1. Chapter 8: Subviral Agents Slide 1/48© Academic Press, 2000. Subviral Agents: Genomes Without Viruses, Viruses Without Genomes Learning Objectives: On completing this session, you should be able to: Understand the concept of subviral agents. Explain the differences between satellites & viroids. Summarize the present state of knowledge about transmissible spongiform encephalopathies.

2. Chapter 8: Subviral Agents Slide 2/48© Academic Press, 2000. Satellites & Viroids Satellites are small RNA molecules which are absolutely dependent on the presence of another virus for multiplication. Even viruses have their own parasites! Most satellites are associated with plant viruses, but a few are associated with bacteriophages or animal viruses, e.g. the Dependovirus genus, which are satellites of adenoviruses. Two classes of satellites can be distinguished, satellite viruses, which encode their own coat proteins, & satellite RNAs (or 'virusoids'), which use the coat protein of the helper virus.

3. Chapter 8: Subviral Agents Slide 3/48© Academic Press, 2000. Satellites Typical properties of satellites are: A genome of approximately 500-2000 nucleotides of single-stranded RNA. Unlike defective virus genomes, there is little or no nucleotide sequence similarity between the satellite & the helper virus genome. They cause distinct disease symptoms in plants which are not seen with the helper virus alone. Replication of satellites usually interferes with the replication of the helper virus (unlike most defective virus genomes). Satellites replicate in the cytoplasm using an RNA-dependent RNA polymerase, an enzymatic activity found in plant but not animal cells. Examples of satellites: Barley yellow dwarf virus satellite RNA: Helper - Luteovirus Tobacco ringspot virus satellite RNA: Helper - Nepovirus Subterranean clover mottle virus satellite RNA: Helper - Sobemovirus

4. Chapter 8: Subviral Agents Slide 4/48© Academic Press, 2000. Viroids Viroids are very small (200-400nt), rod-like RNA molecules with a high degree of secondary structure. They have no capsid or envelope & consist only of a single nucleic acid molecule. Viroids are associated with plant diseases & are distinct from satellites in a number of ways. The first viroid to be discovered & the best studied is potato spindle tuber viroid (PSTVd - viroid names are abbreviated 'Vd' to distinguish them from viruses). Viroids do not encode any proteins & are replicated by host cell RNA polymerase II, or possibly by the product of an RNA dependent RNA polymerase gene in some eukaryotic cells. The details of replication are not understood, but it is likely to occur by a rolling circle mechanism followed by autocatalytic cleavage & self-ligation to produce the mature viroid.

5. Chapter 8: Subviral Agents Slide 5/48© Academic Press, 2000. A Typical Viroid:

6. Chapter 8: Subviral Agents Slide 6/48© Academic Press, 2000. Viroids There is considerable sequence variation between different viroids & this is used as an arbitrary classification to divide viroids into genera & species. All viroids share a common feature, a conserved central region believed to be involved in their replication. One group of viroids is capable of forming a hammerhead structure, giving them the enzymatic properties of a ribozyme (an autocatalytic, self-cleaving RNA molecule). This activity is used to cleave the multimeric structures produced during the course of replication. Other viroids use unknown host nuclear enzymes to achieve this objective. Some viroids (e.g. cadang-cadang coconut viroid, CCCVd) cause severe & lethal disease in their host plants. Others range from no apparent pathogenic effects (e.g. hop latent viroid, HLVd) to mild disease symptoms (e.g. apple scar skin viroid, ASSVd).

