Robert A. Somerville
BBsrc & MRC Neuropathogenesis Unit, Institute for Animal Health, Edinburgh.
David C. Bolton, Ph.D.
Department of Molecular Biology, New York State Institute for Basic Research
The nature of the diseases
"Scrapie" is the old Scottish shepherds' name for a disease of their sheep
which has been known for several centuries. It is the original example of a
group of diseases, known as the "transmissible spongiform encephalopathies"
(TSE), sometimes known as the "prion" diseases. The diseases include
Creutzfeldt Jakob disease (CJD) in humans and bovine spongiform encephalopathy
(BSE) or "mad cow disease." They affect the brain, disrupting or destroying
neurons in large numbers, which inevitably leads to the death of the infected
In many respects the diseases are unusual. For example there is a long period,
the incubation period, after an animal is infected before signs of the disease
can be detected. The incubation period is controlled by a gene of the infected
animal which makes a protein called PrP. There is no apparent reaction to
infection in the animal - no immune response. And most notably, the cause of
the disease, the "infective agent" has unusual properties. These unusual
properties have prompted much speculation and debate about what the "infective
agent" is, and how it works. Despite its unusual properties, is it like other
infectious particles, (i.e. viruses), is it a bit different from other
infectious agents, or is it completely different from any other infectious
particle, even perhaps have biological properties which have never been
described before? This question has been debated for three decades and still
has not been resolved.
The debate revolves around two issues: Firstly, is a nucleic acid (DNA or RNA)
a part of the agent which determines what the agent does. If there is one, why
can we not find it? If there is no nucleic acid, how are agent properties
specified? Secondly, PrP (sometimes called prion protein) , is associated with
the agent somehow - but what does it do? This debate matters because no life
form, including any virus, has been found which does not have nucleic acid as
the molecule which encodes the chemical information for its existence. There is
no model which we can use to compare the T.S.E. agent, if it does not have a
Do "prions" exist? The word "prion" is used in different ways. It is used to
describe the TSE group of diseases but it is also associated with the
"protein-only" hypothesis discussed below. I find it helpful not to use the
word prion to describe the diseases, to avoid confusion with the hypothesis.
The word is also used to describe the infective agent and I think nowadays it
is generally accepted that by "prion" scientists mean a PrP-protein-only agent,
without nucleic acid or other molecule encoding the agent's information. Until
we know better what the structure of the agent is and whether it has a nucleic
acid or not, I find it helpful to use the word "agent". Until a nucleic acid is
found or a mechanism for protein-only replication demonstrated, prion is most
usefully used to describe the protein-only hypothesis.
The nucleic acid challenge: arguments for and against a nucleic acid agent.
So far nobody has found a nucleic acid which could be part of the agent. Also,
several experiments have been performed to look indirectly for evidence that a
nucleic acid is present. Infective material has been treated with chemicals,
enzymes, or with radiation, all of shich which should destroy nucleic acids. If
there were a nucleic acid, these treatments should also destroy the
infectivity. Most of these treatments don't affect infectivity, or do so with
great difficulty. But what do these experiments mean? Some say that there is no
evidence from these experiments that there is a nucleic acid; we need to forget
about nucleic acid as an explanation of the properties of the agent and look
for another explanation. But others argue that it is too soon to draw this
conclusion. We can't find the nucleic acid - not because it isn't there - but
because we don't have the right techniques for finding it. Maybe there is very
little of it to find. Maybe it has some unusual feature that hides it from us.
Perhaps we can't destroy the nucleic acid in infective material because it is
protected from the chemicals and enzymes.
A further argument comes from the different kinds or "strains" of TSEs
themselves. For many years, scrapie from different sheep breeds and TSE
diseases from other animals, e.g. scrapie from goats and BSE from cattle, have
been used to infect laboratory mice. In these mice the biological
characteristics of different sources of the disease can be compared. In
particular the incubation periods and the places where the disease has affected
different parts of the brain can be measured. The length of the incubation
period and the distribution and amount of lesions in the brain act as a
fingerprint for each strain. Fifteen different strains have been shown to exist
by these methods, and several other strains have been indicated in other
experiments. Somehow the TSE agents must encode the differences between the
strains and also determine how each strain interacts with the host animal, in
particular with PrP to define how long the incubation period is going to be and
which parts of the brain will be infected. A lot of information is required for
these functions which we know could be carried by a nucleic acid.
It is possible that another biological molecule could carry this information,
but if so, it too has yet to be identified. We do not know of any other
molecule or any other biological system which could do so. Whatever the
information—carrying molecule is made of, and it is presumably nucleic acid,
it will require protection which could be provided by a host protein, PrP. This
is the essence of the "virino" hypothesis.
To summarize - a lot of information is needed for the infective agent to be
capable of causing a TSE disease. A nucleic acid is the only biological
molecule we know about which could encode all this information. However we have
not discovered it yet, perhaps because we scientists have not yet come up with
the right experiments to find it. A nucleic acid encoding its protective
protein would make an infective agent similar to other viruses. An
informational molecule (presumably nucleic acid) protected by a host protein,
PrP, is the prediction of the virino hypothesis.
