Vertebrate Prions

Chapter Version: ICTV Ninth Report; 2009 Taxonomy Release

Prions are proteinaceous infectious particles that lack nucleic acids. In mammals, prions are composed largely, if not entirely, of a pathogenic isoform of the host-encoded cellular prion protein (PrP). Neither prion-specific nucleic acids nor virus-like particles have been detected in highly purified, infectious preparations.

In fungi, evidence for nine different prions has been accumulated. Those studies are summarized in the section on Fungal Prions.

General description of the mammalian prions

The mammalian prions cause scrapie and other related neurodegenerative diseases of animals and humans (Table 1). The prion diseases are also referred to as the transmissible spongiform encephalopathies (TSEs). TSEs are inevitable fatal and no treatment is available for the afflicted species.

Prions are composed of an abnormal, pathogenic PrP isoform, denoted PrP^Sc. The “Sc” superscript was initially derived from the term scrapie, because scrapie was the prototypic prion disease. Since all of the known prion diseases (Table 1) of mammals involve aberrant metabolism of PrP similar to that observed in scrapie, the “Sc” superscript was suggested for all pathogenic PrP isoforms. In this context, the “Sc” superscript is used to designate the scrapie-like isoform of PrP; for those who desire a more general derivation “Sc” can equally well be derived from the term “prion sickness” (see Table 2).

A post-translational process converts the normal cellular isoform of the protein, designated PrP^C, into PrP^Sc. Attempts to identify posttranslational modifications that distinguish PrP^Sc from PrP^C have not been successful; moreover, PrP^C and PrP^Sc are encoded by the same single copy chromosomal gene. The conformations of the two PrP isoforms are profoundly different; PrP^C has little β-sheet while PrP^Sc has a high β-sheet content.

Like viruses, prions are infectious because they stimulate a process by which more of the pathogen is produced. As prions or viruses accumulate in an infected host, they eventually cause disease. Both prions and viruses exist in different varieties or subtypes that are called strains. But many features of prion structure and replication distinguish them from viruses and all other known infectious pathogens.

Prions differ from viruses and viroids since they lack a nucleic acid genome that directs the synthesis of their progeny. Prions are composed of an abnormal isoform of a cellular protein whereas most viral proteins are encoded by the viral genome and viroids are devoid of protein. Prions can exist in multiple molecular forms, whereas viruses exist in a single form with a distinct ultrastructural morphology. Prions are non-immunogenic, in contrast to viruses, which almost always provoke an immune response. An enlarging body of evidence argues that strains of prions are enciphered in the conformation of PrP^Sc; in contrast, strains of viruses and viroids have distinct nucleic acid sequences that produce pathogens with different properties.

Table 1 The prion diseases

Table 2 Glossary of prion terminology

Mammalian prion diseases

Scrapie was reported for the first time 250 years ago in Great Britain and it affects mainly sheep and goats. The most efficient proof for the transmissibility of the disease was demonstrated when more than 1,500 sheep came down after vaccination against looping-ill virus with a formalin-treated vaccine derived from ovine lymphoid tissue unknowingly infected with scrapie. More recently, there has also been a recrudescence of scrapie outbreaks among European sheep flocks. The mechanism on how the disease can be transmitted from one animal to another is still unclear. Chronic inflammation seems to alter the tropism of prion infectivity to organs hitherto believed prion-free, like mammary glands. This raised concerns about scrapie transmission through milk.

The commonest prion disease is the bovine spongiform encephalopathy, more commonly known as “mad cow disease”, which appeared for the first time in Great Britain in 1986. Since then more than 280,000 cattle have died from this disease. The source of the disease was attributed to animal-derived food supplements that were fed as a nutritional supplement to cattle. The disease has become quite rare since 2003 and only 37 cases were reported in the UK in 2008 (http://www.oie.int/).

Another animal prion disease that has evolved in the United States of America is Chronic Wasting Disease (CWD) in captive deer, with the first case observed in 1967 in a research facility in Fort Collins. Since 1980, cases involving free-ranging animals in Colorado and Wyoming have been reported and to date CWD has been observed in 14 states and two Canadian province. The known natural hosts of CWD are mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), Rocky Mountain elk (Cervus elaphus nelsoni) and Shira’s moose (Alces alces Shirasi). The disease is believed to be horizontally transmitted between cervids with high efficiency.

Transmissible mink encephalopathy (TME), feline spongiform encephalopathy (FSE) and exotic ungulate encephalopathy (EUE) are all thought to occur after the consumption of prion-infected materials.