7. Chapter 8: Subviral Agents Slide 7/48© Academic Press, 2000. Organization of a Viroid Genome

8. Chapter 8: Subviral Agents Slide 8/48© Academic Press, 2000. Viroid Pathogenesis It is not clear how viroids cause pathogenic symptoms, but obviously these must result from some perturbation of the normal host cell metabolism. They show some similarities with certain eukaryotic host cell sequences, in particular with an intron found between the 5.8S & 25S ribosomal RNAs & with the U3 snRNA which is involved in splicing. Therefore, it has been suggested that viroids may interfere with post-transcriptional RNA processing in infected cells. In vitro experiments with purified mammalian protein kinase PKR have shown that the kinase is strongly activated (phosphorylated) by viroid strains that cause severe symptoms, but far less by mild strains. Activation of a plant homologue of PKR could be the triggering event in viroid pathogenesis

9. Chapter 8: Subviral Agents Slide 9/48© Academic Press, 2000. Propagation & Origin of Viroids Most viroids are transmitted by vegetative propagation, i.e. division of infected plants, although a few can be transmitted by insect vectors (non-propagative) or mechanically. Because viroids do not have the benefit of a protective capsid, viroid RNAs would be expected to be at extreme risk of degradation in the environment. However, their small size & high degree of secondary structure protects them to a large extent & they are able to persist in the environment for a sufficiently long period to be transferred to another host. The origins of viroids are obscure. One theory is that they may be the most primitive type of RNA genome - possibly leftovers from the 'RNA world' believed to have existed during the era of prebiotic evolution. Alternatively, they may have evolved at a much more recent time as the most extreme type of parasite.

10. Chapter 8: Subviral Agents Slide 10/48© Academic Press, 2000. Hepatitis Delta Virus (HDV) Hepatitis delta virus (HDV) is a unique chimeric molecule with some of the properties of a satellite virus & some of a viroid which causes disease in humans. HDV requires hepatitis B virus (HBV) as a 'helper virus' for replication & is transmitted by the same means as HBV, benefiting from the presence of a protective coat composed of lipid plus HBV proteins. Virus preparations from HBV/HDV-infected animals contain heterologous particles distinct from those of HBV but with an irregular, ill-defined structure. These particles are composed of HBV antigens & contain the covalently closed circular HDV RNA molecule in a branched or rod-like configuration similar to that of other viroids.

11. Chapter 8: Subviral Agents Slide 11/48© Academic Press, 2000. The Hepatitis Delta Virus Genome

12. Chapter 8: Subviral Agents Slide 12/48© Academic Press, 2000. Hepatitis Delta Virus Unlike all other viroids, HDV encodes a protein, the  antigen, which is a nuclear phosphoprotein. Post-transcriptional RNA editing results in the production of two slightly different forms of the protein,  Ag-S (195 amino acids) which is necessary for HDV replication &  Ag-L (214 amino acids) which is necessary for the assembly & release of HDV-containing particles. The HDV genome is thought to be replicated by host cell RNA polymerase II using a "rolling circle" mechanism which produces linear concatemers which must be cleaved for infectivity. The cleavage is carried out by a ribozyme domain present in the HDV RNA, the only known example of a ribozyme in an animal virus genome.

13. Chapter 8: Subviral Agents Slide 13/48© Academic Press, 2000. Hepatitis Delta Virus HDV is found worldwide wherever HBV infection occurs. The interactions between HBV & HDV are difficult to study, but HDV seems to potentiate the pathogenic effects of HBV infection. Fulminant hepatitis (with a mortality rate of about 80%) is 10 times more common in co-infections than with HBV infection alone. Because HDV requires HBV for replication, it is being controlled by HBV vaccination.

14. Chapter 8: Subviral Agents Slide 14/48© Academic Press, 2000. Prions There are a group of transmissible, chronic, progressive infections of the nervous system which show common pathological effects & are invariably fatal. Their pathology is reminiscent of amyloid diseases such as Alzheimer's syndrome & to distinguish them from such conditions, they are known as transmissible spongiform encephalopathies (TSE). The earliest record of any TSE dates from several centuries ago, when a disease called scrapie was first observed in sheep (see TSE in Animals). Long considered to be caused by viruses, the first doubts about the nature of the infectious agent involved in TSEs arose in the 1960s. In 1967, Tikvah Alper was the first to suggest that the agent of scrapie might replicate without nucleic acid & in 1982, Stanley Prusiner coined the term prion (proteinaceous infectious particle), which according to Prusiner is pronounced "pree-on".

