by conditional cre-lox knockout about 7–8 weeks after intracerebral inoculation accumulated vast amounts of infectivity and PrPSc in astrocytes without exhibiting clinical symptoms or evidence of neuronal damage106. All these findings indicate that neurons that are devoid of PrPC are not affected by PrPSc. www.nature.com/reviews/micro REVIEWS Toxicity due to a PrP conformer other than PrPSc has been considered. Targeting of PrP to the cytosol resulted in rapidly lethal neurodegeneration (albeit without accumulation of PrPSc), and proteasome inhibition induced a slightly protease-resistant cytoplasmic PrP species in cultured cells107,108. Therefore, prion toxicity was proposed to start with retrotranslocation of PrPC from the endoplasmic reticulum to the cytosol, in conjunction with impaired proteasomal function. However, other studies have found that cytosolic PrP retains its secretory leader peptide and does not contain a GPI anchor, suggesting that it never enters the endoplasmic reticulum109. Moreover, the toxicity of cytosolic PrP has been contested110,111. Lingappa and co-workers found that PrPC assumes a transmembrane topology (CtmPrP), the concentration of which correlates with neurotoxicity112,113. These data have been taken to indicate that CtmPrP represents an important toxic moiety. PrP proteinopathies At least twenty human diseases are associated with the deposition of β-sheet-rich protein aggregates, or 114,115 AMYLOID . They are frequently designated ‘conformational diseases’ or ‘proteinopathies’, although it is not always clear whether, or to what extent, the misfolded proteins are the cause of the disease rather than the consequence. TSEs or prion diseases should be distinguished from PrP proteinopathies14,116,117. To qualify as a TSE or prion disease, transmissibility within the same or a different species must be demonstrated, a hurdle that cannot always be overcome, particularly in human disease. Although all human familial CJD cases co-segregate with PRNP mutations, it is possible that some PRNP mutations cause neurodegenerative diseases that are not transmissible and are therefore proteinopathies rather than prion diseases; many such examples have been described in the mouse116 and are exemplified by the octapeptide repeat expansion mutants of both mouse118 and man117,119,120. 1. 2. 3. 4. 5. 6. 7. Prusiner, S. B. Novel proteinaceous infectious particles cause scrapie. Science 216, 136–144 (1982). A historically important paper providing a major breakthrough in the understanding of spongiform encephalopathies. Oesch, B. et al. A cellular gene encodes scrapie PrP 27-30 protein. Cell 40, 735–746 (1985). A historically important paper showing that the gene encoding what is believed to be the infectious molecule is encoded by the host. Chesebro, B. et al. Identification of scrapie prion proteinspecific messenger RNA in scrapie-infected and uninfected brain. Nature 315, 331–333 (1985). Basler, K. et al. Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46, 417–428 (1986). Stahl, N. et al. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32, 1991–2002 (1993). Prusiner, S. B. Prions causing degenerative neurological diseases. Annu. Rev. Med. 38, 381–398 (1987). Hsiao, K. et al. Linkage of a prion protein missense variant to Gerstmann–Sträussler syndrome. Nature 338, 342–345 (1989). Established the first genetic link between a familial prion disease and the PrP gene. NATURE REVIEWS | MICROBIOLOGY 8. 9. 10. 11. 12. 13. 