Human prion diseases

The human prion diseases comprise Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker (GSS) syndrome, Fatal Familial Insomnia (FFI) and Kuru. Epidemiologically, CJD is classified as sporadic (sCJD), familial (fCJD), iatrogenic (iCJD) and variant (vCJD). Even the most frequent form, sCJD, is very rare, with an incidence of 0.4–1.8 person in one million people each year worldwide. Typically, humans with an age between 55 and 70 years are affected. The source of the disease is still unknown, but might have its origin in a spontaneous conversion of PrP^C into PrP^Sc as a rare stochastic event or in somatic mutations in the PRNP gene that encodes the prion protein.

Iatrogenic CJD cases are rare. These cases originate from inoculation of prions through human pituitary-derived growth hormone, cornea transplants, duramater grafts, or cerebral electrode implants. vCJD was recently identified in humans and has affected ~200 victims worldwide since 2006. The histopathology of this disease is reminiscent of that of BSE indicating that this disease has originated by the transmission of BSE to humans. Kuru occurred among the people of the Fore Tribe in Papua New Guinea and was horizontally transmitted between humans by cannibalistic rituals. Since this practice was stopped by the government no new cases have been reported in individuals born after that date.

Familial forms of CJD as well as all cases of GSS and FFI are caused by mutations in PRNP.

Nomenclature of the mammalian prions

The terminology for prions is still evolving (Table 2). While some terms are borrowed from infectious diseases caused by viruses, others are taken from genetics and still others from the biology of protein structure as well as neuropathology. This new area of biological investigation, which has such diverse roots, creates some unique problems with terminology.

Prion properties

Physicochemical and physical properties

The mature human prion protein consists of 208 amino acids. The N-terminal signal sequence that targets the protein into the endoplasmic reticulum is cleaved off in the mature form and a C-terminal polypeptide of 23 amino acids is replaced by the addition of a glycosylphospatidylinositol anchor (GPI) to the C-terminal serine residue. The protein also contains a single disulfide bridge which is formed between residues Cys-179 and Cys-214 and two N-linked glycosylation sites at positions Asn-181 and Asn-197 (Figure 1A).

The N-terminal part of the prion protein contains several Gly-Pro-rich tandem repeats, which vary in number, length and composition between species and are flanked by two positively charged clusters of amino acids 23–27 (CC1) and 95–110 (CC2). The center of the protein contains a hydrophobic stretch of amino acids 111–134 with the palindrome sequence AGAAAAGA. This region is highly conserved between species and is even present in fish and another member of the prion protein family named shadoo. This indicates that this region has gained an important, hitherto unknown physiological function during the evolution of the prion protein.

PrP^Sc

Both PrP^Sc and PrP^C are encoded by the same host gene. Although the amino acid sequence of PrP^Sc and PrP^C are identical the proteins differ from each other in their tertiary structure and their biochemical and physicochemical properties. Low and high resolution structural methods showed that PrP^C is rich in α-helical secondary structure. To date, no structural data at the atomic level exist for PrP^Sc, but optical measurements and Fourier-transform infrared spectroscopy showed an increase in β-sheet structure for PrP^Sc when compared to PrP^C.

In view of their physicochemical differences, PrP^C is monomeric and soluble in nondenaturing detergents, whereas PrP^Sc forms oligomers and aggregates that cannot be dissolved in detergents. Another characteristic is that PrP^C is sensitive to Proteinase K digestion, whilst PrP^Sc can only be partially degraded by Proteinase K leading to an N-terminally truncated resistant core with a size of 27–30 kDa. This fragment is termed PrP^27-30. Diglycosylated, GPI-anchored PrP^Sc has a size of 33–35 kDa as judged by its electrophoretic mobility on a SDS-PAGE.

Nucleic acid

No prion-specific nucleic acid has been detected.

Proteins

The three-dimensional structures of recombinant PrP from a wide variety of species have been solved by nuclear magnetic resonance spectroscopy in solution. The overall architecture of the proteins is nearly identical containing an N-terminally flexible tail of residues 23–125 and a globular folded C-terminal domain of residues 126–231. The C-terminal domain is an autonomously folding unit and contains three α-helices and a short antiparallel β-sheet of residues 128–131 and 161–164 (Figure 1B). A comparison of the recombinant protein with PrP^C purified from calf brain showed that the fold of the recombinant prion proteins is identical with the natural cellular protein and that the posttranslational modifications of PrP^C do not affect the fold.

More recently, a special structural feature was identified in the loop β2–α2 between the second β-sheet and the second α-helix showing high conformational variability between species. Whereas the loop β2–α2 is flexible disordered in the prion proteins of most species, it is extremely well-ordered in the prion proteins from elk, bank vole and tammar wallaby.

In addition, crystal structures have been determined for the C-terminal domain in complex with monoclonal antibodies or FAB fragments, indicating a similar fold for PrP^C as obtained by NMR spectroscopy. The only exception represents the crystal structure of a domain-swapped dimer in which the third α-helices are swapped and the disulfide bridge is newly arranged between the two molecules.

