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Biochemical approaches to scrapie research
TIBS - October I977
220
Biochemical approaches scrapie research
to
Richard H. Kimberlin Scrapie always has been a difficult disease to study. Many of the standard techniques of virology are inapplicable to scrapie research and much reliance has been placed on the use of biochemical methods. Recent studies have revealed some problems and some new leads.
Slow virus diseases are a particularly difficult group of conditions to classify because they involve a wide variety of viruses and many quite different types of virus-host interaction underly the development of clinical disease [ 11. The outstanding features they have in common are a very long incubation period (between infection and disease) and a regular, protracted clinical course which usually ends in death. They are important because they include a number of animal diseases of economic significance, for example, Aleutian disease of ranch-reared mink, maedi and scrapie in sheep. Scrapie is relatively common in some parts of Britain and although it is impossible to calculate the total financial losses caused by this disease one can get some idea of its importance from the disastrous effect it has had on British exports of pedigree sheep, particularly to countries such as Australia and New Zealand which are currently free from scrapie. A number of chronic, fatal conditions of man are also commonly classified as slow virus diseases, for example, progressive multifocal leucoencephalopathy, subacute sclerosing panencephalitis and Creutzfeldt-Jakob disease. All three diseases are rare but it has been pointed out that these few may be just the tip of the iceberg and it is strongly suspected that many among the long list of chronic diseases of man, about which little is known, may be a consequence of a viral infection that occurred years or possibly decades earlier [2]. Multiple sclerosis is a good example of a debilitating disease of man which occurs with tragic frequency and which many believe is caused by a virus, despite the lack of conclusive evidence for this. Ethical considerations obviously restrict many studies of chronic disease in man, and even in these cases where a virus is known to be involved it is often extremely difficult to investigate how the disease R.H.K. is a Principal Scientific Officer and the leader of the Scrapie Project Group at the Agricultural Research Council’s Institute for Research on Animal Diseases, Compton. Newbury, Berkshire, U.K.
develops and what treatments can be used to prevent or cure it; in most cases a vaccine against the virus is not the answer. Hence the importance of animal models of human disease. Scrapie is an excellent example of a disease which is a problem in its natural host, the sheep, but useful as a model of at least two human diseases; Creutzfeldt-Jakob disease which although rare has been described in many countries throughout the world; and kuru, a disease found only in New Guinea but one that was killing the indigenous population at a rate of 1% per annum in the late 1950s.
It is clear, therefore, that scrapie research has become important to both veterinary and human medicine. Scrapie is a slow disease of the central nervous system that occurs naturally in sheep and goats. It is caused by a virus-like agent but genetic factors have a major controlling effect on the development of clinical disease [3]. Early attempts to develop an experimental model of scrapie in sheep were frustrated by the inability to control genetic variation in the host and by ‘the long incubation period (between infection and disease) which is rarely less than lOO120 days. The first problem was overcome by establishing a number of experimental models of the disease in mice but the second problem remains with us because the fastest mouse model of scrapie still takes over 100 days to develop. Scrapie research has been more seriously impeded by the inapplicability of many of the standard techniques for studying virus diseases. For example, the agent has not been identified ultrastructurally and electron microscopy is therefore an inappropriate technique for such studies as the localisation of the agent in cells. There is no reproducible method for
LiCl
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150 I NCUBATION
175 PERIOD
200 ( DAYS 1
225
Fig. 1. Dose-response curves for untreated (--C) extracts of scrapie mouse brain and for extracts treated with 0.5% Na dodecylsuphate (---C--) or 6 M LiCl (---a---). Whole brain was homogenised in 0.32 M sucrose and centrifuged lightly to remove nuclei, myelin and unbroken ceils. The supernatant was centrtfuged at 100,OOOg for 1 h to sediment particulate material with scrapie infectivity. The pellet was resuspended in saline (at a concentration equivalent to a 10% suspension of whole brain) and aliquots incubated with saline, SDS or LiClfor 2 h at room temperature. Ten-fold dilutions were prepared in saline and injected intracerebrally into mice. The curves represent pooled data from 6 experiments. The horizontal bars represent standard errors from a total of II-24 mice. ‘P’indicates probability of a statistically significance difference by Student’s ‘t-test’. Unpublished data of Kimberlin and Walker.