15. Chapter 8: Subviral Agents Slide 15/48© Academic Press, 2000. Pathology of Prion Diseases All prion diseases share a similar underlying pathology, although there are significant differences between various conditions. A number of different diseases are characterized by the deposition of abnormal protein deposits in various organs, e.g. kidney, spleen, liver or brain. These 'amyloid' deposits consist of accumulations of various proteins in the form of plaques or fibrils depending on their origin, e.g. Alzheimer's disease is characterized by the deposition of plaques & 'tangles' composed of  -amyloid protein. None of the 'conventional' amyloidoses are infectious diseases & extensive research has shown that they cannot be transmitted to experimental animals; they result from endogenous errors in metabolism caused by a variety of largely unknown factors. Amyloid deposits appear to be inherently cytotoxic.

16. Chapter 8: Subviral Agents Slide 16/48© Academic Press, 2000. Pathology of Prion Diseases Although the molecular mechanisms involved in cell death are unclear, it is this effect which gives the 'spongiform encephalopathies' their name owing to the characteristic holes in thin sections of affected brain tissue viewed under the microscope; these holes are caused by neuronal loss & gliosis. Thus deposition of amyloid is end stage which links conventional amyloidoses & TSEs & explains the tissue damage seen in both types of disease, but does not reveal anything about their underlying causes. Definitive diagnosis of TSE cannot be made on clinical grounds alone & requires demonstration of PrP deposition by immunohistochemical staining of post-mortem brain tissue, molecular genetic studies, or experimental transmission to animals, as discussed in the following sections.

17. Chapter 8: Subviral Agents Slide 17/48© Academic Press, 2000. TSE in Animals A number of TSEs have been observed & intensively investigated in animals. In particular, scrapie is the paradigm for understanding of human TSEs. Some of these diseases are naturally occurring & have been known about for centuries, whereas others have only been observed more recently & are almost certainly causally related to one another.

18. Chapter 8: Subviral Agents Slide 18/48© Academic Press, 2000. Scrapie Scrapie is a naturally occurring disease of sheep found in many parts of the world, although not universally distributed, which was first described more than 200 years ago. Scrapie appears to have originated in Spain & subsequently spread throughout Western Europe. The export of sheep from Britain in the nineteenth century is thought to have helped scrapie spread around the world. Scrapie is primarily a disease of sheep although it can also affect goats. Infected sheep show severe & progressive neurological symptoms such as abnormal gait, often repeatedly scraping against fences or posts, from which behaviour the disease takes its name. The incidence of the disease increases with the age of the animals. Some countries, such as Australia & New Zealand, have eliminated scrapie by slaughtering infected sheep & the imposition of rigorous import controls. Work in Iceland has shown that the land on which infected sheep graze may retain the condition & infect sheep up to three years later.

19. Chapter 8: Subviral Agents Slide 19/48© Academic Press, 2000. Scrapie The incidence of scrapie in a flock is related to the breed of sheep. Some breeds are relatively resistant to the disease while others are prone to it, indicating genetic control of susceptibility. The natural mode of transmission between sheep is unclear. Lambs of scrapie infected sheep are more likely to develop the disease, but the reason for this is unclear. Symptoms of scrapie are not seen in sheep of less than one & a half years old, which indicates that the incubation period of scrapie is at least this long. The first traces of infectivity can be detected in the tonsils, mesenteric lymph nodes & intestines of sheep 10-14 months old, which suggests an oral route of infection. The infective agent is present in the membranes of the embryo but it has not been demonstrated in colostrum or milk or in the tissues of the newborn lambs.

20. Chapter 8: Subviral Agents Slide 20/48© Academic Press, 2000. Transmissible Mink Encephalopathy (TME) TME is a rare disease of farmed mink caused by exposure to a scrapie-like agent in feed. The disease was first identified in Wisconsin, USA, in 1947 & has also been recorded in Canada, Finland, Germany & Russia. Like other TSEs, TME is a slow progressive neurological disease. Early symptoms include changes in habits & cleanliness as well as difficulty in eating or swallowing. TME-infected mink become hyperexcitable & begin arching their tails over their backs, ultimately losing locomotor coordination. Natural TME has a minimum incubation period of 7-12 months & although exposure is generally through oral routes, horizontal mink-to-mink transmission cannot be ruled out. The origin of the transmissible agent in TME appears to be contaminated foodstuffs.