Morphology

High resolution structural studies using X-ray crystallography and solution NMR spectroscopy for PrP^Sc are more challenging due to the limited solubility of PrP^Sc. First morphological characterizations for PrP were obtained from rod-shaped particles that were isolated from detergent and proteinase K-treated microsomal fractions which were enriched for prion infectivity. These rod-shaped particles have a width of 10–20 nm and mean lengths of 100–200 nm. The rods are smooth, almost ribbon-like, and infrequently are twisted. The fine structure of these aggregates was originally designated by Mertz as scrapie-associated fibrils (SAF). These partially purified rods show the ultrastructural and tinctorial properties of amyloid, including Thioflavin T positivity and the green–gold birefringence obtained under cross-polarized light after Congo Red staining, indicating the presence of cross β-sheet secondary structure. The purified fractions of the rods also contained 2D crystals, which were used for crystallographic studies and structural models for PrP^Sc based on these data were created.

Several in vitro conversion model systems to obtain PrP fibers from prion proteins produced in Escherichia coli were also used to study their morphological and structural aspects. Amyloid fibers obtained with these assays under physiological conditions appear as long and unbranched fibers in negatively stained electron micrographs (Figure 1C). Low resolution structural data are also available for recombinant PrP and PrP fragments.

Soluble oligomers rather than insoluble fibrils have been hypothesized in other amyloid diseases such as Alzheimer`s disease to be the relevant toxic species and the formation of large aggregates might have a protective effect by sequestering the more toxic oligomers. Solubilization of PrP^27-30 into liposomes indicates that large fibrillar structures are not required for infectivity and that PrP^Sc oligomers are the relevant molecules for infectivity. To investigate the relationship between size and infectivity in prion diseases, purified PrP^res was partially disaggregated to obtain a spectrum of different-sized aggregates. It turned out that particles with a size of 17–27 nm which corresponds to a molecular weight of 300–600 kDa and an association of 14–28 PrP molecules are the most infectious particles. These particles are non-fibrillar and appear as small amorphous ellipsoidal and spherical particles with a diameter of 20–25 nm in transmission electron micrographs.

Lipids

The prion protein is attached by a glycosylphosphatidylinositol anchor (GPI) in cholesterol rich lipid rafts in the outer leaflet of the cell membrane of neurons to its carboxy-terminal serine residue. The glycan core of the GPI-anchor consists of mannose-mannose-mannose-(N-acetyl neuraminic acid-galactose-N-acetylgalactosamine)-mannose-glucosamine-myo-inositol. As commonly found for GPI anchored proteins, this core is attached to the cell membrane by a phospholipid membrane and to the C-terminus of the GPI-anchored protein through a phosphoethanolamine bridge.

PrP^C can be released from the cell surface by digestion with phosphoinositide-specific phospholipase C (PI-PLC), whereas PrP^Sc cannot be released by the enzyme, which indicates that the cleavage site is not accessible due to the conformational rearrangement of PrP^C. The importance of the GPI anchor for disease was recently demonstrated by the generation of transgenic mice that express a PrP construct lacking the GPI anchor. After infection with RML prions these heterozygous mice developed widely spread plaques in the brain, but without developing any clinical signs of disease. In a follow-up study the authors reported that infection of homozygous transgenic mice expressing anchorless PrP with RML induced a new fatal disease with unique clinical signs and altered neuropathology.

Carbohydrates

PrP also contains two highly conserved Asn-Xaa-Ser/Thr sequons for N-linked glycosylation at Asn-181 and Asn-197 (numeration according to human PrP, resulting in di-, mono- and unglycosylated states of the protein. Mass spectrometry analysis of the two N-linked oligosaccharides of PrP^C and PrP^Sc from hamster and mouse showed that the oligosaccharides consist of different neutral and sialylated bi-, tri- and tetra-antennary oligosaccharides with more complex acidic glycan structures at Asn-196. In addition, a higher content of tri- and tetra-antennary glycans and reduced levels of glycans with bisecting GlcNAc residues were identified for PrP^Sc, leading to the suggestion that the activity of N-acetylglucosaminyltransferase III is reduced. No O-linked glycosylation has been identified.

Genome organization and replication

Genome organization

The PrP gene has been identified in many mammalian species, birds, reptiles and amphibians. PrP-like genes have also been identified in fish. The gene encoding the prion protein consists of three exons, from which exon 3 encodes the complete mature protein. The entire ORF of all known mammalian PrP genes is contained within a single exon. The gene encoding the human prion protein is located on chromosome 20 in a single exon, whereas the gene encoding the murine prion protein is located on the systemic chromosome 2.