The prion challenge: arguments for and against a protein-only agent
Perhaps there is no nucleic acid to find and there is a different, unique way
for TSEs to encode their information. Perhaps, as some have argued, we should
abandon the search for a nucleic acid and look instead for a new mechanism by
which the TSEs could cause disease.
An abnormal form of the PrP protein, (called PrPSc) is found when we try to
purify the agent which causes the disease from infected brain. It was
originally discovered by David Bolton and his colleagues. PrP is also a normal
host protein, in other words it is found in uninfected brains and in other
tissues. In TSE-infected brains PrP changes its biochemical properties which
allows us to distinguish between the two forms. It becomes more difficult to
destroy with enzymes that digest proteins (proteases) and it will sediment in
an ultracentrifuge when spun very fast, although the normal protein continues
to float in solution. This protein probably has something to do with the
infective agent, but what and how is again disputed.
PrP seems to be involved in the disease in three ways. 1. Using special
techniques, the abnormal form can be seen in infected brain as deposits of the
protein. In other words, as a consequence of the disease it becomes a
"pathological product of infection." 2. The protein controls how long the
incubation period of different TSE agent strains will be. Changing the
structure of PrP alters the incubation period or even whether or not the animal
becomes infected. 3. PrP is probably part of the infective agent. If there is a
nucleic acid, the role of PrP would be to protect the nucleic acid from being
destroyed. If there is no nucleic acid, as in the protein-only or prion
hypothesis, the role of PrP would be to encode the information needed for all
the different TSE strains.
How might PrP carry the information? There are several ideas. Originally it
was thought that it might be self-replicating; like nucleic acids it would
determine the order of its own building blocks, i.e. its amino acid sequence.
However it was soon shown that the host animal made PrP, which in infected
animals somehow changed into the abnormal form. In some cases there are
mutations in PrP (differences in its amino acid sequence) which are associated
with disease. However, these are very rare and in most TSEs there is no
mutation of PrP. There is a change in the conformation (or shape) of PrP in the
abnormal form, and there seem to be some differences in the conformations found
between some TSE strains. Perhaps abnormal PrP can act as a "template" and
induce more normal PrP to take up the altered conformations. This has been done
in the test tube. However the big question is whether more infectivity can be
made in the test tube to show that altering PrP makes it infectious. Some
scientific groups are trying to design this experiment at the moment but there
are several technical problems to be overcome before the experiment can be
If abnormal PrP is the sole cause of TSEs, we need to know how it works. There
are many ideas for a mechanism but they all require careful testing to see how
they may be possible. Unless and until we have a mechanism in which PrP acts as
the infectious agent and encodes all the information needed to encode the
different TSE strains, there must be some doubt about the "protein-only
hypothesis", i.e. that the TSE agent does not have a nucleic acid.
Overall I think the existing data about T.S.E. diseases and their causal agents
are best explained by proposing that a nucleic acid or some other informational
molecule does carry all the information required by their agents. The prion
hypothesis does not adequately address the question of how all this information
can be encoded. Although a nucleic acid has not been found, nor is their direct
evidence that one exists, this situation can be explained by conventional
biology. The nucleic acid could be a part of a virus, or an informational
molecule, presumably nucleic acid, could be protected by PrP, a virino. Much
information is required by the different T.S.E. strains. A nucleic acid is the
only type of molecule which we know could carry such information.
What are prions? The name prion was coined in 1981 by Dr. Stanley Prusiner to
identify the agents that cause a novel type of fatal brain diseases. Bovine
spongiform encephalopathy (BSE or mad cow disease), sheep scrapie and
Creutzfeldt-Jakob disease (CJD) of humans are examples of prion diseases. In
this essay, I will use the term prion instead of agent to denote the infectious
particle that causes these diseases. The "protein-only" hypothesis is a
controversial hypothesis that describes what prions are and how they reproduce.
The discussion that follows is necessarily limited to only the most important
evidence in support of the protein-only hypothesis due to space limitations.
What is the protein-only hypothesis?
There are three main features of the protein-only hypothesis. The first is
that the active component in prions is an abnormal protein called prion protein
(abbreviated PrP). Normal animal cells make a form of PrP that is called
cellular PrP (abbreviated PrPC). Animals infected with prions make abnormal
PrP. In scrapie, abnormal PrP is called PrPSc.
The abnormal protein itself directs the conversion of the normal host protein
to the abnormal form. In other words, PrPSc converts PrPC into PrPSc.