14. Evolutionary considerations Prion diseases differ from other proteinopathies in that they are transmissible, not only under experimental conditions but also naturally, predominantly by ingestion. Although in certain cases the inception of an experimental amyloidosis can be accelerated by the injection of amyloid into a predisposed host102, mammalian prions are exceptional in that they are able to enter their hosts by natural portals and make their way from the gut to the brain, using intermediate tissues for amplification. In the case of microorganisms, including viruses, acquisition of such sophisticated entry and transport mechanisms is attributed to evolutionary processes — genomic mutations and selection of mutants that most readily enter their host and find a suitable niche in which to efficiently replicate and/or perpetuate. However, if the prion consists solely of a protein encoded by the genome of its host, what drives the prion to become efficient in replication and invasion of its parent? We can only speculate. For example, the ‘misfolded’ form of PrP might have originated as a ‘messenger’ protein that on the one hand has or had a physiological function, but on the other hand has a malignant potential that is rarely realized and was not selected against because evolutionary pressure does not operate efficiently at post-reproductive age. It has been proposed that, in yeast, a prion-like phenomenon involving Sup35 might confer a selective advantage on yeast growing under fluctuating environmental conditions121. Another possibility is that PrP/PrPSc is derived from an ancient pathogen, the genetic material of which was integrated into the genome of its host and harnessed to fulfil a useful function while its pathogenic potential was minimized. More trivially, but equally improbably, mammalian prion disease could result from the combination of the natural propensity of proteins to assume β-sheet-rich conformations122, a failure of the organism to prevent their formation and accumulation in some cases, and the coincidental ability of the isoform to penetrate organisms and cells through natural portals. Prusiner, S. B. et al. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63, 673–686 (1990). First demonstration that susceptibility to prion disease is modulated by the sequence of the host PrP. Büeler, H. et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577–582 (1992). Büeler, H. et al. Mice devoid of PrP are resistant to scrapie. Cell 73, 1339–1347 (1993). Proof that expression of PrP is essential for prion propagation and pathogenesis. Wickner, R. B. et al. Yeast prions act as genes composed of self-propagating protein amyloids. Adv. Protein Chem. 57, 313–334 (2001). A review of the phenomenon of yeast prions by the person who discovered them. Riesner, D. et al. Prions and nucleic acids: search for ‘residual’ nucleic acids and screening for mutations in the PrP gene. Dev. Biol. Stand. 80, 173–181 (1993). Chesebro, B. Prion protein and the transmissible spongiform encephalopathy diseases. Neuron 24, 503–506 (1999). Manuelidis, L. Transmissible encephalopathies: speculations and realities. Viral Immunol. 16, 123–139 (2003). An overly critical assessment of the ‘protein-only’ hypothesis, but worth looking at. 15. Kimberlin, R. H. Scrapie agent: prions or virinos? Nature 297, 107–108 (1982). 16. Griffith, J. S. Self-replication and scrapie. Nature 215, 1043–1044 (1967). First proposal of the ‘protein-only’ hypothesis. 17. Prusiner, S. B. Molecular biology of prion diseases. Science 252, 1515–1522 (1991). 18. Weissmann, C. A ‘unified theory’ of prion propagation. Nature 352, 679–683 (1991). 19. Meyer, R. K. et al. Separation and properties of cellular and scrapie prion proteins. Proc. Natl Acad. Sci. USA 83, 2310–2314 (1986). 20. Safar, J. et al. Eight prion strains have PrPSc molecules with different conformations. Nature Med. 4, 1157–1165 (1998). Provides evidence that different prion strains are associated with different PrP conformations. 21. Peretz, D. et al. Strain-specified relative conformational stability of the scrapie prion protein. Protein Sci. 10, 854–863 (2001). 22. Neary, K., Caughey, B., Ernst, D., Race, R. E. & Chesebro, B. Protease sensitivity and nuclease resistance of the scrapie agent propagated in vitro in neuroblastoma cells. J. Virol. 65, 1031–1034 (1991). 