In general, PrP genes are composed of three exons, as clearly demonstrated for mouse and sheep. The PrP genes of humans and Syrian hamsters (SHa) appear to have three exons but most HuPrP and SHaPrP mRNAs are spliced from only two exons that are separated by ~10 kb. Exon-1 of the SHaPrP gene encodes a portion of the 5′ untranslated leader sequence while exon-3 encodes the ORF and the 3′-UTR. The ORF of the HuPrP gene encodes a protein of 253 amino acids while the mouse and SHaPrP genes encode proteins of 254 residues. The promoters of the PrP genes of both animals contain three or two repeats, respectively, of G–C nonamers, but are devoid of TATA boxes. These nonamers represent a motif which may function as a canonical binding site for transcription factor Sp1.

The sequence similarity between the ORFs encoding different mammalian PrPs is ~90%: 30–50% identities and around 50% similarities are found between mammals, birds, reptiles and amphibians. The sequence homology between fish and tetrapod PrPs is less than 25%.

Replication

Several hypotheses have been described to explain the unusual nature of the infectious prion agent. The most widely discussed hypotheses are (i) the “protein-only” hypothesis, (ii) the virino hypothesis and (iii) the hypothesis that stoichometric transformation of PrP^C to PrP^Scin vitro requires specific RNA molecules.

Although the “protein-only” hypothesis has long been under debate, it is to date the most widely accepted theory to explain the unusual nature of the infectious agent. The “protein-only” hypothesis postulates that a protein rather than a virus or nucleic acid is the infectious agent which transfers the disease related information from one host to another. The “protein-only” hypothesis was first formulated by Alper and Griffith in 1967, renewed by Prusiner in 1982 and later refined by Weissmann. It postulates that the infectious agent consists solely of a host-encoded protein. The propagation is assumed to be a post-translational conformational process in which the infectious agent causes the refolding of the healthy form of the prion protein into the pathogenic isoform by acting as a template. An increasing number of experiments are in line with the “protein-only” hypothesis. The first strong argument is that prions cannot be transmitted to PrP knockout mice in which the Prnp gene is removed and so these mice do not carry prion infectivity in the brain. Mice overexpressing PrP^C were found to be highly susceptible to scrapie. Futhermore, familial human prion diseases are associated with mutations in the PRNP gene. This group of experiments strongly indicates that either PrP^C or mutations in the gene encoding PrP are needed to develop a prion disease.

Over the past years many efforts have been undertaken to prove the “protein-only” hypothesis by the de novo generation of infectious prions from either noninfectious brain-derived PrP^C or recombinant PrP produced in E. coli. The first evidence consisted of producing partially proteinase K resistant PrP (PrP^res) from radioactivity labeled PrP^C in cell-free systems. However, the amount of the de novo produced PrP^Sc was low compared to the starting material and was not infectious.

Further support for the hypothesis was obtained from several studies using the PMCA (protein misfolding cyclic amplification) technology. This assay in which PrP^res is multiplied by serial amplification steps was developed by Soto and colleagues. In the PMCA, brain homogenate from healthy animals is mixed with small quantities of PrP^res. After several rounds of incubation, sonicating and dilution with fresh brain homogenate from healthy animals, PrP^res can be amplified in large quantities. In one approach, PMCA generated hamster PrP^res could induce scrapie in wild-type hamster. Modified protocols of the PMCA in which either purified PrP^C or recombinant expressed PrP have been used as substrate were also applied. However, the sensitivity was much lower than in the PMCA performed with brain homogenate, suggesting that additional auxiliary factors might be necessary for successful amplification.

A systematic investigation for the minimal compounds needed for efficient amplification was performed by Supattapone and coworkers. In this approach PrP^res was produced from purified PrP^C, copurified lipids and single stranded polyanions. Inoculation of these de novo prions caused TSE in wild-type hamster. The most convincing evidence for the “protein-only” hypothesis comes from a study that was recently published by Ma and coworkers. Here, infectious prions were obtained with the PMCA using PrP^C purified from E. coli extracts, anionic phospholipids (POPG) and RNA obtained from mouse liver. These prions induced a neurologic disease in wild-type mice after 130 days with the classical signs of a transmissible prion disease. In other approaches, recombinant prion proteins without any cofactors were converted into cross-β-sheet aggregates that are capable of inducing a transmissible prion disease in transgenic mice or wild-type hamster.

Genetic evidence that supports the “protein-only” hypothesis was obtained from a study in which transgenic mice expressing two amino acids at positions 170 and 174 from the elk prion protein in the Prnp gene were generated. These two mutations lead to a well-structured loop that connects the second β-sheet and the second a-helix in the C-terminal domain of the protein. Mouse lines expressing these transgenes develop a spontaneous prion disease which is transmissible to transgenic mice overexpressing wild-type PrP and consecutively to wild-type mice.

