- Email: [email protected]
PrP genetics in sheep and the implications for scrapie and BSE
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18 Dean, M. et al. (1996)Science 273, 1856-1862 19 Huang, Y.X. et al. (1996) Nat. Med. 2, 1240-1243 20 Meyer, L. and CarrY,N. (1996) Presse Med. 25,379-384 21 Butcher, G,A. (1992) Parasitol. Today 8, 307-311 22 Dorrucci, M. et al. (1995)J. Infect. Dis. 172, 1503-1508 23 Toure-Baide,A. et al. (1996) Infect. Immun. 64, 744-750 24 Greenberg, A.E. et al. (1991)New Engl. J. Med. 325, 105-109 25 Weiss, R. (1993) Science 260, 1273-1278 26 Lucas, S.B.et al. (1993) AIDS 7, 1569-1579 27 Del Amo, J. et al. (1996) AIDS 10, 1563-1569 28 Pantaleo, G. et al. I1993) Nature 362, 355-358 29 Giorgi, J.V. and Detels, R. (1989) Clin. Immunol. lmmunopathol. 52, 10-18 30 Levacher,M. et aI. (1992) Clin. Exp. Immunol. 90, 376-382 31 Copeland, K.F.T. and Heeney, J.T. (1996) Microhiol. Rev. 60, 722-742 32 Saag, M.S. etal. (1996) Nat. Med. 2, 625-629 33 Huber, B.T., Hsu, P-N. and Sutkowski, N. (1996) Microbiol. Rev. 60, 473-482 34 Westby, M., Manca, F. and Dalgleish, A.G. (1996) Immunol. Today 17, 120-126 35 Dadaglio, D. et al. (1994) J. Exp. Med. 179, 413-424 36 Garcia, S. et aI. (1996) Blood 88, 2151-2161 37 Shearer, G.M. and Clerici, M. I1991) AIDS 5, 245-253 38 Di Rienzo, A.M. et al. (1994) Eur. J. Immunol. 24, 34-40 39 Poccia, F. et al. (19961J. Immunol. 157 449-461 40 Constant, P. et al. (1994) Science 264,267-270 41 Gougeon, M.L. (1995) Cell Death Differ. 2, 1-8 42 Gougeon, M.L. et al. i1996) J. Immunol. 156, 3509-3520 43 Boudet, F., Lecoeur, H. and Gougeon, M.L. (1996)]. Immunol. 156, 2282-2293 44 Amendola, A. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
11057-11062
45 Gougeon, M.L. et al. (1997)J. Immunol. 158, 2964-2976 46 Baruchel,S. and Wainherg, M.A. (1992) J. Leukocyte Biol. 52, 111-114 47 Westendorp, M.O. et al. (1995) EMBO ]. 14, 546-554 48 Ehret, A. et al. (1996) J. Virol. 70, 6502-6507 49 Lopez, O. et al. (1996) Clin. Chim. Acta 247, 181-187 50 Torres-Rocca,J.F. et aI. (1995) Cell Death Differ. 2, 309-319 51 Fultz, P.N. et al. (1992) AIDS Res. Hum. Retroviruses 8, 313-317 52 Villinger, F. et al. (1996) Immunol. Lett. 51, 59-68 53 Ho, D.D. (1992) Lancet 339, 1549 54 Staprans, S.I. et al. (1995)]. Exp. Med. 182, 1727-1737 55 O'Brien, W.A. et al. (1995) Blood 86, 1082-1089 56 Stanley, S.K. et al. (1996) New Engl. ]. Med. 334, 1222-1230 57 Ho, D.D, et al. (1995) Nature 373, 123-126 58 Poll, G. et al. (1994) Res. Immunol. 145, 578-582 59 Nightingale, S.D. et al. (1992)J. Infect. Dis. 165, 1082-1085 60 Wallis, R.S. et al. (1993)J. Infect. Dis. 167, 43-48 61 Goletti, D. et al. (1996) J. lmmunol. 157, 1271-1278 62 Lederman, M.M. et al. (1994) J. AIDS 7, 727-733 63 Zhang, Y. et al. (1995) J. Clin. Invest. 95, 2324 64 Toossi, Z. et al. (1993)J. Exp. Med. 177, 1511-1516 65 Bush, C.E. etal. (1996)J. AIDS Hum. Retroviruses 13, 23-26 66 Lemaitre, M. et al. (1992)Infect. Immun. 60, 742-748 67 Brenner, C., Neyrolles, O. and Blanchard, A. (1996) Front. Biosci. 1, 42-54 68 Lo, S-C. (1992) in Mycoplasmas, Molecular Biology and Pathogenesis (Maniloff,J. et al., eds), pp. 525-545, ASM Press 69 Wang, R.Y.H. et al. (1992) Lancet 340, 1312-1316 70 Grau, O. etal. (1995)]. Infect. Dis. 172, 672-681 71 Kedes, D.H. etaL (1996) Nat. Med. 2, 918-924 72 Simpson, G.R. et al. (1996) Lancet 349, 1133-1138
PrP genetics in sheep and the implications for scrapie and BSE Nora Hunter patterns of incidence of scrapie The strong links between PrP genotype ovine spongiform enin sheep and of some forms of and the occurrence of scrapie in sheep cephalopathy (BSE) is a strengthen evidence supporting the central h u m a n TSE, and there is overrelatively new disease of whelming evidence that the genimportance of the PrP protein in the cattle that has attracted a great etic c o m p o n e n t in these species development of transmissible spongiform deal of publicity recently. It is encephalopathies, despite the fact that the is the PrP gene. Although polyone of a group of related dismorphisms and mutations of cattle PrP gene has, so far, failed to show eases k n o w n collectively as the PrP gene are linked to incuany association between PrP alleles and transmissible spongiform enbation period of experimental susceptibility to BSE. cephalopathies (TSEs), the earliTSE and to the occurrence of est recognized of which is N. Hunter is in the Institute for Animal Health, natural TSE disease in mice, scrapie, which occurs in sheep BBSRC/MRC Neuropathogenesis Unit, sheep, goats and humans, such and goats. The TSEs are all West Mains Road, Edinburgh, UK EH9 3JF. linkage has not yet been demslowly progressive, inevitably tel: +44 131 6675204, fax: +44 131 6 6 8 3872, onstrated in cattle. fatal, neurodegenerative disore-mail: [email protected] PrP consists of ~250 amino ders that are characterized by acids (exact length depends on the species), is glycosylvacuolated brain neurones and the deposition of an ated at either one, or both, of two possible glycosylation abnormal form of a host protein (PrP or prion protein). sites and is attached to the outside of the neuronal cell Most TSEs have also been shown to be experimentally m e m b r a n e by a glycosylphosphatidylinositol (GPI) transmissible. There is a strong genetic component in the