21. Chapter 8: Subviral Agents Slide 21/48© Academic Press, 2000. Feline Spongiform Encephalopathy (FSE) FSE was recognized in the UK in May 1990 as a scrapie- like syndrome in domestic cats resulting in ataxia (irregular & jerky movements) & other symptoms typical of spongiform encephalopathies. By December 1997, a total of 81 cases had been reported in the UK. In addition, FSE has been recorded in a domestic cat in Norway & in three species of captive wild cats (cheetahs, puma & ocelot). Inclusion of cattle offal in commercial pet foods was banned in the UK in 1990, so the incidence of this disease is expected to decline rapidly.

22. Chapter 8: Subviral Agents Slide 22/48© Academic Press, 2000. Chronic Wasting Disease (CWD) A disease similar to scrapie which affects deer & captive exotic ungulates (e.g. nyala, oryx, kudu, etc). CWD was first recognized in captive deer & elk in the Western United States in 1967 & appears to be endemic in origin. The incidence in wild deer & elk is up to 15% in Colorado. This is worrying, since prions taken from the brains of infected deer & elk are able to convert normal human prion to a protease-resistant form, a well-studied test for the ability to cause human disease.

23. Chapter 8: Subviral Agents Slide 23/48© Academic Press, 2000. Bovine Spongiform Encephalopathy (BSE) BSE was first recognized in dairy cattle the UK in 1986 as a typical spongiform encephalopathy. Affected cattle showed altered behaviour & a staggering gait, giving the disease its name in the press of 'mad cow disease'. On microscopic examination, the brains of affected cattle showed extensive spongiform degeneration. It was concluded that BSE resulted from the use of contaminated foodstuffs. To obtain higher milk yields & growth rates, the nutritional value of feed for farmed animals was routinely boosted by the addition of protein derived from waste meat products & bonemeal (MBM) prepared from animal carcasses, including sheep & cows. This practice was not unique to the UK but was widely followed in most developed countries.

24. Chapter 8: Subviral Agents Slide 24/48© Academic Press, 2000. Incidence of BSE in the UK By January 2000, a total of 179,256 cases of BSE had been reported in the UK & 1,392 cases elsewhere:

25. Chapter 8: Subviral Agents Slide 25/48© Academic Press, 2000. Origins of BSE The initial explanation for the emergence of BSE in the UK was as follows: Because scrapie is endemic in Britain, it was assumed that this was the source of the infectious agent in the feed. Traditionally, MBM was prepared by a rendering process involving steam treatment & hydrocarbon extraction, resulting in two products, a protein-rich fraction called 'greaves' containing about 1% fat from which MBM was produced & a fat-rich fraction called 'tallow' which was put to a variety of industrial uses. In the late 1970s, the price of tallow fell & the use of expensive hydrocarbons in the rendering process was discontinued, producing an MBM product containing about 14% fat in which the infectious material may not have been inactivated.

26. Chapter 8: Subviral Agents Slide 26/48© Academic Press, 2000. Origins of BSE However, it is now known that none of the rendering processes used, before or after the 1980s, completely inactivates the infectivity of prions. Therefore cattle would have been exposed to scrapie prions in all countries worldwide where scrapie was present & MBM was used, not just in the UK in the 1980s. BSE is not scrapie.BSE is not scrapie. The biological properties of the scrapie & BSE agents are distinct, e.g. transmissibility to different animal species & pattern of lesions produced in infected animals. There is no evidence to support the assumption that BSE is scrapie in cows. The only feasible interpretation based on present knowledge is that BSE originated as an endogenous bovine (cow) prion which was amplified by the feeding of cattle-derived protein in MBM back to cows. The emergence of BSE in the UK appears to have been due to a chance event compounded by poor husbandry practices, i.e. use of MBM in ruminant feed.