Antigenic properties

The generation of antibodies against PrP^C and PrP^Sc is difficult, because of the strong self-tolerance of PrP^C. These limitations have been overcome by using Prnp^0/0 mice, which are not immune tolerant to PrP^C, and show a strong immune response against PrP^C. These mice have been used for the production of highly efficient monoclonal antibodies, including the “POM” library, which contains antibodies directed against conformational epitopes in the folded C-terminal part of the protein as well as against linear epitopes in the unstructured N-terminal part. These monoclonal antibodies represent valuable high immunoaffinity reagents that can be applied in a variety of techniques for the detection of PrP^C and PrP^Sc.

Several studies showed that anti-PrP antibodies have neutralizing effects by clearing PrP^Sc levels in scrapie-infected cell lines, in cell-free systems and by recognition of PrP^C in transgenic mice on intraperitoneal prion inoculation. Furthermore, passive transfer of anti-PrP monoclonal antibodies delays the onset of scrapie in mice infected with prions by the intraperitoneal route. However, this method requires high levels of the antibodies to cause a significant effect on the survival of prion-infected mice and it has been ineffective in mice showing already clinical signs, which might be due to the blood–brain barrier (BBB) impermeability of the antibodies. Active immunization as antibody-based anti-prion prophylaxis is still difficult to achieve, although intense effort has been made to circumvent the tolerance of the humoral immune system to PrP^C by using different antigens and various antibody retrieval techniques.

The production of antibodies directed against PrP^Sc is more challenging as this requires binding of the antibodies against specific conformational epitopes that are only exposed in PrP^Sc. Antibodies that specifically bind PrP^Sc without binding to PrP^C have been reported, but their affinity seems to be poor and their diagnostic value has awaited confirmation for more than a decade.

Biological properties

Physiological function of PrP^C

Despite extensive research, the physiological function of PrP^C is still unclear. A clarification of the function of PrP^C was expected from the generation of PrP^C knock out mice. However, these transgenic mouse lines show only a subtle phenotype. Since then several biological functions, some of which are controversial, have been attributed to PrP^C, including neuronal signal transduction, lymphocyte function, copper-binding, as well as pro- and anti-apoptotic functions.

In the search for intrinsic functional domains of PrP^C several mouse lines were generated with specific deletions in the PrnP gene. These mice developed several prionopathies characterized by shortened life span and the development of white matter disease in the central nervous system (CNS) as well as neuronal death in the cerebellum.

More recently, PrP^C was shown to play an important role in maintaining peripheral myelin in Schwann cells, indicating that PrP^C acts as a signaling protein in axons necessary to keep peripheral nerves intact. The absence of PrP^C causes otherwise demyelination in the Schwann cells.

Mutations in the human PrP genes

About 10–15% of the CJD cases and virtually all cases of GSS and FFI are autosomal dominantly inherited prion diseases with point mutations, amino acid insertions or deletions in the PRNP gene (Table 3). There is variability in the clinical and pathological findings, the age of onset and the duration depending on the particular PRNP mutation involved. Some PRNP mutations might cause neurodegenerative diseases that are not transmissible and therefore represent a proteinopathy rather than a prion disease.

Most of the point mutations are located in the well-structured C-terminal domain in PrP^C (Table 3; Figure 2A–C) and in the immediate preceding fragment of residues 102–125. Amino acid insertions and deletions are found in the N-terminal part of the protein affecting the length of the octapeptide repeat region.

Table 3 Human PrP gene mutations found in the inherited prion diseases

Point mutations, insertions and deletions identified in the human PRND gene in patients. The polymorphism at amino acid position 129 that is associated with disease is also indicated. Octarepeat inserts are designated as bpi (base pair insertion); Octarepeat deletions as bpd (base pair deletion).

Table based on Aguzzi and Calella (2009). Physiol. Rev., 89, 1105-1152; Aguzzi, Baumann and Bremer (2008). Annu. Rev. Neurosci., 31, 439–477.

Polymorphisms in PrP genes

The M/V polymorphism at codon 129 in HuPrP influences the incidence of sporadic and infectious CJD as well as the age of onset of inherited prion diseases. Homozygosity at codon 129 is more frequent in both sporadic and infectious CJD; it also heralds an earlier age of clinical disease in people carrying pathologic mutations. Those individuals who carry the DI78N mutation develop FFI manifested by insomnia and dysautonomia if they encode M129 on the mutant allele, while those that encode V129 present with fCJD are characterized by dementia (Table 3).

The E/K polymorphism at codon 219 in HuPrP seems to influence resistance to CJD. Individuals with wild-type PrP sequences who are heterozygous for K219 appear to be resistant to CJD. This dominant negative effect is likely to be the results of increased affinity of PrP^C (K219) for protein X which is thought to function like a molecular chaperone in facilitating the conversion of PrP^C into PrP^Sc.