23. McKinley, M. P., Bolton, D. C. & Prusiner, S. B. A proteaseresistant protein is a structural component of the scrapie prion. Cell 35, 57–62 (1983). VOLUME 2 | NOVEMBER 2004 | 8 6 9 REVIEWS 24. Kuczius, T. & Groschup, M. H. Differences in proteinase K resistance and neuronal deposition of abnormal prion proteins characterize bovine spongiform encephalopathy (BSE) and scrapie strains. Mol. Med. 5, 406–418 (1999). 25. Harris, D. A. et al. A transgenic model of a familial prion disease. Arch. Virol. Suppl. 103–112 (2000). 26. Post, K. et al. Rapid acquisition of β-sheet structure in the prion protein prior to multimer formation. Biol. Chem. 379, 1307–1317 (1998). 27. Appel, T. R., Dumpitak, C., Matthiesen, U. & Riesner, D. Prion rods contain an inert polysaccharide scaffold. Biol. Chem. 380, 1295–1306 (1999). 28. Bolton, D. C., Rudelli, R. D., Currie, J. R. & Bendheim, P. E. Copurification of Sp33-37 and scrapie agent from hamster brain prior to detectable histopathology and clinical disease. J. Gen. Virol. 72, 2905–2913 (1991). 29. Weissmann, C. Spongiform encephalopathies. The prion’s progress. Nature 349, 569–571 (1991). 30. Manson, J. C. et al. A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy. EMBO J. 18, 6855–6864 (1999). An interesting example of how a single amino acid change in PrP can affect pathogenesis in prion disease. 31. Lasmezas, C. I. et al. Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science 275, 402–405 (1997). 32. Collinge, J. et al. Transmission of fatal familial insomnia to laboratory animals. Lancet 346, 569–570 (1995). 33. Manuelidis, L., Sklaviadis, T. & Manuelidis, E. E. Evidence suggesting that PrP is not the infectious agent in Creutzfeldt–Jakob disease. EMBO J. 6, 341–347 (1987). 34. Tzaban, S. et al. Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes. Biochemistry 41, 12868–12875 (2002). 35. Tremblay, P. et al. Mutant PrPSc conformers induced by a synthetic peptide and several prion strains. J. Virol. 78, 2088–2099 (2004). 36. Prusiner, S. B. & Scott, M. R. Genetics of prions. Annu. Rev. Genet. 31, 139–175 (1997). 37. Liemann, S. & Glockshuber, R. Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. Biochemistry 38, 3258–3267 (1999). 38. Jarrett, J. T. & Lansbury, P. J. Seeding ‘one-dimensional crystallization’ of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell 73, 1055–1058 (1993). 39. Orgel, L. E. Prion replication and secondary nucleation. Chem. Biol. 3, 413–414 (1996). 40. Vanik, D. L., Surewicz, K. A. & Surewicz, W. K. Molecular basis of barriers for interspecies transmissibility of mammalian prions. Mol. Cell 14, 139–145 (2004). 41. Chernoff, Y. O., Lindquist, S. L., Ono, B., Inge-Vechtomov, S. G. & Liebman, S. W. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science 268, 880–884 (1995). 42. Wickner, R. B. et al. Prions of yeast as heritable amyloidoses. J. Struct. Biol. 130, 310–322 (2000). 43. Enari, M., Flechsig, E. & Weissmann, C. Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. Proc. Natl Acad. Sci. USA 98, 9295–9299 (2001). 44. Montrasio, F. et al. B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice. Proc. Natl Acad. Sci. USA 98, 4034–4037 (2001). 45. Telling, G. C. et al. Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83, 79–90 (1995). 46. Peretz, D. et al. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412, 739–743 (2001). 47. Priola, S. A. & Chesebro, B. A single hamster PrP amino acid blocks conversion to protease-resistant PrP in scrapieinfected mouse neuroblastoma cells. J. Virol. 69, 7754–7758 (1995). 48. Hunter, N. in Prion Diseases (eds Baker, H. F. & Ridley, R. M.) 211–221 (Humana Press, New Jersey, 1996). 49. Perrier, V. et al. Dominant-negative inhibition of prion replication in transgenic mice. Proc. Natl Acad. Sci. USA 23, 23 (2002). 50. Windl, O. et al. Genetic basis of Creutzfeldt–Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic variation in the PRNP gene. Hum. Genet. 