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infecting cells with scrapie in vitro, which precludes the use of cultured cells for infectivity assays of the agent. Immunological techniques for detecting the agent in tissues are not applicable to scrapie research because the host does not mount a conventional immune response to infection and the agent has not been purified sufficiently to permit the use of methods based on specific antisera [4]. At the moment the only way of detecting the presence of infectious agent is by animal inoculation. This has been developed into a quantitative titration technique which measures the number of infectious units in a tissue homogenate or extract. The assay is extremely sensitive and reasonably accurate [5]. It suffers from the disadvantage that, even with the shortest incubation models of scrapie, it takes at least 200-300 days to complete an assay and a logical sequence of 4 or 5 experiments can occupy up to 4 years!
The two major areas of scrapie research are firstly, the chemical structure of the agent [6] (and of the various strains which are known to exist) and secondly, the nature of the interactions between agent and host, from infection to the development of clinical disease [7-91. Both areas of research have successfully exploited the biological assay for scrapie irifectivity. However the paucity of additional methods of study has resulted in the extensive use of biochemical techniques, particularly in brain which is the source of the largest amounts of agent as well as the tissue that is damaged in scrapie. This article discusses a few of the recent applications of biochemistry to scrapie research. Nature of the agent Our present understanding of the scrapie agent is‘still very hazy but the working hypothesis accepted by most workers can be briefly summarised as follows [4-61.
Scrapie infectivity is closely associated with the cell membranes of tissues such as brain and spleen. All attempts to purify scrapie agent have failed and it is considered likely that it does not exist as a discrete entity, that is there is no virion. The operational size of the agent (the smallest membrane fragments with high scrapie activity) is very roughly 20-30 nm with a mol. wt. of well over 107. Despite the lack of any direct evidence, it is considered likely that membranes with scrapie activity ‘contain’ a specific scrapie nucleic acid. The results of irradiation studies with y-rays and U.V. light indicate that the putative scrapie nucleic acid may have a mol. wt. of only 105. Moreover it has been suggested that this may be the only scrapiespecific part of the infectious complex, particularly as the coding potential of so small a nucleic acid is very limited. The problem now facing us is how do we develop this hypothesis experimentally and establish it as fact? Chemical treatment of scrapie agent
INDIVIDUAL
GLYCOSIDASES (NUMBERED 1- 11)
Fig. 2. Projiles of glycosidase activity in brain. (A) Chandler source of scrapie in Compton white mice (--C) and in hamsters (--A--) ; data from [lb’] and [23]. (3) Chandler scrapie I--C) and cuprizone toxicity (--0--) in Compton mice; datafrom [I8]. (C) 22C (-0-) and 79 A (--@--I strains ofscrapie in Balblc mice: unpublished data of Kimberlin and Dickinson. Bars represent standard errors of 3 experiments. (D) Chandler scrapie (-*-_I and A774 strain of Semliki Forest virus (--O--) in A2G mice. Unpublished data of Kimberlin. Glycosidase activities were measured on fresh brain from intracerebrally infected animals in the clinical stage of scrapie, from mice fed cuprizone for 6 weeks andfrom mice killed II days after intraperitoneal Iinfection with SF virus [21]. Key to enzymes: I. a-fucosidase; 2. a-galactosidase; 3. N-acetyl-~-D-glucosaminidase; 4. &galactosidase; 5. a-mannosidase (pH 4.1) ; 6. a-glucosidase; 7. N-acetyl-/I-D-galactosaminidaFe; 8. B-glucosidme; 9. B_glucuronidase; 10. a-mannosidase (pH 6.0) ; II. j%xylosidase.
One way of probing the structure of the infectious complex is to expose it to reagents which have some specificity of action and measure their effects on infectivity titres. For example, the sensitivity of viruses to sodium dodecyl sulphate (SDS) has. been used to assess the relative importance of protein-nucleic acid and proteinprotein links in stabilising virus structure [lo]. Many experiments of this type have been carried out using concentrated salt solutions, detergents and organic solvents to reduce scrapie infectivity [6]. However it must be remembered that these scrapie experiments are carried out with very crude tissue extracts, not with purified agent. One consequence of this is that the effects of a given treatment on the nonscrapie components in the inoculum may affect the way the inoculum interacts with the host on subsequent titration, that is, the efficiency of infection may be altered. It is emphasised that the titration assay measures infectivity and this can only be used to indicate relative amounts of agent if the efficiency of infection remains constant. Fig. 1 illustrates one simple experiment carried out to investigate this problem. Treatment of scrapie brain with 6 M LiCl or 0.5 % SDS reduces infectivity by about 90-99x [6]. Let us assume that this loss of infectivity is caused solely by inactivation of agent. Then the curves that relate incubation period to the number of infectious units (dose-response curves) should be identical for treated and untreated homogenates. This is clearly not the case. Both the treated curves are shifted to the right and the shape is considerably altered.