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scrapie and BSE (Ref. 7). Genotypes encoding QQw! are more susceptible to scrapie. For example, in Suffolk Normal Abnormal sheep the genotype AA136RR!s4QQ171 is most susceptible, although with Name PrPc PrPsc less than 100% incidence: it is quite PK Sensitive Partially resistant a common genotype among healthy Detergent Soluble Insoluble animals, even in infected flocks 9J°. Length ~250 amino acids ~250 amino acids Structure (z helix and loops [3 sheet PrP genetic variation in Suffolk Glycosylated Two sites Two sites sheep is much less than in the soMolecular weight(-PK) 3 3 - 3 5 kDa 33-35 kDa called 'valine breeds'. Breeds such as Molecular weight (+PK) Degraded 27-30 kDa Cheviots, Swaledales and Shetlands Antigen!city Bind to same antibodies Bind to same antibodies encode PrP gene alleles with valine Location Cell surface, GPI-anchored Fibrils, deposits at codon 136. The rare genotype Expression Many tissues Brain, CNS, lymph VVI36RRI54QQ171 appears to be exnodes, spleen, tonsils tremely susceptible to scrapie 4,H and, Expression in disease Protein levels constant Protein levels increase when it does occur, it is almost always Turnover Rapid Slow in scrapie-affected sheep: it has thereScrapie infectivity Does not co-purify Co-purifies fore been suggested that scrapie may Abbreviations:CNS, central nervoussystem; GPI,glycosylphosphatidylinositol;PK, proteinase K. simply be a genetic disease!2. However, healthy animals of this genotype can live up to 8 years of age (around the maximum age for sheep in commercial flocks), which anchor !. The protein was first discovered in an abnormal conformation (PrP sc) and is the constituent of is well past the usual 2-4 years seen in scrapie-affected animals 4,~3.Thus, the genetic disease hypothesis seems scrapie-associated-fibrils (SAF), a general characteristic of TSEs (Table 1 ). It was quickly realized that naturally less likely than an etiology that involves host genetic control of susceptibility to an infecting agent. In some occurring polymorphic or variant forms of the normal valine breed sheep flocks affected by scrap!e, there is protein (PrP c) are associated with differences in incua survival advantage if genotypes encode certain PrP bation period of experimental scrapie in laboratory mice 2 and in sheep 3. This area of study has blossomed, alleles, such as AI36H154Q171 and A136R154R171. Thus, and there are now many such disease-associated poly- despite having a high risk allele such as V136Rls4Q171, animals are unlikely to develop scrapie if their genomorphisms and mutations in sheep and in humans. This article will concentrate on what is known about types are VA!36HRIs4QQrfor VA136RR154RQ171(Refs 4,14). However, this does not occur in all outbreaks; ruminants. for example, VA136RRls4RQ17! sheep can sometimes be susceptible to scrapie ~s. Natural scrapie and PrP genotype Studies of natural scrapie have confirmed the importance of three codons (136, 154 and 171) (see Table 2) Scrapie strains in the sheep PrP gene4-6 that were originally shown to be There are some intriguing differences between flocks associated with differing incubation periods following and between breeds. Why, for example, is the genotype experimental challenge of sheep with different sources that is most susceptible in Suffolks (AAI~6RRIs4QQJ7~) of scrapie and BSE (Refs 3,7). Although there are breed usually found to be resistant to natural scrapie in differences in PrP allele frequencies and in disease- Cheviots (a valine breed)? In addition, why do some flocks of valine breeds show scrapie only in animals associated alleles, some clear 'rules' have emerged from this work. The most resistant genotype is AA136RR154 encoding the V136R154Q171allele (VV136RRl~4QQI71 and RR171. Only one out of hundreds of scrap!e-affected VA13~RRl.s4QQI71), whereas in other flocks, despite sheep worldwide has been reported with this genotype the existence of Vl.~6R154Q171-encoding sheep, scrapie appears to target only QQI7~ genotypes and occurs in - a Japanese Suffolk sheepL AA136RR154RR!71animals both VA136RR154QQ17~and AA136RRls4QQn sheep6? are also resistant to experimental challenge with both The answers could come from studies of experimental TSE in sheep, where different sources of scrapie and Table 2. Sheep PrP gene -~ the three most important BSE apparently target sheep according to their genodisease-related polymorphisms type at either codon 136 or codon 171. Following challenge by injection, the scrapie source SSBP/1 affects Codon Amino acid alternatives Single-letter code Cheviot sheep encoding the V136Rls4QI7! allele, whereas the scrapie source CH1641, as well as BSE, targets 136 Valine V136 sheep primarily according to the codon 171 genotype, Alanine A136 producing disease with the shortest incubation period 154 Arginine R154 in sheep that are QQ171 (Ref. 7). By extending these Histidine H154 findings to naturally affected sheep, it is possible that 171 Arginine R~r~ there are also various types or strains of natural scrapie Glutamine Q~z~ that target particular sheep breeds and/or different PrP Table 1. Normal and abnormal PrP
