Lecture 34: Prions. PRION Prion diseases can occur through: Infectious

Lecture 34: Prions.
PRION = Proteinaceous Infectious particle
Prion diseases can occur through:
1. Infection: Infectious
2. As a dominantly inherited genetic disorder: Familial
3. Consequent to a spontaneous mutation: Sporadic
Mammalian Prion diseases.
Characterized as Spongiform
Invariably fatal.
Characterized by amyloid plaques or spongy
(spongiform) appearance of the affected
areas of the brain, due to accumulation of
vesicular structures in the brains of
infected organisms.
Vacuoles are seen in one neuron
and in the neuropiles. Astrocytes
with small nucleus proliferate. No
inflammatory cells infiltrae in the brain.
•For many years, prion diseases were thought to be caused
by slow-acting viruses, and were referred to as ‘slow virus
diseases’, ‘transmissible spongiform encephalopathies’, or
‘unconventional viral diseases’.
•First discovered by showing that injection of a brain
extract from a patient into a chimpanzee could result in
infection. Incubation times to clinical disease are long
•Inherited forms tend to appear in mid-life.
Human Prion Diseases
Human prion disease should be considered in any patient who develops a
progressive subacute or chronic decline in cognitive or motor function. Typically
adults between 40 and 80 years of age.
Creutzfeldt-Jakob disease (CJD).
Characterized by a progressive dementia
• iCJD (infectious)
– injection of brain matter from a CJD patient into chimps suggested that CJD was a
result of an infectious agent.
– Patients have been infected iatrogenically from injections of human growth hormone
derived from human pituitary gland extracts.
fCJD: (familial).
sCJD (spontaneous)
– Dominantly inherited trait, i.e. heterozygous individuals develop fCJD.
– Penetrance is 100%, i.e. if the carriers live long enough, they will all eventually
develop prion disease.
– Inherited forms have been demonstrated in a number of families. Genealogic
investigations from 4 southern English families show that they are related and argue
for the existence of a single founder born more than 200 years ago.
– Lybian jews. Had been thought to be due to the consumption of lightly cooked/raw
sheep brain and eyeballs. Later shown to be due to a specific mutation.
– Similar mutation found in people originating from Orava in North Central Slovakia, in a
cluster of families from Chile, and a large German family living in the US.
– CJD can occur in the absence of a familial history or infection. Can be due to a
spontaneous mutation on the PrP gene, or even in the presence of only wild-type genes:
a hint into the nature of the process.
Human Prion Diseases
• Gerstmann-Steussler-Scheinker disease (GSS)
– The first of the spongiform encephalopathies that was
described as a genetically inherited trait.
– Linkage analysis from GSS families demonstrated that
PrP was the responsible gene.
– GSS may also arise spontaneously.
• Fatal Familial Insommnia
– FFI: characterized by adults generally over age 50 who
present with a progressive sleep disorder who die within
about a year of onset.
– More than 30 families worldwide have been identified.
• FSI (fatal sporadic insomnia). The sporadic form
of FFI.
• Kuru
– Affects the Fore people of New Guniea. Transmitted
through ritual cannabalism.
Regional distribution of PrPSc in transgenic mice inoculated with
brain extracts from humans who died of prion disease
Mouse inoculated with
brain extract from FFI
Mouse inoculated with
brain extract from fCJD
(E200K) patient
Ventral posterior lateral
thalamic nucleus
Animal Prion Diseases
• The classic spongiform encephalopathy of sheep
• Characterized by progressive loss of motor control
• Propensity of infected animals to obsessively rub or scrape
themselves against things (fenceposts, sides of enclosures,
etc) to the point of scraping off all their hair and rubbing
their skin raw.
• Horizontally transmissible through the herd.
Animal Prion Diseases
Mad Cow disease (Bovine Spongiform
Encephalopathy or BSE)
• Characterized by progressive loss of motor control.
• Transmissible.
• Long incubation period is dose dependent: 2-10 years with
a mean period of 5 years.
• Route of infection has been linked infective sheep and
cattle tissue in meat and bone meal, a component of feed
for cattle and other domestic livestock.
• A change in the traditional feed-rendering process was
linked to the rise in BSE. The traditional includes an
extraction step with organic solvents, which is thought to
have extracted out the prion protein. In Great Britian in
the early 1980's a new cost effective method was
implemented that omitted the organic extraction step.
The result was the BSE epidemic of the mid- to late-80's
(thanks to Maggie Thacher).
Animal Prion Diseases
BSE (Con’t)
• Following the introduction of the ruminant feed
ban in July1988, and the wholesale slaughter of
infected herds, BSE is declining in Great Britian.
• Small epidemic in France linked to human growth
hormone derived from cadavers
• BSE cow found in Canada in 2003
• BSE cow found in USA in 2004 – came from
Canadian herd.