TIBS - October
222 Even more dramatic changes in doseresponse curves can be produced simply by dry heating the ME7 strain of scrapie to temperatures of around 160”C (DickinThis son, personal communication). means that the treatments have modified scrapie pathogenesis in some way (see also [ 111) and it is therefore quite likely that the change of infectivity may reflect an alteration in the efficiency of infection as well as in the amount of agent. In further experiments, normal brain was treated with SDS and mixed with untreated scrapie brain prior to titration. The results shown in Table I indicate that scrapie infectivity was actually increased by this procedure ! Therefore these studies illustrate the complexity of effects caused by SDS (and presumably other reagents) which make it very difficult to interpret altered infectivity titres in terms of agent structure, particularly when titre differences are relatively small. Purification
of agent
If current thinking on the nature of scrapie agent is correct then purification means the removal from scrapie membranes of as much host material as possible, without losing infectivity. Brain microsomes from scrapie mice are a convenient source of high infectivity. It has been shown that treatment with mild detergents, such as lysolecithin, can remove up to 98 % of membrane protein and 90 % of phospholipids to give a lOO-fold increase in specific scrapie infectivity compared to whole brain [6]. However it is emphasised that the final extract is still very complex biochemically, and in view of the results with SDS (Table I), it is possible that the amounts of agent are actually reduced by lysolecithin treatment but the efficiency of infection may be increased to give no apparent loss of infectivity.
I977
TABLE I Alteration of the infectivity titre of a preparation of scrapie brain by the addition of SDS-treated normal brain. Normal and scrapie extracts were prepared as described in the legend to Fig. 1. A 10% suspension of normal membranes was incubated at 37°C for 2 h with 0.5% SDS. The mixture was diluted in saline to give l:/, normal membranes and 0.05 y0 SDS. Scrapie membranes were added and dilutions for inoculation into mice were performed immediately so that the exposure of agent to SDS was minimal. The data show individual titrations of 2 control and 4 treated preparations performed as a single experiment. Unpublished results of Kimberlin and Walker. Group
Infectivity
Controls Treated
7.50.7.88 8.00,8.25 8.36,8.36
titres (-log,,,
brains of scrapie mice. These changes are thought to be secondary consequences of brain damage [8] and conceivably, the single-stranded DNA could also be the result of brain damage. To prove otherwise may be difficult. The only direct approach is to show that the injection of the singlestranded DNA into mice can cause scrapie. This is unlikely to succeed unless the nucleic acid can at least be protected from nucleases in vivo. Certainly all previous attempts to produce scrapie by the injection of nucleic acids from scrapie tissues have failed [ 161. An alternative approach would be to see if there are any sequences in host nucleic acids which are complementary to the single-stranded DNA or to an RNA copy of it, made in vitro. If the answer was no, and contaminating microbial agents could be eliminated, then this would provide good circumstantial evidence that the single-stranded DNA was part of the infectious scrapie agent. Biochemical
changes in scrapie brain
Although an extraneural phase of agent replication takes place in such lymphoid
LD,,
i.c. units/O.03 Mean Mean
g brain) = 7.73 = 8.27
organs as spleen [9], scrapie is a disease that affects the CNS and it is here that histopathological lesions of scrapie are found [7]. There has been intensive study of the biochemistry of scrapie brain in an attempt to understand how the disease develops. The problems of interpretation are considerable [8]. One of the most important has been to find ways of distinguishing primary events in scrapie pathogenesis from secondary changes. The most throughly studied biochemical change in scrapie brain is the increased activity of a selected group of glycosidases (Fig. 2A). Early studies showed that the altered profile of glycosidase activity in scrapie had many of the expected features of a primary event; for example progressive development in the latter half of the incubation period in parallel with the multiplication of agent in brain and the progressive development of histological lesions [ 171. However this interpretation was changed dramatically when studies were broadened to include scrapie in hamster, and other pathological conditions.