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codons. Currently, the best way to investigate strains is by passage of natural scrapie sources by injection into a panel of mouse lines where characteristics of incubation periods and brain area vacuolation give distinct profiles or patterns .6. As not all natural scrapie sources transmit easily to mice, an important objective for future research will be to facilitate this process by creating transgenic mice that carry various sheep PrP gene alleles. Cattle PrP genotype and BSE When BSE was found in cattle, the cattle PrP gene was investigated for similar markers of resistance or susceptibility to those found in sheep. The cattle gene, however, is remarkably invariant compared with the sheep and human PrP genes. There has been one major polymorphism described - that of a difference in the numbers of an octapeptide repeat (either five or six copies) within the PrP protein 17. In humans, many variations (4-14) in the octapeptide repeat number have been found, some of which show clear linkage to the incidence of human TSE (Ref. 18). In cattle, the most common number of octapeptide repeats is six, and three genotypes have been described: 5:5, 6:6 and the heterozygote 6:5. In a study comparing the frequencies of these genotypes in BSE-affected and healthy cattle, there was no difference between the two groups, with about 90% of animals being 6:6 and 10% being 6:5. The genotype 5:5 was rare (<1%) and was not found in the BSE-affected animalsC It is possible that the 5:5 genotype is associated with resistance because all BSE cattle bad at least one copy of the six-repeat allele, but this remains to be established by direct experimental challenge of either 5:5 cattle or transgenic mice expressing bovine PrP gene alleles. Cattle appear to be unusual in that a PrP-genotyperelated link with TSE incidence has not, as yet, been demonstrated for them. BSE challenge does show such differences in other species, including mice 16, sheep v and goats 2° (where intracerebral challenge with BSE gives incubation period differences related to the goats' genotypes at codon 142). It may be that all cattle would be susceptible to BSE if they received a high enough dose of infection, or it may be that a polymorphism outside the protein-coding region could provide such a link. This possibility is currently under investigation. Scrapie transmission and etiology As the association of PrP genotype and response to scrapie is now so well understood in sheep, it is now possible to design better experiments aimed at revealing the etiology of the disease. Although there have been many such studies in the past, the sheep used were of unknown PrP genotype and susceptibility to scrapie, and the results were often contradictory. However, infectivity has been found in both the alimentary tract and placental tissue of sheep, potentially implicating horizontal and vertical transmission of natural scrapie. Since the discovery of the PrP protein, it has been found that its abnormal, aggregated form (PrP so) is so closely associated with infection that it can be used as
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a marker for the presence of infectivity. One view is that PrP sc itself is the infectious agent or prion 21, but others suggest that infectivity is a separate entity that nevertheless relies on PrP for successful infection and is closely associated with PrP sc (Ref. 22). So how would these ideas fit with the genetics of PrP as it is understood in sheep? The conversion of the normal host protein, PrP c, to the aggregated form, PrP so, is fundamental to the disease process, and it may be that the rate of conversion (which can be persuaded to occur in vitro > ) is allele dependent. Thus, in sheep, the protein produced by the allele linked to the highest incidence of scrapie (V136Rls4QlTl) might convert easily to the PrP sC form and result in a relatively rapid onset of disease. In contrast, the other alleles that are associated less strongly with disease or with longer survival times might produce PrP c that converts less easily, whereas the allele associated most strongly with resistance (A136Rls4R17|) might produce a form of PrP c that does not convert at all or does so with extreme difficulty. Recent support for this idea has come from in vitro experiments 24. In this hypothesis, the more PrP sc aggregating in the brain of an affected animal, the more likely it is that scrapie disease will occur; indeed, PrP sc is believed to be toxic to neurones 2s. Symptoms could also be produced through the sudden loss of the 'normal' function of PrP c in the adult animal. What this normal function might be is still not clear; however, transgenic mice that have a