Risks to human health
• New variant of CJD (vCJD) began to appear in
humans 8 years after the first known case of BSE
in cattle. Although there have only been 21
confirmed cases of vCJD to date, the young ages
of the patients and the lack of mutant PrP alleles
suggests that they developed disease via an
infectious route.
Animal Prion Diseases
Other mammalian prion diseases
• Transmissible Mink Encephalopathy (TME): Mink
are carnivores, raised on farms, infection results
from being fed prion-containing meat and bone
• Feline Spongiform Encephalopathy (FSE):
Infection results from being fed prion-containing
• Exotic Ungulate Encephalopathy (EUE): occurs in
the greater kudu, nyala, oryx, i.e. ungulates housed
in zoos. Infection results from being fed prioncontaining meat and bone meal.
• Chronic Wasting Disease (CWD). Occurs in mule
deer and elk. Unknown etiology, perhaps due to
contact with infected farm animals. Concern in
Wyoming that it will spread to cattle.
Etiology, molecular biology & biochemistry
The Prion Protein (PrP).
•Discovery of the prion protein used a mouse scrapie
•Pruisner and co-workers infected mice by
intracerebellar injection with brain extracts from
Scrapie infected sheep.
•Provided a bioassay that could be used to identify the
prion-causing agent.
•The infectious fraction is:
•Nuclease resistant.
•Resistant to UV irradiation at 254 nm.
•Sensitive to proteases. Suggests that the agent is a
•Strongly suggests that the agent is not a nucleic acid.
The Prion Protein (PrP).
Amyloid plaque
stained with Congo
Bifefringence under
polarized light
The Prion Protein
•Infectious agent purified to a single protein species
•Named the Prion Protein (PrP).
•Infectious dose: ~105 PrPSc molecules correspond to one
ID50 unit of prions.
•This leaves open the formal possibility of some nucleic
acid contamination, and that the infectious agent is a
small protein-associated nucleic acid.
•No difference in the quantity of PrP between normal and
diseased brains.
•Constitutively expressed in brains of adult animals.
•Highly regulated during development.
•Cellular functions
•Long term survival of Purkinje neurons??
•Circadian activity rhythms and patterns??
Molecular characterization of the PrP
• Protein sequence analysis was used to clone
the PrP gene.
• Highly conserved gene among all
Prion protein gene structure.
• PrP is encoded by a single open reading
frame (ORF) encoding 253 amino acids in
all known mammalian and avian PrP genes.
Rules out the possibility that the infective
form arises from alternative RNA splicing.
Molecular characterization of the PrP gene.
Alignment of the 44 known PrP sequences shows a
striking degree of consevation between the
mammalian sequences, suggestion the retention of
an important function through evolution.
• However, PrP-null mice are fine. Therefore, PrP is
not an essential protein.
PrP protein structure
• PrP post-translationally processed to remove:
– a 22 amino acid NH2-terminal signal peptide
• at the COOH-terminus, 23 residues are removed
during the addition of a glycophosphatidyl
inositol (GPI) moiety that anchors the protein to
the cell membrane.
• NH2-Terminal sequence repeats
• Amino terminal domain of mammalian PrP contains
5 copies of a P(H/Q)GGG(G)WGQ octarepeat
• Insertions of octarepeats have been linked to
Human disease
• Deletions of octarepeats do not appear to cause
disease, however, deletions do not prevent
PrP protein structure
• Conserved Ala-Gly region
• Between A113 to Y128 is a highly conserved Glyand Ala-rich region.
• Structurally, the sequence appears to have been
selected for its properties of flexibility, and
perhaps for the ability to undergo conformational
• Closest protein sequence is spider dragline silk, a
highly β-sheet rich protein that is capable of
forming filamentous polymers.
• A single point mutation A117V is linked to GSS.
• Two β-sheet (S12 and S2) and three α-helix (A, B &
C) forming regions are conserved
PrP protein structure
Human mutations
•extra copies of the
•Homozygosity for
•A117V (the
polymorphism that
was used to
demonstrate the
inherited nature of
GSS and prion
•Requires double
mutation: D178N +
Structural studies
• PrPc is protease-sensitive;
• PrPsc is protease-resistant
• PrPc is α-helix rich: 40% a-helix, little β-sheet;
• PrPsc is β-sheet rich: 30% a-helix, 45% β-sheet
• No differences in postranslational chemical
modifications were found between the two forms
of the protein.
• Rules out this as a possible cause of prion
Structural studies
•PrPc is soluble
•PrPsc forms insoluble filaments
45,000X magnification electron micrograph
of yeast prion protein
fibers formed in the test tube. The rigid
fibers are similar to those
observed in amyloid diseases of mammals.