Isolation of a single stranded DNA
The problems with agent purification can be circumvented by trying to isolate the putative scrapie nucleic acid from starting materials that have been depleted of host nucleic acids. One recent study involved the preparation of synaptic plasma membranes from scrapie brain followed by detergent treatment with lysolecithin and Triton X-100 [12]. Minute amounts of a small single-stranded DNA were isolated from this material which were not found in similar preparations of normal brain [13]. The DNA had an apparent mol. wt. of 60,000; close to the expected size of the putative scrapie nucleic acid. However it is well known that there is an abnormal synthesis of DNA [14] and an increased activity of DNAase [ 151in the
The name scrapie originates from the way many animals affected with the disease compulsively rub or scrape themselves against fixed objects. The photograph illustrates the severe loss of wool that can result from prolonged rubbing in a Herdwick sheep experimentally affected with scrapie. Thephotograph is by courtesy ofI. H. Pattbon.
TIBS - October 1977
Secondary changes in scrapie
Fig. 2A shows that the enzyme profiles for hamsters and mice are quite different, suggesting that the shape of the profile is more a feature of the host than the agent. Cuprizone (biscyclohexanone oxaldihydrazone) toxicity mimics many of the biochemical changes of scrapie brain [18] (including the abnormal DNA synthesis and the increased DNAase activity) and Fig. 2B shows that it produces in mice a glycosidase profile almost identical to that found in mouse scrapie. Interestingly, the profile produced by cuprizone in hamsters closely resembles that of hamster scrapie [9], again suggesting that the shape of the profile is characteristic of the host. One of the main histopathological lesions of cuprizone toxicity in mice is vacuolation of the white matter [20], a feature shared by the Chandler strain of scrapie (which was used in the scrapie experiments). This suggested that the similar glycosidase profiles of scrapie and cuprizone brain might be associated with white matter vacuolation. Fig. 2C indicates that’ this suggestion is incorrect because the profiles found with the 22C strain of scrapie, which produces no white matter vacuolation, were almost identical to those of the 79A strain, which produces a great deal of white matter damage [7]. Further attempts to define the underlying basis of the glycosidase changes in scrapie andcuprizone toxicity were confounded by the results of another experiment which showed that remarkably similar profiles could be produced by infection with an avirulent strain of Semliki Forest virus. This is an intriguing finding because there is evidence that the enzyme changes in the latter infection are immunologically mediated [21] whereas all the evidence for scrapie indicates just the opposite ! Polyadenylated
RNA
A useful by-product of these experiments has been the development of controls, such as cuprizone toxicity, which can be used to evaluate other biochemical findings in scrapie brain, for example the recently reported [22] decrease in amounts of nuclear polyadenylated RNA (poly(A)RNA). This is a particularly interesting finding because the reduction in poly(A)RNA can be detected within a few weeks of infection. A similar change was found in spleen, possibly indicating an association with the multiplication of agent in this tissue. But reduced amounts of poly(A)RNA were also found in liver. This organ contains very little scrapie infectivity [9], so clearly, alternative explanations must be sought. However it is important to have found a biochemical change which occurs
223 earlier in the incubation period of scrapie than any other abnormality; it is the early changes which are most likely to be of primary importance in understanding the disease ! References 1 Kimberlin, R.H. (1976) in SIow Virus Diseases R. H., ed) pp. of Animals and Man (Kimberlin, 3-13, North-Holland, Amsterdam 2 Kimberlin, R.H. (1976) New Sci. 72, 381-382 3 Dickinson, A.G. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R. H., ed.), pp. 209-241, North-Holland, Amsterdam 4 Kimberlin, R. H. (1976) Sci. Prog. Oxf 63, 461b 481 5 Kimberlin, R. H. (1976) Scrapie in the Mouse, pp. 7-10, Meadowfield Press, Co. Durham, U.K. 6 Millson, G.C., Hunter, G.D. and Kimberlin, R.H. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R.H., ed.), pp. 243-266, North-Holland, Amsterdam 7 Fraser, H. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R. H., ed.), pp. 267305, North-Holland, Amsterdam 8 Kimberlin, R.H. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R. H., ed.), pp. 307-323, North-Holland, Amsterdam 9 Outram, G. W. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R.H., ed.), pp. 3255357, North-Holland, Amsterdam