lifelong absence of PrP expression (PrP-null mice) have impaired neuronal electrophysiological characteristics 26,2v and lack sleep continuity 28. H o w then is the disease passed from one animal to another? One idea is that the PrP s< in the inoculum acts as a template for the deposition of more aggregated PrP protein in the new host. This idea is supported by recent experiments in yeast where heritable information may be passed on to daughter cells via an autocatalytic alteration in the conformation of a normal protein 29. But however interesting and relevant these ideas are, many scientists think that an additional factor other than PrP conformation may still be required for disease. For example, in about 55% of cases in transmission studies, BSE caused disease in C57BL/6 mice without producing the disease-related form of PrP (Ref. 30). This has led to the conclusion that, although PrP is of major importance, some further unidentified agent may be involved. Comparison with other diseases Scrapie injection does not cause disease in mice in which the PrP gene has been knocked out (PrP-null mice) 31. The crucial importance of PrP gene expression in disease occurrence is strikingly similar to the relationship between expression of viral receptors and the ability of associated viruses to infect cells 32,33.Mutations of such receptors have also been shown to be linked to resistance to disease 34. This does not necessarily mean that TSEs are caused by a virus, but suggests that a crucial first step in the infectious process may involve a recognition and binding reaction: PrP sc must, after all, come into direct or indirect contact with PrP c (expressed on
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toms is now without question. However, future experiments must address the more subtle details of the biology of TSEs and the findings that the PrP genotype hypothesis does not explain. Only in this way will the great debate about the nature of the TSE agent finally be settled.
Q u e s t i o n s for future research • What is the nature of the infectious agent that causes scrapie? • Does the disease result simply from the conversion of PrP c to PrP sc, or is there another factor involved? • What is the significance of PrP gene variation and its association with disease incidence, and how does this operate at the molecular level? • Do different strains of natural scrapie exist? • Is there a non-protein-coding region polymorphism or mutation that is associated with the incidence of BSE in cattle?
References
cell surfaces, just like viral receptors) to convert it to more PrP sc and cause disease. However, this idea does not immediately explain all the experimental results. For example, a mutation linked to the development of Gerstmann-Str~iussler syndrome (GSS) in humans causes disease spontaneously in transgenic mice overexpressing the mutated GSS/PrP transgene 3s. In this case, the mutated protein expressed from the GSS/PrP transgene may immediately fold into the PrP sc form without any further molecular interaction. In disease terms, it may not matter how PrP sc enters the body (via inoculation or mutated gene), as long as it does SO.
Whatever the nature of the TSE agents, their modes of transmission are not understood. It is not known, for example, whether resistant sheep are truly resistant and are able to fight off infection or whether they still harbour infectivity, but the infectious agents have an incubation period that is longer than the sheep lifespan. If the latter idea is correct, these animals could act as sources of infection (vectors) for other sheep. The idea of hidden, yet still transmissible, infection is not without precedent; for example, slowly progressive measles infection can, rarely, pass to the brain causing degeneration after an apparently healthy gap of several years 36. If scrapie behaves in a similar fashion, symptomless carriers could be the means by which susceptible individuals acquire infection. TSE research continues to produce intriguing results. The critical importance of the PrP gene and its many variant forms in the appearance of disease symp-
1 Hope,J. (1993)Arch. Virol. 7, 201-214 2 Carlson,G.A.et al. (1986) Cell 46, 503-511 3 Goldmann,W. etal. (1991)J. Gen. Virol. 72, 2411-2417 4 Hunter, N. et al. (1996)Arch. Virol. 141,809-824 5 Belt,P.B.G.M.etal. (1995)J. Gen. Virol. 76, 509-517 6 Clouscard,C. et al. (1995)J. Gen. Virol. 76, 2097-2101 7 Goldmann,W. et al. (1994)J. Gen. Virol. 75, 989-995 8 Ikeda,T. et al. (1995)]. Gen. Virol. 76, 2577-2581 9 Westaway,D. et al. (1994) Genes Dev. 8, 959-969 10 Hunter, N. et al. (1997) Vet. Rec. 140, 59-63 11 Hunter,N. et al. (1994)Arch. Virol. 137, 171-177 12 Ridley,R.M. and Baker,H.F. (1995)Br. Med. J. 311, 1071-1075 13 Hunter,N. et al. (1997)Nature 386, 137 14 Laplanche,J.L. et al. (1993) Genomics 15, 30-37 15 Hunter,N. et al. Vet. Rec. (in press) 16 Bruce,M. et al. (1994) Philos. Trans. R. Soc. London Set. B 343, 405-411 17 Goldmann,W. et al. (1991)]. Gen. ViroL 72,201-204 18 Poulter,M. et al. (1992) Brain 115, 675-685 19 Hunter, N. et al. (1994) Vet. Rec. 135,400-403 20 Goldmann,W. et al. (1996)J. Gen. Virol. 