98, 259–264 (1996). 870 | NOVEMBER 2004 | VOLUME 2 51. Collinge, J. & Rossor, M. A new variant of prion disease. Lancet 347, 916–917 (1996). 52. Peden, A. H., Head, M. W., Ritchie, D. L., Bell, J. E. & Ironside, J. W. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 364, 527–529 (2004). 53. Race, R. E., Fadness, L. H. & Chesebro, B. Characterization of scrapie infection in mouse neuroblastoma cells. J. Gen. Virol. 68, 1391–1399 (1987). 54. Rubenstein, R., Carp, R. I. & Callahan, S. M. In vitro replication of scrapie agent in a neuronal model: infection of PC12 cells. J. Gen. Virol. 65, 2191–2198 (1984). 55. Schatzl, H. M. et al. A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. J. Virol. 71, 8821–8831 (1997). 56. Vorberg, I., Raines, A., Story, B. & Priola, S. A. Susceptibility of common fibroblast cell lines to transmissible spongiform encephalopathy agents. J. Infect. Dis. 189, 431–439 (2004). 57. Vilette, D. et al. Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein. Proc. Natl Acad. Sci. USA 98, 4055–4059 (2001). 58. Bosque, P. J. & Prusiner, S. B. Cultured cell sublines highly susceptible to prion infection. J. Virol. 74, 4377–4386 (2000). 59. Kloehn, P.-C., Stoltze, l., Flechsig, E., Enari, M. & Weissmann, C. A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions. Proc. Natl Acad. Sci. USA 100, 11666–11671 (2003). 60. Nishida, N. et al. Successful transmission of three mouseadapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein. J. Virol. 74, 320–325 (2000). 61. Fischer, M. et al. Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J. 15, 1255–1264 (1996). 62. Kaneko, K. et al. Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc. Natl Acad. Sci. USA 94, 10069–10074 (1997). 63. Kuczius, T., Haist, I. & Groschup, M. H. Molecular analysis of bovine spongiform encephalopathy and scrapie strain variation. J. Infect. Dis. 178, 693–699 (1998). 64. Kocisko, D. A. et al. Cell-free formation of protease-resistant prion protein. Nature 370, 471–474 (1994). First demonstration that PrPC can be converted to PrPSc in a cell-free system. 65. Saborio, G. P., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813 (2001). 66. Deleault, N. R., Lucassen, R. W. & Supattapone, S. RNA molecules stimulate prion protein conversion. Nature 425, 717–720 (2003). 67. Legname, G. et al. Synthetic mammalian prions. Science 305, 673–376 (2004). 68. Bruce, M. E., Fraser, H., McBride, P. A., Scott, J. R. & Dickinson, A. G. in Prion Diseases of Humans and Animals (eds Prusiner, S. B., Collinge, J., Powell, J. & Anderton, B.) 497–508 (Ellis Horwood, New York, London, 1992). 69. Bessen, R. A. & Marsh, R. F. Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. J. Virol. 66, 2096–2101 (1992). First demonstration that different prion strains are associated with different forms of PrPSc. 70. Telling, G. C. et al. Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science 274, 2079–2082 (1996). 71. Collinge, J., Sidle, K. C., Meads, J., Ironside, J. & Hill, A. F. Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 383, 685–690 (1996). Provides biochemical evidence that the agents causing BSE and vCJD are related. 72. Safar, J. G. et al. Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice. Nature Biotechnol. 20, 1147–1150 (2002). 73. Bellon, A. et al. Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity. J. Gen. Virol. 84, 1921–1925 (2003). 74. Caughey, B. et al. Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems. An update. Mol. Biotechnol. 13, 45–55 (1999). 75. Tanaka, M., Chien, P., Naber, N., Cooke, R. & Weissman, J. S. Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323–8. (2004). 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. Elegant demonstration that two different fibrillar conformations of a yeast protein generated in vitro are propagated unchanged in yeast and underlie two different phenotypic strains. See also REF. 76. King, C. Y. & Diaz-Avalos, R. Protein-only transmission of three yeast prion strains. Nature 428, 319–323 (2004). Kimberlin, R. H., Cole, S. & Walker, C. A. Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters. J. Gen. Virol. 68, 1875–1881 (1987). DeArmond, S. J. et al. Selective neuronal targeting in prion disease. Neuron 19, 1337–1348 (1997). Miller, M. W., Williams, E. S., Hobbs, N. T. & Wolfe, L. L. Environmental sources of prion transmission in mule deer. Emerg. Infect. Dis. 10, 1003–1006 (2004). Pattison, I. H. in NINDB Monograph No. 2, Slow, Latent and Temperate Virus Infections (eds Gajdusek, D. C., Gibbs, C. J. & Alpers, M.) 249–257 (1965). Hill, A. F. et al. Species-barrier-independent prion replication in apparently resistant species. Proc. Natl Acad. Sci. USA 97, 10248–10253 (2000). Race, R., Raines, A., Raymond, G. J., Caughey, B. & Chesebro, B. Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt–Jakob disease in humans. J. Virol. 75, 10106–10112 (2001). Asante, E. A. et al. BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein. EMBO J. 21, 6358–6366 (2002). Scott, M. R. et al. Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice. Proc. Natl Acad. Sci. USA 94, 14279–14284 (1997). Lloyd, S. E. et al. Identification of multiple quantitative trait loci linked to prion disease incubation period in mice. Proc. Natl Acad. Sci. USA 98, 6279–6283 (2001). Stephenson, D. A. et al. Quantitative trait loci affecting prion incubation time in mice. Genomics 69, 47–53 (2000). Moreno, C. R., Lantier, F., Lantier, I., Sarradin, P. & Elsen, J. M. Detection of new quantitative trait loci for susceptibility to transmissible spongiform encephalopathies in mice. Genetics 165, 2085–2091 (2003). Manolakou, K. et al. Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice. Proc. Natl Acad. Sci. USA 98, 7402–7407 (2001). Wickner, R. B. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 264, 566–569 (1994). The seminal paper linking extra-chromosomal inheritance in yeast with the self-propagating conformational variant of a protein. Wickner, R. B., Edskes, H. K., Roberts, B. T., Pierce, M. & Baxa, U. Prions of yeast as epigenetic phenomena: high protein ‘copy number’ inducing protein ‘silencing’. Adv. Genet. 46, 485–525 (2002). Glover, J. R. et al. Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell 89, 811–819 (1997). Support for the seeding hypothesis. Blättler, T. et al. PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389, 69–73 (1997). Shows that PrP is required not only for susceptibility to prion infection but also for prion transport through the organism. Brandner, S. et al. Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system. Proc. Natl Acad. Sci. USA 93, 13148–13151 (1996). Huang, F. P., Farquhar, C. F., Mabbott, N. A., Bruce, M. E. & MacPherson, G. G. Migrating intestinal dendritic cells transport PrPSc from the gut. J. Gen. Virol. 83, 267–271 (2002). Klein, M. A. et al. PrP expression in B lymphocytes is not required for prion neuroinvasion. Nature Med. 4, 1429–1433 (1998). Kitamoto, T., Muramoto, T., Mohri, S., Dohura, K. & Tateishi, J. Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with Creutzfeldt–Jakob disease. J. Virol. 65, 6292–6295 (1991). Mackay, F. & Browning, J. L. Turning off follicular dendritic cells. Nature 395, 26–27 (1998). Montrasio, F. et al. Impaired prion replication in spleens of mice lacking functional follicular dendritic cells. Science 288, 1257–1259 (2000). Mabbott, N. A., Mackay, F., Minns, F. & Bruce, M. E. Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie. Nature Med. 6, 719–720 (2000). www.nature.com/reviews/micro REVIEWS 100. Mabbott, N. A., Young, J., McConnell, I. & Bruce, M. E. Follicular dendritic cell dedifferentiation by treatment with an inhibitor of the lymphotoxin pathway dramatically reduces scrapie susceptibility. J. Virol. 77, 6845–6854 (2003). 