Prions do not contain a nucleic acid genome
J.S. Griffith first proposed the protein-only theory in 1967 to explain how
prions could replicate if they were made of protein but did not contain nucleic
acids. He did this fifteen years before the discovery of PrPSc and PrPC. Many
have called the theory heretical because it describes replication of a
pathogenic agent without a nucleic acid genome. (Genes are made of nucleic
acids. The nucleic acids store and transmit genetic information in all known
organisms.) In fact, the hypothesis is based upon known properties of proteins
with the added wrinkle that a protein molecule folded in an abnormal way can
alter the folding of another protein molecule and thereby change its biological
properties. To quote from J.S. Griffith's 1967 paper, "the occurrence of a
protein agent would not necessarily be embarrassing although it would be most
Disproving (not proving) hypotheses
People often assume that scientists are in the business of trying to prove
hypotheses or theories. This assumption is incorrect because hypotheses can
never be proved; they can only be disproved. A hypothesis that fails one or
more tests is considered disproved and it is discarded. If it is not disproved
after being tested in many different ways, we become more confident that it is
correct. A hypothesis is valid as long as it explains the behavior of the
system it describes, but it is always possible that it will have to be revised
or discarded based on new results.
Proteins are chains of chemicals called amino acids linked together like beads
on a string. There are 20 different amino acids (imagine 20 differently
colored beads) and each amino acid has a different chemical behavior. The
prion protein has about 250 amino acids. The amino acid string does not remain
linear once it is made, however, and the properties of the different amino
acids make the protein fold into a specific shape or conformation. The
conformation of a protein determines its function. Different amino acid
sequences produce proteins with different conformations and functions. Genes
determine the sequence of amino acids in a protein. Changes in the gene
(mutations) can change the amino acid sequence of the protein and alter its
conformation and function.
The prion protein (PrPSc) fulfills all the necessary criteria to be the active
component of the infectious particle. First, infectious prions isolated from
brain tissue contain PrPSc. A process called purification removes molecules
that are not part of the prion. The purity of a prion preparation is judged by
how much infectivity is present for each gram of protein or nucleic acid.
PrPSc is the only protein found in the best-purified preparations. Scientists
have looked in these preparations for specific nucleic acids (e.g., virus
genes) but have not found one despite searching for more than 30 years. Thus,
the only molecule identified in the infectious particle is PrPSc. PrPSc is
involved in all known prion diseases. In some cases, PrPSc molecules have a
normal sequence but an abnormal conformation. In other cases, a change in the
PrP gene sequence (mutation) causes PrP to fold incorrectly.
All mammals appear to have prion protein genes and the gene sequences are
similar, but not identical, in related species. Differences in the PrP amino
acid sequence play an important role in determining whether prions from one
species can infect hosts of another species. This behavior is difficult to
explain if prions are not made of prion protein.
PrPSc molecules can bind to PrPC molecules in the test tube and convert them to
the abnormal (PrPSc) conformation. The sequences of the PrPSc and PrPC
molecules must be similar for the conversion to work, and thus the behavior of
the PrP molecules in the test tube parallels the behavior of prions in nature.
Sometimes prions from different cases of prion disease vary in the way they
affect the brain, giving rise to different prion strains. The variation in
strain behavior correlates with differences in the conformation of their PrP
molecules. Prions isolated from certain new cases of CJD in the United Kingdom
that are thought to be caused by BSE prions show unique strain characteristics.
Those prions have a PrP conformation that is similar to that of the PrP
molecule from BSE prions, but different from that in conventional CJD prions or
Adding, changing or inactivating PrP genes in normal mice creates genetically
engineered mice called transgenic mice. Hamster prions can not infect normal
mice but they can infect transgenic mice that have copies of the hamster PrP
gene. Infecting the transgenic mice with hamster prions produces new prions
that contain hamster PrPSc and can infect hamsters. Conversely, the transgenic
mice produce only mouse prions and mouse PrPSc when infected with mouse prions.
Those results show that prions prefer to convert PrP molecules that have the
same sequence. This is consistent with the protein-only hypothesis but is
difficult to explain if prions are not composed of PrP.
Other mice were given extra copies of a hybrid (or chimeric) PrP gene that had
portions of the hamster amino acid sequence and portions of the mouse amino
acid sequence. Transgenic mice with those genes produced chimeric prions (and
chimeric PrPSc) when infected with either mouse or hamster prions. The
chimeric prions (prions with chimeric PrPSc) could infect both normal mice and
hamsters, but preferentially infected the transgenic hosts having the chimeric
PrP gene. Thus, changing the PrP sequence produced a novel prion that did not
previously exist in nature. Those results are very difficult to explain by
theories other than the protein-only hypothesis.
Another type of transgenic mouse, called a PrP "knockout" mouse has its PrP
gene disrupted or made inactive. PrP knockout mice do not make PrP. These
mice do not become diseased and do not produce prions when exposed to prions
from any known source. When brain cells are transplanted from a normal mouse
into the brain of a PrP knockout mouse, the normal brain cells can be infected,
make prions and become diseased, but the neighboring cells from the PrP
knockout host can not. Thus, the PrP protein is required for prions to
replicate and cause disease.
The protein-only hypothesis remains controversial because it breaks new
conceptual ground. Those who have worked in this field under other paradigms
(like the virus or virino hypotheses) are reluctant to accept this new
paradigm. Scientists from other fields are more receptive to this hypothesis,
however, and thus it has gained broad support. This hypothesis best explains
all of the observations about these agents and the diseases they cause. If at
some point it fails to do so, the hypothesis will need to be revised or
rejected in favor of a better hypothesis. That is the nature of science.