77, 2885-2891 21 Prusiner,S.B.et al. (1990) Cell 63, 673-686 22 Somerville,R.A. (1991) Rev. Med. Virol. 1,131-139 23 Caughey,B. et al. (1995) Chem. Biol. 2, 807-817 24 Bossers,A. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4931-4936 25 Hope,J. et al. (1996)Neurodegeneration 5, 1-11 26 Collinge,J. et al. (1994) Nature 370, 295-297 27 Manson,J.C. et al. (1995) Neurodegeneration 4, 113-115 28 Tobler,I. et al. (1997)J. Neurosci. 17, 1869-1879 29 Tuite, M. and Lindquist,S. (1996) Trends Genet. 12, 467-471 30 Lasmezas,C. et al. (1997) Science 275,402-405 31 Bueler,H. et al. (1993) Cell 73, 1339-1347 32 Ren, R. etal. (1990) Ceil 63,353-362 33 Montgomery,R. et al. (1996) Cell 87, 427-436 34 Liu, R. etal. (1996) Cell 86, 367-377 35 Hsiao, K.K.et al. (1990) Science 250, 1587-1590 36 Primrose,N. and Dimmock,S. (1994) Introduction to Modern Virology (4th edn), BlackwellScience
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18 Dean, M. et al. (1996)Science 273, 1856-1862 19 Huang, Y.X. et al. (1996) Nat. Med. 2, 1240-1243 20 Meyer, L. and CarrY,N. (1996) Presse Med. 25,379-384 21 Butcher, G,A. (1992) Parasitol. Today 8, 307-311 22 Dorrucci, M. et al. (1995)J. Infect. Dis. 172, 1503-1508 23 Toure-Baide,A. et al. (1996) Infect. Immun. 64, 744-750 24 Greenberg, A.E. et al. (1991)New Engl. J. Med. 325, 105-109 25 Weiss, R. (1993) Science 260, 1273-1278 26 Lucas, S.B.et al. (1993) AIDS 7, 1569-1579 27 Del Amo, J. et al. (1996) AIDS 10, 1563-1569 28 Pantaleo, G. et al. I1993) Nature 362, 355-358 29 Giorgi, J.V. and Detels, R. (1989) Clin. Immunol. lmmunopathol. 52, 10-18 30 Levacher,M. et aI. (1992) Clin. Exp. Immunol. 90, 376-382 31 Copeland, K.F.T. and Heeney, J.T. (1996) Microhiol. Rev. 60, 722-742 32 Saag, M.S. etal. (1996) Nat. Med. 2, 625-629 33 Huber, B.T., Hsu, P-N. and Sutkowski, N. (1996) Microbiol. Rev. 60, 473-482 34 Westby, M., Manca, F. and Dalgleish, A.G. (1996) Immunol. Today 17, 120-126 35 Dadaglio, D. et al. (1994) J. Exp. Med. 179, 413-424 36 Garcia, S. et aI. (1996) Blood 88, 2151-2161 37 Shearer, G.M. and Clerici, M. I1991) AIDS 5, 245-253 38 Di Rienzo, A.M. et al. (1994) Eur. J. Immunol. 24, 34-40 39 Poccia, F. et al. (19961J. Immunol. 157 449-461 40 Constant, P. et al. (1994) Science 264,267-270 41 Gougeon, M.L. (1995) Cell Death Differ. 2, 1-8 42 Gougeon, M.L. et al. i1996) J. Immunol. 156, 3509-3520 43 Boudet, F., Lecoeur, H. and Gougeon, M.L. (1996)]. Immunol. 156, 2282-2293 44 Amendola, A. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
11057-11062
45 Gougeon, M.L. et al. (1997)J. Immunol. 158, 2964-2976 46 Baruchel,S. and Wainherg, M.A. (1992) J. Leukocyte Biol. 52, 111-114 47 Westendorp, M.O. et al. (1995) EMBO ]. 14, 546-554 48 Ehret, A. et al. (1996) J. Virol. 70, 6502-6507 49 Lopez, O. et al. (1996) Clin. Chim. Acta 247, 181-187 50 Torres-Rocca,J.F. et aI. (1995) Cell Death Differ. 2, 309-319 51 Fultz, P.N. et al. (1992) AIDS Res. Hum. Retroviruses 8, 313-317 52 Villinger, F. et al. (1996) Immunol. Lett. 51, 59-68 53 Ho, D.D. (1992) Lancet 339, 1549 54 Staprans, S.I. et al. (1995)]. Exp. Med. 182, 1727-1737 55 O'Brien, W.A. et al. (1995) Blood 86, 1082-1089 56 Stanley, S.K. et al. (1996) New Engl. ]. Med. 334, 1222-1230 57 Ho, D.D, et al. (1995) Nature 373, 123-126 58 Poll, G. et al. (1994) Res. Immunol. 145, 578-582 59 Nightingale, S.D. et al. (1992)J. Infect. Dis. 165, 1082-1085 60 Wallis, R.S. et al. (1993)J. Infect. Dis. 167, 43-48 61 Goletti, D. et al. (1996) J. lmmunol. 157, 1271-1278 62 Lederman, M.M. et al. (1994) J. AIDS 7, 727-733 63 Zhang, Y. et al. (1995) J. Clin. Invest. 95, 2324 64 Toossi, Z. et al. (1993)J. Exp. Med. 177, 1511-1516 65 Bush, C.E. etal. (1996)J. AIDS Hum. Retroviruses 13, 23-26 66 Lemaitre, M. et al. (1992)Infect. Immun. 60, 742-748 67 Brenner, C., Neyrolles, O. and Blanchard, A. (1996) Front. Biosci. 1, 42-54 68 Lo, S-C. (1992) in Mycoplasmas, Molecular Biology and Pathogenesis (Maniloff,J. et al., eds), pp. 525-545, ASM Press 69 Wang, R.Y.H. et al. (1992) Lancet 340, 1312-1316 70 Grau, O. etal. (1995)]. Infect. Dis. 172, 672-681 71 Kedes, D.H. etaL (1996) Nat. Med. 2, 918-924 72 Simpson, G.R. et al. (1996) Lancet 349, 1133-1138
PrP genetics in sheep and the implications for scrapie and BSE Nora Hunter patterns of incidence of scrapie The strong links between PrP genotype ovine spongiform enin sheep and of some forms of and the occurrence of scrapie in sheep cephalopathy (BSE) is a strengthen evidence supporting the central h u m a n TSE, and there is overrelatively new disease of whelming evidence that the genimportance of the PrP protein in the cattle that has attracted a great etic c o m p o n e n t in these species development of transmissible spongiform deal of publicity recently. It is encephalopathies, despite the fact that the is the PrP gene. Although polyone of a group of related dismorphisms and mutations of cattle PrP gene has, so far, failed to show eases k n o w n collectively as the PrP gene are linked to incuany association between