101. Glatzel, M., Heppner, F. L., Albers, K. M. & Aguzzi, A. Sympathetic innervation of lymphoreticular organs is rate limiting for prion neuroinvasion. Neuron 31, 25–34 (2001). 102. Race, R., Oldstone, M. & Chesebro, B. Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration: role of prion protein expression in peripheral nerves and spleen. J. Virol. 74, 828–833 (2000). 103. Mallucci, G. R. et al. Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration. EMBO J. 21, 202–210 (2002). 104. Büeler, H. et al. High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene. Mol. Med. 1, 19–30 (1994). 105. Brandner, S. et al. Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379, 339–343 (1996). 106. Mallucci, G. et al. Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 302, 871–874 (2003). 107. Ma, J., Wollmann, R. & Lindquist, S. Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science, 1781–1785 (2002). 108. Ma, J. & Lindquist, S. Conversion of PrP to a selfperpetuating PrPSc-like conformation in the cytosol. Science 298, 1785–1788 (2002). 109. Drisaldi, B. et al. Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. J. Biol. Chem. 278, 21732–21743 (2003). NATURE REVIEWS | MICROBIOLOGY 110. Heller, U., Winklhofer, K. F., Heske, J., Reintjes, A. & Tatzelt, J. Post-translational import of the prion protein into the endoplasmic reticulum interferes with cell viability: a critical role for the putative transmembrane domain. J. Biol. Chem. 278, 36139–36147 (2003). 111. Roucou, X., Guo, Q., Zhang, Y., Goodyer, C. G. & LeBlanc, A. C. Cytosolic prion protein is not toxic and protects against Bax-mediated cell death in human primary neurons. J. Biol. Chem. 278, 40877–40881 (2003). 112. Hegde, R. S. et al. Transmissible and genetic prion diseases share a common pathway of neurodegeneration. Nature 402, 822–826 (1999). 113. Hegde, R. S. et al. A transmembrane form of the prion protein in neurodegenerative disease. Science 279, 827–834 (1998). 114. Carrell, R. W. & Lomas, D. A. Conformational disease. Lancet 350, 134–138 (1997). 115. Lansbury, P. T. Jr. Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis and disease. Proc. Natl Acad. Sci. USA 96, 3342–3324 (1999). 116. Flechsig, E., Manson, J. C., Barron, R., Aguzzi, A. & Weissmann, C. in Prion Biology and Diseases (ed. Prusiner, S. B.) 373–434 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2004). 117. Tateishi, J., Kitamoto, T., Hoque, M. Z. & Furukawa, H. Experimental transmission of Creutzfeldt–Jakob disease and related diseases to rodents. Neurology 46, 532–537 (1996). 118. Chiesa, R. et al. Molecular distinction between pathogenic and infectious properties of the prion protein. J. Virol. 77, 7611–7622 (2003). Discusses the difference between a PrP proteinopathy and prion disease. 119. Goldfarb, L. G. et al. Transmissible familial Creutzfeldt–Jakob disease associated with five, seven, and eight extra octapeptide coding repeats in the Prnp gene. Proc. Natl Acad. Sci. USA 88, 10926–10930 (1991). 120. Tateishi, J. & Kitamoto, T. Inherited prion diseases and transmission to rodents. Brain Pathol. 5, 53–59 (1995). 121. True, H. L. & Lindquist, S. L. A yeast prion provides a mechanism for genetic variation and phenotypic diversity. Nature 407, 477–483 (2000). 122. Chiti, F. et al. Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc. Natl Acad. Sci. USA 96, 3590–3594 (1999). 123. Weissmann, C. Molecular biology of prion diseases. Trends Cell Biol. 4, 10–14 (1994). 124. Weissmann, C., Enari, M., Klohn, P. C., Rossi, D. & Flechsig, E. Transmission of prions. Proc. Natl Acad. Sci. USA 14, 14 (2002). Competing interests statement The author declares no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Infectious Disease Information: http://www.cdc.gov/ncidod/diseases/index.htm Bovine spongiform encephalopathy OMIM: http://www.ncbi.nlm.nih.gov/Omim/ Creutzfeldt–Jacob disease | kuru Access to this links box is available online. VOLUME 2 | NOVEMBER 2004 | 8 7 1