27. Chapter 8: Subviral Agents Slide 27/48© Academic Press, 2000. Human TSEs There are four recognized human TSEs: Creutzfeldt-Jakob disease (CJD):Creutzfeldt-Jakob disease (CJD): Spongiform encephalopathy in cerebral and/or cerebellar cortex and/or subcortical grey matter, or encephalopathy with prion protein (PrP) immunoreactivity (plaque and/or diffuse synaptic and/or patchy/perivacuolar types). 3 forms: sporadic, iatrogenic (recognised risk, e.g. neurosurgery), familial (same disease in first degree relative). Familial fatal insomnia (FFI):Familial fatal insomnia (FFI): Thalamic degeneration, variable spongiform change in cerebrum. Occurs in families with PrP 178 asp- asn mutation. Gerstmann-Straussler-Scheinker disease (GSS):Gerstmann-Straussler-Scheinker disease (GSS): Encephalo(myelo)pathy with multicentric PrP plaques. Occurs in families with dominantly inherited progressive ataxia and/or dementia. Kuru:Kuru: Characterized by large amyloid plaques. Occurs in the Fore population of New Guinea due to ritual cannibalism, now eliminated.

28. Chapter 8: Subviral Agents Slide 28/48© Academic Press, 2000. Sources of Human TSE There are believed to be three sources from which human TSEs originate: Sporadic:Sporadic: Creutzfeldt-Jakob disease (CJD) arises spontaneously at a frequency of about one in a million people per year with little variation worldwide. This category accounts for 90% of all human TSE, but only about 1% of sporadic CJD cases are transmissible to mice. Iatrogenic/Acquired TSE:Iatrogenic/Acquired TSE: This occurs due to recognized risks, e.g. neurosurgery, transplantation, etc. Familial:Familial: Approximately 10% of human TSEs are familial, i.e. inherited. There are a number of mutations in the human PrP gene which are known to give rise to TSE as an autosomal dominant trait acquired by hereditary transmission.

29. Chapter 8: Subviral Agents Slide 29/48© Academic Press, 2000. Mutations in the Human Prp Gene Known to Give Rise to TSE

30. Chapter 8: Subviral Agents Slide 30/48© Academic Press, 2000. Kuru Kuru occurred primarily in 169 villages occupied by the Fore tribes in the highlands of New Guinea. The first cases were recorded in the 1950s & involved progressive loss of voluntary neuronal control, followed by death less than 1 year after the onset of symptoms. The key to the origin of the disease was provided by the profile of its victims - it was never seen in young children, rarely in adult men & was most common in both male & female adolescents & in adult women. The Fore people practised ritual cannibalism as a rite of mourning for their dead. Women & children participated in these ceremonies but adult men did not take part, explaining the age/sex distribution of the disease. The incubation period for kuru can be in excess of 30 years, but in most cases is somewhat shorter. The practice of ritual cannibalism was discouraged in the late 1950s & the incidence of kuru declined dramatically.

31. Chapter 8: Subviral Agents Slide 31/48© Academic Press, 2000. vCJD In April 1996, a paper was published which described a new variant of CJD (vCJD) in the UK. These cases share unusual features which distinguish them from other forms of CJD: An early age of onset or death (average 27, c.f. 65 for CJD). A prolonged duration of illness (average 13 months, c.f. 3 months for CJD). A predominantly psychiatric presentation including anxiety, depression, withdrawal & behavioural changes rather than neurological symptoms. The subsequent development of a cerebellar syndrome with ataxia. Forgetfulness & memory disturbance develop & progress to severe cognitive impairment. Myoclonus (involuntary muscular contractions) develops in the majority of patients. Typical EEG appearances of CJD are absent. Neuropathologic spongiform change, neuronal loss & astrocytic gliosis most evident in the basal ganglia & thalamus. Amyloid plaque formation reminiscent of that seen in kuru extensively distributed throughout the cerebrum & cerebellum.