PrP alleles and transmissible spongiform enbation period of experimental susceptibility to BSE. cephalopathies (TSEs), the earliTSE and to the occurrence of est recognized of which is N. Hunter is in the Institute for Animal Health, natural TSE disease in mice, scrapie, which occurs in sheep BBSRC/MRC Neuropathogenesis Unit, sheep, goats and humans, such and goats. The TSEs are all West Mains Road, Edinburgh, UK EH9 3JF. linkage has not yet been demslowly progressive, inevitably tel: +44 131 6675204, fax: +44 131 6 6 8 3872, onstrated in cattle. fatal, neurodegenerative disore-mail: [email protected] PrP consists of ~250 amino ders that are characterized by acids (exact length depends on the species), is glycosylvacuolated brain neurones and the deposition of an ated at either one, or both, of two possible glycosylation abnormal form of a host protein (PrP or prion protein). sites and is attached to the outside of the neuronal cell Most TSEs have also been shown to be experimentally m e m b r a n e by a glycosylphosphatidylinositol (GPI) transmissible. There is a strong genetic component in the
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scrapie and BSE (Ref. 7). Genotypes encoding QQw! are more susceptible to scrapie. For example, in Suffolk Normal Abnormal sheep the genotype AA136RR!s4QQ171 is most susceptible, although with Name PrPc PrPsc less than 100% incidence: it is quite PK Sensitive Partially resistant a common genotype among healthy Detergent Soluble Insoluble animals, even in infected flocks 9J°. Length ~250 amino acids ~250 amino acids Structure (z helix and loops [3 sheet PrP genetic variation in Suffolk Glycosylated Two sites Two sites sheep is much less than in the soMolecular weight(-PK) 3 3 - 3 5 kDa 33-35 kDa called 'valine breeds'. Breeds such as Molecular weight (+PK) Degraded 27-30 kDa Cheviots, Swaledales and Shetlands Antigen!city Bind to same antibodies Bind to same antibodies encode PrP gene alleles with valine Location Cell surface, GPI-anchored Fibrils, deposits at codon 136. The rare genotype Expression Many tissues Brain, CNS, lymph VVI36RRI54QQ171 appears to be exnodes, spleen, tonsils tremely susceptible to scrapie 4,H and, Expression in disease Protein levels constant Protein levels increase when it does occur, it is almost always Turnover Rapid Slow in scrapie-affected sheep: it has thereScrapie infectivity Does not co-purify Co-purifies fore been suggested that scrapie may Abbreviations:CNS, central nervoussystem; GPI,glycosylphosphatidylinositol;PK, proteinase K. simply be a genetic disease!2. However, healthy animals of this genotype can live up to 8 years of age (around the maximum age for sheep in commercial flocks), which anchor !. The protein was first discovered in an abnormal conformation (PrP sc) and is the constituent of is well past the usual 2-4 years seen in scrapie-affected animals 4,~3.Thus, the genetic disease hypothesis seems scrapie-associated-fibrils (SAF), a general characteristic of TSEs (Table 1 ). It was quickly realized that naturally less likely than an etiology that involves host genetic control of susceptibility to an infecting agent. In some occurring polymorphic or variant forms of the normal valine breed sheep flocks affected by scrap!e, there is protein (PrP c) are associated with differences in incua survival advantage if genotypes encode certain PrP bation period of experimental scrapie in laboratory mice 2 and in sheep 3. This area of study has blossomed, alleles, such as AI36H154Q171 and A136R154R171. Thus, and there are now many such disease-associated poly- despite having a high risk allele such as V136Rls4Q171, animals are unlikely to develop scrapie if their genomorphisms and mutations in sheep and in humans. This article will concentrate on what is known about types are VA!36HRIs4QQrfor VA136RR154RQ171(Refs 4,14). However, this does not occur in all outbreaks; ruminants. for example, VA136RRls4RQ17! sheep can sometimes be susceptible to scrapie ~s. Natural scrapie and PrP genotype Studies of natural scrapie have confirmed the importance of three codons (136, 154 and 171) (see Table 2) Scrapie strains in the sheep PrP gene4-6 that were originally shown to be There are some intriguing differences between flocks associated with differing incubation periods following and between breeds. Why, for example, is the genotype experimental challenge of sheep with different sources that is most susceptible in Suffolks (AAI~6RRIs4QQJ7~) of scrapie and BSE (Refs 3,7). Although there are breed usually found to be resistant to natural scrapie in differences in PrP allele frequencies and in disease- Cheviots (a valine breed)? In addition, why do some flocks of valine breeds show scrapie only in animals associated alleles, some clear 'rules' have emerged from this work. The most resistant genotype is AA136RR154 encoding the V136R154Q171allele (VV136RRl~4QQI71 and RR171. Only one out of hundreds of scrap!e-affected VA13~RRl.s4QQI71), whereas in other flocks, despite sheep worldwide has been reported with this genotype the existence of Vl.