10 Boatman, S. and Kaper, J. M. (1976) Virology 70, l-16 I I Dickinson, A.G. and Fraser, H. (1969) Nature 222, 892-893 12 Somerville, R.A., Millson, G.C. and Hunter, G.D. (1976) Biochem. Sot. Trans. 4, 1112~1113 13 Corp, C. R. and Somerville, R. A. (I 976) Biochem. Sot. Trans. 4, 111~1112 14 Kimberlin, R.H. (1972) J. Neurochem. 19, 27672778 15 Millson, G.C. (1965) J. Neurochem. 12,461468 16 Hunter, G.D., Collis, S.C., Millson, G.C. and Kimberlin, R. H. (1976)J. Gen. Viral. 32,157-162 I7 Kimberlin, R. H., Millson, G. C. and Mackenzie, A. (1971) J. Comp. Pathol. 81,469477 18 Kimberlin, R. H., Millson, G. C., Bountiff, L. and Collis, S.C. (1974) J. Comp. Pathol. 84, 2633270 19 Kimberlin, R. H., Colhs, SC. and Walker, C. A. (1976) J. Comp. Pathol. 86, 135-142 20 Pattison, I. H. and Jebbett, J. N. (1971) Res. Vet. Sci. 12, 378-380 21 Suckling, A.J., Bateman, S. and Webb, H.E. (1976) Biochem. Sot. Trans. 4, 326-328 22 Corp, C. R. and Kimberlin, R. H. (1976) Biochem. Sot. Trans. 4, 113221133 23 Kimberlin, R.H. and Marsh, R.F. (1975) J. Infecf. Dis. 131,977103
On the existence of trimeric enzymes W.A. Wood 2-Keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida has been shown to be a trimeric oligomer, and recently trimeric structures have been reported for a few other enzymes. This development is discussed in the context of the belief that odd-numbered arrangements are rare andprobably not suited to efficientfunction or evolutionary survival.
In the past 15 years there has been a remarkable evolution of thinking about the types of oligomeric structures of proteins which possess suitable properties to function efficiently and to survive in Nature. Initially, as methods for probing subunit composition became available, small oligomers of both odd- and evennumbered protomer composition were considered possible and examples with n= 1 to 6 were reported [l]. However, by the late 1960s a widely held belief had developed that protomer structures which allowed odd-numbered arrangements would not survive evolutionary pressures. This feeling seems to have been generated not from any known inherent chemical instability characteristics of such oligomers, but mainly from implications of the Monod-Wyman-Changeux model of allosteric enzymes. W. A. Wood is Professor of Biochemistry at Michigan State University, East Lansing, MI 48824, U.S.A.
The Monod model [2] requires that symmetry of an oligomer be conserved when one protomer changes conformational state. Monod et al. [2] described two symmetrical arrangements : (a) isologous associations among protomers involving a binding domain with two identical binding sets, and a two-fold rotational axis of symmetry; and (b) heterologuos associations in which the binding domain is made up of two different binding sets. It was further developed that isologous, or isologous plus heterologous associations ‘give rise to evennumbered oligomers’, whereas exclusive use of heterologous domains of binding could lead to oligomers containing any number of protomers greater than two. ‘On this basis, the apparently rather wide prevalence of dimers and tetramers among oligomeric enzymes suggest rather strongly that the quaternary structures of these proteins are mostly built up by isologous polymerization’ [2]. In such consi-
220
Biochemical approaches scrapie research
to
Richard H. Kimberlin Scrapie always has been a difficult disease to study. Many of the standard techniques of virology are inapplicable to scrapie research and much reliance has been placed on the use of biochemical methods. Recent studies have revealed some problems and some new leads.
Slow virus diseases are a particularly difficult group of conditions to classify because they involve a wide variety of viruses and many quite different types of virus-host interaction underly the development of clinical disease [ 11. The outstanding features they have in common are a very long incubation period (between infection and disease) and a regular, protracted clinical course which usually ends in death. They are important because they include a number of animal diseases of economic significance, for example, Aleutian disease of ranch-reared mink, maedi and scrapie in sheep. Scrapie is relatively common in some parts of Britain and although it is impossible to calculate the total financial losses caused by this disease one can get some idea of its importance from the disastrous effect it has had on British exports of pedigree sheep, particularly to countries such as Australia and New Zealand which are currently free from scrapie. A number of chronic, fatal conditions of man are also commonly classified as slow virus diseases, for example, progressive multifocal leucoencephalopathy, subacute sclerosing panencephalitis and Creutzfeldt-Jakob disease. All three diseases are rare but it has been pointed out that these few may be just the tip of the iceberg and it is strongly suspected that many among the long list of chronic diseases of man, about which little is known, may be a consequence of a viral infection that occurred years or possibly decades earlier [2]. Multiple sclerosis is a good example of a debilitating disease of man which occurs with tragic frequency and which many believe is caused by a virus, despite the lack of conclusive evidence for this. Ethical considerations obviously restrict many studies of chronic disease in man, and even in these cases where a virus is known to be involved it is often extremely difficult to investigate how the disease R.H.K. is a Principal Scientific Officer and the leader of the Scrapie Project Group at the Agricultural Research Council’s Institute for Research on Animal Diseases, Compton. Newbury, Berkshire, U.K.