The Q/R polymorphism at codon 171 in OvPrP seems to govern the resistance of sheep to natural scrapie. Sheep with wild-type PrP sequences that are heterozygous for R171 appear to be resistant to natural scrapie, but scrapie can be transmitted to some of these animals by inoculation of prions. This dominant negative effect is also likely to be the result of increased affinity of PrP^C (R171) for protein X. A polymorphism at codon 136 is also thought to influence the susceptibility of some breeds of sheep to scrapie.

The S/F polymorphism at codon 225 in mule deer influences susceptibility to CWD. In addition, elk have a polymorphism at codon 132 (M/L) of PrnP corresponding to the polymorphic codon 129 (M/V) in humans. Elk with codon 132LL experimentally infected with CWD were resistant to infection for at least 4 years, whereas 132MM or 132ML elk developed terminally clinical disease after 23 or 40 months. A reduced susceptibility to CWD was reported in white-tailed deer with G/S and Q/H polymorphisms at codons 96 and 95, respectively.

Prion strains

One of the most intriguing phenomena in prion diseases is the appearance of different strains. Prion strains are defined as infectious isolates that, when transmitted to identical hosts, exhibit distinct disease phenotypes. Prion strains can be discriminated by specific incubation times, histopathological lesion profiling and clinical signs. Most phenotypes of strains are relatively stable during serial transmission. Often distinct new strains are observed upon transmission of prions across a transmission barrier or into animals that express specific polymorphisms of the prion gene, a phenomenon sometimes referred to as a “strain mutation”. The transmission barrier is explained by differences in the amino acid sequence between two species whereby single amino acid changes can lead to significant changes in the incubation times.

To explain the appearance of different prion strains within the framework of the “protein-only” hypothesis it was suggested that the information for the strain-specific characteristics and phenotypes must be enciphered in the PrP^Sc conformation itself or in other words PrP^Sc of each strain exhibits a different conformation. A possible molecular explanation for the appearance of different conformations was recently provided from a systematic structural study on fibril-forming peptides derived from prion and other amyloid proteins. These peptides form small β-sheets and adopt distinct crystalline polymorphisms according to their relative orientation and packing arrangements. Each different crystalline polymorph could thereby retain and transfer the strain-specific information from one species to another. Experimental evidence for strain-specific PrP^Sc conformations include the different electrophoretic mobilities of different strains, the immunological reactivity to amino-proximal antibodies upon proteinase K digestion and the different susceptibilities of PrP^Sc derived from different strains to chaotropic salts. In addition, strains can be discriminated by their accessibility of specific epitopes to monoclonal antibodies. These epitopes are fully accessible in PrP^C, but buried in PrP^Sc and only become exposed after treatment with defined concentrations of chaotropic salts.

Optical tools to classify cross-β-sheet amyloid deposits are historically small amyloidotropic dyes such as Congo red and Thioflavin T. However, the use of these dyes is limited to the detection of total amyloid per se and different amyloid morphologies as present in prion strains cannot be separated with these dyes. More sophisticated tools would therefore be of high interest for the diagnosis of amyloid deposits with distinct morphologies. Luminescent conjugated polymers (LCPs) are a class of novel amyloidotropic dyes that have recently been proven to overcome these limitations. LCPs are small fluorescent probes that were originally synthesized for use in electrical devices. These dyes exhibit a highly flexible backbone that adopts a distinct conformation upon binding to the ordered structure of amyloid fibrils and generates a fluorescent signature that is specific for the structural symmetry of these fibrils. The spectral properties of the LCPs were used for the analysis of brain sections from mice that were infected with distinct prion strains (Figure 3). The LCPs could discriminate different prion strains due to individual staining patterns and could even detect prion deposits that were negative for Congo red and Thioflavin T. These findings provide additional evidence that PrP^Sc strains might be enciphered in the conformation of the prion aggregate.

Taxonomic considerations of mammalian prions

A listing of the different mammalian prions is given in Table 1. Although the prions that cause TME and BSE are referred to as TME prions and BSE prions; this may be unjustified, because both are thought to originate from the oral consumption of scrapie prions in sheep-derived foodstuffs and because many lines of evidence argue that the only difference among the various prions is the sequence of PrP which is dictated by the host and not the prion itself. The human prions present a similar semantic conundrum. Transmission of human prions to laboratory animals produces prions carrying PrP molecules with sequences dictated by the PrP gene of the host, not that of the inoculum.