~6R154Q171-encoding sheep, scrapie appears to target only QQI7~ genotypes and occurs in - a Japanese Suffolk sheepL AA136RR154RR!71animals both VA136RR154QQ17~and AA136RRls4QQn sheep6? are also resistant to experimental challenge with both The answers could come from studies of experimental TSE in sheep, where different sources of scrapie and Table 2. Sheep PrP gene -~ the three most important BSE apparently target sheep according to their genodisease-related polymorphisms type at either codon 136 or codon 171. Following challenge by injection, the scrapie source SSBP/1 affects Codon Amino acid alternatives Single-letter code Cheviot sheep encoding the V136Rls4QI7! allele, whereas the scrapie source CH1641, as well as BSE, targets 136 Valine V136 sheep primarily according to the codon 171 genotype, Alanine A136 producing disease with the shortest incubation period 154 Arginine R154 in sheep that are QQ171 (Ref. 7). By extending these Histidine H154 findings to naturally affected sheep, it is possible that 171 Arginine R~r~ there are also various types or strains of natural scrapie Glutamine Q~z~ that target particular sheep breeds and/or different PrP Table 1. Normal and abnormal PrP
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codons. Currently, the best way to investigate strains is by passage of natural scrapie sources by injection into a panel of mouse lines where characteristics of incubation periods and brain area vacuolation give distinct profiles or patterns .6. As not all natural scrapie sources transmit easily to mice, an important objective for future research will be to facilitate this process by creating transgenic mice that carry various sheep PrP gene alleles. Cattle PrP genotype and BSE When BSE was found in cattle, the cattle PrP gene was investigated for similar markers of resistance or susceptibility to those found in sheep. The cattle gene, however, is remarkably invariant compared with the sheep and human PrP genes. There has been one major polymorphism described - that of a difference in the numbers of an octapeptide repeat (either five or six copies) within the PrP protein 17. In humans, many variations (4-14) in the octapeptide repeat number have been found, some of which show clear linkage to the incidence of human TSE (Ref. 18). In cattle, the most common number of octapeptide repeats is six, and three genotypes have been described: 5:5, 6:6 and the heterozygote 6:5. In a study comparing the frequencies of these genotypes in BSE-affected and healthy cattle, there was no difference between the two groups, with about 90% of animals being 6:6 and 10% being 6:5. The genotype 5:5 was rare (<1%) and was not found in the BSE-affected animalsC It is possible that the 5:5 genotype is associated with resistance because all BSE cattle bad at least one copy of the six-repeat allele, but this remains to be established by direct experimental challenge of either 5:5 cattle or transgenic mice expressing bovine PrP gene alleles. Cattle appear to be unusual in that a PrP-genotyperelated link with TSE incidence has not, as yet, been demonstrated for them. BSE challenge does show such differences in other species, including mice 16, sheep v and goats 2° (where intracerebral challenge with BSE gives incubation period differences related to the goats' genotypes at codon 142). It may be that all cattle would be susceptible to BSE if they received a high enough dose of infection, or it may be that a polymorphism outside the protein-coding region could provide such a link. This possibility is currently under investigation. Scrapie transmission and etiology As the association of PrP genotype and response to scrapie is now so well understood in sheep, it is now possible to design better experiments aimed at revealing the etiology of the disease. Although there have been many such studies in the past, the sheep used were of unknown PrP genotype and susceptibility to scrapie, and the results were often contradictory. However, infectivity has been found in both the alimentary tract and placental tissue of sheep, potentially implicating horizontal and vertical transmission of natural scrapie. Since the discovery of the PrP protein, it has been found that its abnormal, aggregated form (PrP so) is so closely associated with infection that it can be used as