Allosteric model of Prion
Prion form is “enciphered” thru protein conformation
Subunit interaction model for
prion formation
Crystal seed model
Propagation of the prion form of
the proteins
Prion formation in vitro (yeast
prion system)
Prion formation in vitro (Human prion system)
A major technical hurdle was that nobody had been able to establish an
in vitro prion propagation system using PrP. This was accomplished
(Jackson, G.S.. et al., 1998. Science 283: 1935 – 37).
Overcoming the species
barrier…implications for hBSE
Advantages of the prion model
•Self-encoded protein explains the lack of lymphocytic infiltration
•Epidemiology is best explained by the prion model:
•Spontaneous forms are due to the rare, stochastic conversion of
native, wild-type protein to prion form.
•Inherited form is due to the PrP gene mutations that make the
conformational conversion more energetically favorable.
•Infectious because infectious form also makes the conformational
conversion of endogenous PrPc more energetically favorable.
•Incubation times. Once the species barrier has been crossed, of the
host species PrPc to PrPsc becomes more energetically favorable.
•Strain differences, e.g. FFI vs CJD, and deposition patterns are
explained by the prion hypothesis.
Problems with the prion model
•Cannot make or isolate completely
pure PrPsc .
•Therefore, the argument can be
made that infectious innoculum
contains a small amount of an
infectious nucleic acid.
Q: Does requirement for transmissability limit our
conceptual understanding of prion disease?
By the allosteric model, any protein that can template a
conformational change should also fit the definition.
Opens up new possibilities, e.g.
Altzheimers, Sickle Cell disease
Early-Onset Familial Alzheimer Disease With Coexisting-Amyloid and Prion Pathology (JAMA.
Figure. Double Immunostaining for -Amyloid and Prion Protein (PrP) in the Frontal Cortex
Senile plaques immunopositive for -amyloid40 (arrowheads in panels A and C), PrP106-126(arrowheads in
B), and for -amyloid40 plus PrP106-126 (double arrowheads in C) are shown. Twodifferent chromogens
were used, first diaminobenzidine dihydrochloride to reveal the -amyloid peptide (reddish-brown) and
second, benzidine dihydrochloride to reveal the PrP106-126 (blue). When both signals are superposed,
the blue signal appears dark and is localized in the center of the plaque.
Scale bar is 50 µm for panels A and B, and 75 µm for panel C.
Fundamental significance of
• Molecular signals that induce host polymerase to accept
viroids as templates for
transcription (missing from cellular RNAs?)
• Molecular mechanisms of viroid replication (operative in
uninfected cells?)
• How do viroids induce disease?
• Why are viroids restricted to higher plants?
• How did viroids originate?
T.O. Diener (1987)
First report of viroid disease
- Schultz and Folsom (1923)
Viroid disease symptoms
Potato spindle tuber viroid
stunt viroid
Apple viroid disease
Sunblotch Viroid
Coconut cadang-cadang viroid
•Smallest known agents of
infectious disease
•Discovered by T.O. Diener
•Small, covalently closed circular,
single stranded RNAs
•No protein coding capacity (246399 nt)
•Autonomous replication (no
helper virus)
•29 species (8 genera, 2 families)
Viroid classification
• Host range may be broad
(HSVd) or narrow (CCCVd)
• Mechanically transmissable
• Not insect transmitted
• Most important vector = man
Viroid pathogenicity
Host contribution
• Induction of “pathogenesis related” proteins
• Protein kinases / signaling cascades
Viroid contribution
• Role of pathogenicity domain
• Induction of RNA silencing
Viroid Movement, i.e.
spread within infected plant
• Nuclear / chloroplast import
• Cell-to-cell movement
• Long distance via phloem
Movement of viroids and viruses
Infected leaf
Source-to-sink movement
Moves from infected leaf to roots, up
phloem to actively photosynthesizing
leaves, then back down the plant
Developmental controls on movement
as well
Origin of viroids
Three pieces of evidence that viroids are relics of a precellular RNA world:
•Structural periodicity / polyploid genomes: Suggests that they are constructed
from very ancient functional RNA domains.
• Minimal ribozymes / genomic tags: represent ancient enzymes
• Viroids / introns / transposons: all ancient and phylogenetically related to one
Phylogeny of subviral RNAs:
They’re genetically related to one another
HDV: hepatitis delta virus
Plant Satellite
100 nt
Elena et al. 1991. Proc. Natl. Acad. Sci. USA 88, 5631-5634.
Hepatitis delta
virus: a human
• Rod-like secondary structure
• Replicates in nucleus
• RNA polymerase II
• Ribozyme-mediated cleavage
• Protein coding region /RNA editing
Some interesting musings…
•Viroids can only be propagated
•Cannot be propagated vertically, do not make it
through meiosis.
•Origins of sex?
•Implications for animal cloning?