To simplify the terminology, the generic term PrP^Sc is suggested in place of such terms as PrP^CJD, PrP^BSE and PrP^res. To distinguish PrP^Sc found in humans or cattle from that found in other animals, we suggest HuPrP^Sc or BoPrP^Sc instead of PrP^CJD or PrP^BSE, respectively (Table 1). Once human prions and thus HuPrP^Sc molecules have been passaged into animals, then the prions and PrP^Sc are no longer of the human species unless they were formed in an animal expressing a HuPrP transgene. In the case of mutant PrPs, the mutation and any important polymorphism can be denoted in parentheses following the particular PrP isoform. For example, in FFI, the pathogenic PrP isoform would be referred to as PrP^Sc or HuPrP^Sc; alternatively, if it were important to identify the mutation, then it would be written as HuPrP^Sc (D178N, M129) (Table 3). The term PrP^res or PrP-res is derived from the protease-resistance of PrP^Sc but protease-resistance, insolubility, and high β-sheet content should be only considered as surrogate markers of PrP^Sc since one or more of these may not always be present. Whether PrP^res is useful in denoting PrP molecules that have been subjected to procedures that modify their resistance to proteolysis, but have not been demonstrated to convey infectivity or to cause disease remains debatable.

The term PrP* has been used in two different ways. First, it has been used to identify a fraction of PrP^Sc molecules that are infectious. Such a designation is thought to be useful since there are ~10⁵ PrP^Sc molecules per infectious unit. Second, PrP* has been used to designate a metastable intermediate of PrP^C that is bound to protein X. It is noteworthy that neither a subset of biologically active PrP^Sc molecules nor a metastable intermediate of PrP^C has been identified, to date.

In mice, the PrP gene denoted Prnp is now known to be identical with two genes denoted Sinc and Prn-i that control the length of the incubation time in mice inoculated with prions. These findings permit a welcome simplification. A gene designated Pid-1 on mouse chromosome 17 also appears to influence experimental CJD and scrapie incubation times but information on this locus is limited.

Distinguishing among CJD, GSS and FFI has grown increasingly difficult with the recognition that CJD, GSS and FFI are autosomal dominant diseases caused by mutations in the PRNP gene. Initially, it was thought that a specific PrP mutation was associated with a particular clinico-neuropathologic phenotype but an increasing number of exceptions are being recognized. Multiple examples of variations in the clinico-neuropathologic phenotype within a single family where all affected members carry the same PrP mutation have been recorded. Most patients with a PrP mutation at codon 102 present with ataxia and have PrP amyloid plaques; such patients are generally given the diagnosis of GSS, but some individuals within these families present with dementia, a clinical characteristic that is usually associated with CJD. One suggestion is to label these inherited disorders as “prion disease” followed by the mutation in parenthesis while another is the use the terms fCJD and GSS followed by the mutation. In the case of FFI, describing the Dl78N mutation and M129 polymorphism seems unnecessary since this is the only known mutation-polymorphism combination that gives the FFI phenotype.

Derivation of name

Prion: sigla from proteinaceous and infectious particle.

PrP orthologs: Doppel and Shadoo

Two other proteins, the Doppel and Shadoo, have been identified that belong to the PrP family. In contrast to PrP, these two proteins are not infectious. The Doppel protein (a German synonym of double; Dpl) was originally identified by the observation of phenotypic discrepancies in PrP0/0 mice. Two mouse lines, ZHI-Prnp^0/0 mice and Edbg Prnp^−/− mice, showed no obvious phenotype, whereas ZHII-Prnp^0/0, Ngsk-Prnp^0/0 and Rcm0-Prnp^0/0 developed late onset ataxia caused by cerebellar Purkinje cell degeneration. The identification of a novel gene locus (Prnd) 16 kb downstream of the Prnp gene and its product, Dpl, provided an explanation for the different phenotypes.

The amino acid identity between PrP and Dpl is about 20%. The human Dpl consists of 179 amino acids and contains two glycosylation sites. Compared to PrP, Dpl lacks the octarepeats, the charged clusters and the hydrophobic stretch and contains an additional disulfide bridge. The N-terminal tail, residues 24–52, of Dpl is significantly shorter due to the lack of the octarepeats, but the C-terminal domain of residues 52–149 has a similar structural organization as seen for PrP. The well-structured C-terminal domain of the human Dpl contains four α-helices comprising residues 72–80 (α1), 101–115 (α2a), 117–121 (α2b) and 127–141 (α3) and a short antiparallel β-sheet of residues 58–60 and 88–90. The two disulfide bridges in the protein are formed between Cy109–Cys143, connecting helices α2 and α3, and between Cys95–Cys148, connecting the loop β2–α2 with the C-terminal end of Dpl.

Dpl is mainly expressed in testis and heart, to some smaller extent in other peripheral organs and at a very low expression rate in the brains of adult wild-type mice. Due to the high biochemical and structurally similarities between PrP and Dpl it was assumed that the proteins also exhibit similar physiological functions.