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a marker for the presence of infectivity. One view is that PrP sc itself is the infectious agent or prion 21, but others suggest that infectivity is a separate entity that nevertheless relies on PrP for successful infection and is closely associated with PrP sc (Ref. 22). So how would these ideas fit with the genetics of PrP as it is understood in sheep? The conversion of the normal host protein, PrP c, to the aggregated form, PrP so, is fundamental to the disease process, and it may be that the rate of conversion (which can be persuaded to occur in vitro > ) is allele dependent. Thus, in sheep, the protein produced by the allele linked to the highest incidence of scrapie (V136Rls4QlTl) might convert easily to the PrP sC form and result in a relatively rapid onset of disease. In contrast, the other alleles that are associated less strongly with disease or with longer survival times might produce PrP c that converts less easily, whereas the allele associated most strongly with resistance (A136Rls4R17|) might produce a form of PrP c that does not convert at all or does so with extreme difficulty. Recent support for this idea has come from in vitro experiments 24. In this hypothesis, the more PrP sc aggregating in the brain of an affected animal, the more likely it is that scrapie disease will occur; indeed, PrP sc is believed to be toxic to neurones 2s. Symptoms could also be produced through the sudden loss of the 'normal' function of PrP c in the adult animal. What this normal function might be is still not clear; however, transgenic mice that have a lifelong absence of PrP expression (PrP-null mice) have impaired neuronal electrophysiological characteristics 26,2v and lack sleep continuity 28. H o w then is the disease passed from one animal to another? One idea is that the PrP s< in the inoculum acts as a template for the deposition of more aggregated PrP protein in the new host. This idea is supported by recent experiments in yeast where heritable information may be passed on to daughter cells via an autocatalytic alteration in the conformation of a normal protein 29. But however interesting and relevant these ideas are, many scientists think that an additional factor other than PrP conformation may still be required for disease. For example, in about 55% of cases in transmission studies, BSE caused disease in C57BL/6 mice without producing the disease-related form of PrP (Ref. 30). This has led to the conclusion that, although PrP is of major importance, some further unidentified agent may be involved. Comparison with other diseases Scrapie injection does not cause disease in mice in which the PrP gene has been knocked out (PrP-null mice) 31. The crucial importance of PrP gene expression in disease occurrence is strikingly similar to the relationship between expression of viral receptors and the ability of associated viruses to infect cells 32,33.Mutations of such receptors have also been shown to be linked to resistance to disease 34. This does not necessarily mean that TSEs are caused by a virus, but suggests that a crucial first step in the infectious process may involve a recognition and binding reaction: PrP sc must, after all, come into direct or indirect contact with PrP c (expressed on
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toms is now without question. However, future experiments must address the more subtle details of the biology of TSEs and the findings that the PrP genotype hypothesis does not explain. Only in this way will the great debate about the nature of the TSE agent finally be settled.
Q u e s t i o n s for future research • What is the nature of the infectious agent that causes scrapie? • Does the disease result simply from the conversion of PrP c to PrP sc, or is there another factor involved? • What is the significance of PrP gene variation and its association with disease incidence, and how does this operate at the molecular level? • Do different strains of natural scrapie exist? • Is there a non-protein-coding region polymorphism or mutation that is associated with the incidence of BSE in cattle?
References
cell surfaces, just like viral receptors) to convert it to more PrP sc and cause disease. However, this idea does not immediately explain all the experimental results. For example, a mutation linked to the development of Gerstmann-Str~iussler syndrome (GSS) in humans causes disease spontaneously in transgenic mice overexpressing the mutated GSS/PrP transgene 3s. In this case, the mutated protein expressed from the GSS/PrP transgene may immediately fold into the PrP sc form without any further molecular interaction. In disease terms, it may not matter how PrP sc enters the body (via inoculation or mutated gene), as long as it does SO.
Whatever the nature of the TSE agents, their modes of transmission are not understood. It is not known, for example, whether resistant sheep are truly resistant and are able to fight off infection or whether they still harbour infectivity, but the infectious agents have an incubation period that is longer than the sheep lifespan. If the latter idea is correct, these animals could act as sources of infection (vectors) for other sheep. The idea of hidden, yet still transmissible, infection is not without precedent; for example, slowly progressive measles infection can, rarely, pass to the brain causing degeneration after an apparently healthy gap of several years 36. If scrapie behaves in a similar fashion, symptomless carriers could be the means by which susceptible individuals acquire infection. TSE research continues to produce intriguing results. The critical importance of the PrP gene and its many variant forms in the appearance of disease symp-
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