32. Chapter 8: Subviral Agents Slide 32/48© Academic Press, 2000. vCJD The official UK Spongiform Encephalopathy Advisory Committee concluded that vCJD is "a previously unrecognised & consistent disease pattern" & that "although there is no direct evidence of a link, on current data & in the absence of any credible alternative the most likely explanation at present is that these cases are linked to exposure to BSE". By June 2000, 70 people had died of vCJD in the UK & several more in other countries. Current investigations & modelling of the epidemic suggest that the maximum number of deaths from vCJD in the UK is likely to be 14,000 or less - hardly a comforting thought.

33. Chapter 8: Subviral Agents Slide 33/48© Academic Press, 2000. Molecular Biology of Prions The evidence that prions are not conventional viruses is based on the fact that nucleic acid is not necessary for infectivity since they show: Resistance to heat inactivation - infectivity is reduced but not eliminated by high temperature autoclaving (135 o C for 18 min). Some infectious activity is even retained after treatment at 600 o C, suggesting that an inorganic molecular template is capable of nucleating the biological replication of the agent. Resistance to radiation damage - infectivity was found to be resistant to short-wave ultraviolet radiation & to ionizing radiation. These treatments inactivate infectious organisms by causing damage to the genome. There is an inverse relationship between the size of target nucleic acid molecule & the dose of radioactivity or ultraviolet light needed to inactivate them, i.e. large molecules are sensitive to much lower doses than are smaller molecules.

34. Chapter 8: Subviral Agents Slide 34/48© Academic Press, 2000. Resistance to Radiation Damage

35. Chapter 8: Subviral Agents Slide 35/48© Academic Press, 2000. Molecular Biology of Prions The scrapie agent was found to be highly resistant to both ultraviolet light & ionizing radiation, indicating that any nucleic acid present must be less than 80 nucleotides. Resistance to DNAse & RNAse treatment, to psoralens & to Zn 2+ catalysed hydrolysis - all of which treatments inactivate nucleic acids. Sensitivity to urea, SDS, phenol & other protein-denaturing chemicals.

36. Chapter 8: Subviral Agents Slide 36/48© Academic Press, 2000. Molecular Biology of Prions The properties of prions indicate an agent with the properties of a protein rather than a virus. A protein of 254 amino acids, PrP Sc, is associated with scrapie infectivity. Biochemical purification of scrapie infectivity results in preparations highly enriched in PrP Sc & purification of PrP Sc results in enrichment of scrapie activity. In 1984, Prusiner determined the sequence of 15 amino acids at the end of purified PrP Sc. This led to the discovery that all mammalian cells contain a gene (Prnp) which encodes a protein identical to PrP Sc, termed PrP C. No biochemical differences between PrP C & PrP Sc have been determined, although unlike PrP C, PrP Sc is partly resistant to protease digestion, resulting in the formation of a 141 amino acid protease-resistant fragment which accumulates as fibrils in infected cells.

37. Chapter 8: Subviral Agents Slide 37/48© Academic Press, 2000. The PrP Protein

38. Chapter 8: Subviral Agents Slide 38/48© Academic Press, 2000. Molecular Biology of Prions Only a proportion of the total PrP in diseased tissue is present as PrP Sc, but is the infectious form of the PrP protein, since highly purified PrP Sc is infectious when used to inoculate experimental animals. Like other infectious agents, there is a dosage effect which gives a strong correlation between the amount of PrP Sc in an inoculum & the incubation time until the development of disease. TSEs, which behave like infectious agents, appear to be caused by an endogenous gene/protein.

39. Chapter 8: Subviral Agents Slide 39/48© Academic Press, 2000. TSE Pathology

40. Chapter 8: Subviral Agents Slide 40/48© Academic Press, 2000. PrP Susceptibility of a host species to prion infection is co-determined by the prion inoculum & the Prnp gene. Disease incubation times for individual prion isolates vary in different strains of inbred mice but for a given isolate in a particular strain are remarkably consistent. These observations have resulted in two important concepts: 1) Prion strain variation: At least 15 different strains of PrP Sc have been recognized. These can be determined from each other by the incubation time to the onset of disease & the type & distribution of lesions within the CNS in inbred strains of mice. Thus prions can be 'fingerprinted' & BSE can be distinguished from scrapie or CJD. 2) The species barrier: When prions are initially transmitted from one species to another, disease develops only after a very long incubation period, if at all. On serial passage in the new species, the incubation time often decreases dramatically & then stabilizes. This species barrier can be overcome by introducing a PrP transgene from the prion donor, e.g. hamster, into the recipient mice.