To elucidate the biological function of Dpl, Prnd knockout mice were generated by homozygous targeted disruption of the Prnd gene. The Prnd^0/0 mice survive until adulthood like the Prnp knockout mice. In contrast to the PrP^0/0, the Prnd^0/0mice show a specific phenotype. Whereas females lacking Dpl were viable and fertile, males without Dpl suffer from male sterility. In addition, the Prnd gene was disrupted in the ataxic Zurich II mice by using transallelic targeted meiotic recombination to generate Prnp/Prnd double deletion mice. These mice did not show any ataxia, proving the causal role for Dpl overexpression in the Zurich II phenotype.

The PrP-like protein Shadoo (Sho) (for shadow of the prion protein) was identified in 2003 by intense database search for nucleotide sequences that are similar to PrnP. Shadoo has been shown to present in many mammals and fish. The related gene Sprn is located on chromosomes 7 and 10 in mice and humans, respectively, which are distinct from Prnp and Prnd, indicating that Sprn has evolved separately. The entire open reading frame of Sprn is contained in a single exon. Sho seems to be expressed in the central nervous system, and to be neuroprotective by preventing cerebellar degeneration mediated by CNS-expressed Dpl and N-terminal deletion mutants of PrP.

The mature protein consists of 98 amino acids and is presumably gycosylated at one site. Similarly to PrP, Sho contains the highly conserved valine- and alanine-rich hydrophobic stretch, the N-terminal signal sequence for targeting the protein into the endoplasmatic reticulum, and the GPI anchor. On the other hand, it lacks the octarepeats and has no cysteine residues. In addition, Shadoo does not contain a C-terminal domain and far-UV CD spectra indicate that the protein is unfolded.

Further reading

Note: A version of this text with full cited references is available online on ScienceDirect®, www.sciencedirect.com

Aguzzi and Calella, 2009 A. Aguzzi, A.M. Calella, Prions: protein aggregation and infectious diseases. Physiol. Rev. 89 (2009) 1105–1152.

Aguzzi and Polymenidou, 2004 A. Aguzzi, M. Polymenidou, Mammalian prion biology: one century of evolving concepts. Cell. 116 (2004) 313–327.

Aguzzi et al., 2008 A. Aguzzi, F. Baumann, J. Bremer, The prion's elusive reason for being. Annu. Rev. Neurosci. 31 (2008) 439–477.

Aguzzi et al., 2007 A. Aguzzi, M. Heikenwalder, M. Polymenidou, Insights into prion strains and neurotoxicity. Nat. Rev. Mol. Cell Biol. 8 (2007) 552–561.

Aguzzi et al., 2008 A. Aguzzi, C. Sigurdson, M. Heikenwalder, Molecular mechanisms of prion pathogenesis. Annu. Rev. Pathol. 3 (2008) 11–40.

Collinge, 1997 J. Collinge, Human prion diseases and bovine spongiform encephalopathy (BSE). Hum. Mol. Genet. 6 (1997) 1699–1705.

Falsig et al., 2008 J. Falsig, K.P. Nilsson, T.P. Knowles, A. Aguzzi, Chemical and biophysical insights into the propagation of prion strains. HFSP J. 2 (2008) 332–341.

Heppner and Aguzzi, 2004 F.L. Heppner, A. Aguzzi, Recent developments in prion immunotherapy. Curr. Opin. Immunol. 16 (2004) 594–598.

Prusiner, 1982 S.B. Prusiner, Novel proteinaceous infectious particles cause scrapie. Science. 216 (1982) 136–144.

Watts and Westaway, 2007 J.C. Watts, D. Westaway, The prion protein family: diversity, rivalry, and dysfunction. Biochim. Biophys. Acta. 1772 (2007) 654–672.

Weissmann, 1991 C. Weissmann, A “unified theory” of prion propagation. Nature. 352 (1991) 679–683.

Weissmann, 1996 C. Weissmann, The Ninth Datta Lecture. Molecular biology of transmissible spongiform encephalopathies. FEBS Lett. 389 (1996) 3–11.

Weissmann and Aguzzi, 2005 C. Weissmann, A. Aguzzi, Approaches to therapy of prion diseases. Annu. Rev. Med. 56 (2005) 321–344.

Weissmann and Flechsig, 2003 C. Weissmann, E. Flechsig, PrP knock-out and PrP transgenic mice in prion research. Br. Med. Bull. 6 (2003) 43–60.

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Contributed by

Aguzzi, A. and Hornemann, S., including contributions to the Eighth ICTV Report by Prusiner, S.B., Baron, H., Carlson, G., Cohen, F.E., DeArmond, S.J., Gabizon, R., Gambetti, P., Hope, J., Kitamoto, T., Kretzschmar, H.A., Laplanche, J.-L., Tateishi, J., Telling, G. and Will, R.