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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.
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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.
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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
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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
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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
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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.
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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
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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.
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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
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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.
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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
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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.
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8.
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Competing interests statement
The author declares no competing financial interests.
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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/
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