41. Chapter 8: Subviral Agents Slide 41/48© Academic Press, 2000. The Species Barrier

42. Chapter 8: Subviral Agents Slide 42/48© Academic Press, 2000. PrP PrP C & PrP Sc are not post-translationally modified & the genes which encode them are not mutated, i.e. this is distinct from mendelian inheritance of familial forms of CJD. How such apparently complex behaviour can be 'encoded' by a 254 amino acid protein has not been firmly established, but there is evidence that the fundamental difference between the infectious, pathogenic form (PrP Sc ) & the endogenous form (PrP C ) results from a change in the conformation of the folded protein, which adopts a conformation rich in  -sheet.

43. Chapter 8: Subviral Agents Slide 43/48© Academic Press, 2000. PrP c & PrP Sc

44. Chapter 8: Subviral Agents Slide 44/48© Academic Press, 2000. PrP Transgenic 'knockout' Prnp 0/0 mice which do not possess an endogenous prion gene are immune to the effects of PrP Sc & do not propagate infectivity to normal mice, indicating that: Production of endogenous PrP C is an essential part of the disease process in TSEs The infectious inoculum of PrP Sc does not replicate. These experiments have given few clues to the normal role of PrP C.

45. Chapter 8: Subviral Agents Slide 45/48© Academic Press, 2000. PrP Most Prnp 0/0 mice are developmentally normal & do not have CNS abnormalities, suggesting that loss of normal PrP C function is not the cause of TSE & that the accumulation of PrP Sc is responsible for disease symptoms. One strain of Prnp 0/0 mouse was found to develop late- onset ataxia & neurological degeneration. This observation led to the discovery of another gene called Prnd close to the Prnp gene which encodes a 179 amino acid PrP-like protein designated doppel (Dpl), overexpression of which appears to cause neurodegeneration. Like Prnp, this gene is conserved in vertebrates including humans & may have arisen from Prnp by gene duplication. It is suspected that there may be other members of the Prn gene family.

46. Chapter 8: Subviral Agents Slide 46/48© Academic Press, 2000. Other Prions The URE3 protein of the yeast Saccharomyces cerevisiae has properties very reminiscent of PrP. Other PrP-like proteins are also known, e.g. PSI in yeast & Het-s* in the fungus Podospora. URE3 modifies a cellular protein Ure2p causing altered nitrogen metabolism; similarly, the PSI phenotype involves a self-propagating aggregation of Ure2p & the cellular protein Sup35p. Cells 'infected' with URE3 can be 'cured' by treatment with protein-denaturing agents such as guanidium, which is believed to cause refolding of URE3 to the Ure2p conformation.

47. Chapter 8: Subviral Agents Slide 47/48© Academic Press, 2000. Prions The prion hypothesis is revolutionary & has justifiably met with a somewhat sceptical reception. The prion story is not yet complete. It is still possible to construct numerous alternative theories of varying degrees of complexity (and plausibility!) to fit the experimental data. Science progresses by the construction of experimentally verifiable hypotheses. For many years, research into spongiform encephalopathies has been agonizingly slow because each individual experiment has taken at least one & in some cases many years to complete. With the advent of molecular biology, this has now become a fast-moving & dynamic field.

48. Chapter 8: Subviral Agents Slide 48/48© Academic Press, 2000. Summary A variety of novel infectious agents cause disease in plants, in animals & in humans. Several types of non-viral, sub-cellular pathogens have disease-causing potential. These include satellites, viroids & prions. Conventional strategies to combat virus infections, such as drugs & vaccines, have no effect on these unconventional agents. A full understanding of the biology of these novel infectious entities will be necessary before means of treatment for the diseases they cause will become available.