develops and what treatments can be used to prevent or cure it; in most cases a vaccine against the virus is not the answer. Hence the importance of animal models of human disease. Scrapie is an excellent example of a disease which is a problem in its natural host, the sheep, but useful as a model of at least two human diseases; Creutzfeldt-Jakob disease which although rare has been described in many countries throughout the world; and kuru, a disease found only in New Guinea but one that was killing the indigenous population at a rate of 1% per annum in the late 1950s.
It is clear, therefore, that scrapie research has become important to both veterinary and human medicine. Scrapie is a slow disease of the central nervous system that occurs naturally in sheep and goats. It is caused by a virus-like agent but genetic factors have a major controlling effect on the development of clinical disease [3]. Early attempts to develop an experimental model of scrapie in sheep were frustrated by the inability to control genetic variation in the host and by ‘the long incubation period (between infection and disease) which is rarely less than lOO120 days. The first problem was overcome by establishing a number of experimental models of the disease in mice but the second problem remains with us because the fastest mouse model of scrapie still takes over 100 days to develop. Scrapie research has been more seriously impeded by the inapplicability of many of the standard techniques for studying virus diseases. For example, the agent has not been identified ultrastructurally and electron microscopy is therefore an inappropriate technique for such studies as the localisation of the agent in cells. There is no reproducible method for
LiCl
125
150 I NCUBATION
175 PERIOD
200 ( DAYS 1
225
Fig. 1. Dose-response curves for untreated (--C) extracts of scrapie mouse brain and for extracts treated with 0.5% Na dodecylsuphate (---C--) or 6 M LiCl (---a---). Whole brain was homogenised in 0.32 M sucrose and centrifuged lightly to remove nuclei, myelin and unbroken ceils. The supernatant was centrtfuged at 100,OOOg for 1 h to sediment particulate material with scrapie infectivity. The pellet was resuspended in saline (at a concentration equivalent to a 10% suspension of whole brain) and aliquots incubated with saline, SDS or LiClfor 2 h at room temperature. Ten-fold dilutions were prepared in saline and injected intracerebrally into mice. The curves represent pooled data from 6 experiments. The horizontal bars represent standard errors from a total of II-24 mice. ‘P’indicates probability of a statistically significance difference by Student’s ‘t-test’. Unpublished data of Kimberlin and Walker.
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infecting cells with scrapie in vitro, which precludes the use of cultured cells for infectivity assays of the agent. Immunological techniques for detecting the agent in tissues are not applicable to scrapie research because the host does not mount a conventional immune response to infection and the agent has not been purified sufficiently to permit the use of methods based on specific antisera [4]. At the moment the only way of detecting the presence of infectious agent is by animal inoculation. This has been developed into a quantitative titration technique which measures the number of infectious units in a tissue homogenate or extract. The assay is extremely sensitive and reasonably accurate [5]. It suffers from the disadvantage that, even with the shortest incubation models of scrapie, it takes at least 200-300 days to complete an assay and a logical sequence of 4 or 5 experiments can occupy up to 4 years!
The two major areas of scrapie research are firstly, the chemical structure of the agent [6] (and of the various strains which are known to exist) and secondly, the nature of the interactions between agent and host, from infection to the development of clinical disease [7-91. Both areas of research have successfully exploited the biological assay for scrapie irifectivity. However the paucity of additional methods of study has resulted in the extensive use of biochemical techniques, particularly in brain which is the source of the largest amounts of agent as well as the tissue that is damaged in scrapie. This article discusses a few of the recent applications of biochemistry to scrapie research. Nature of the agent Our present understanding of the scrapie agent is‘still very hazy but the working hypothesis accepted by most workers can be briefly summarised as follows [4-61.
Scrapie infectivity is closely associated with the cell membranes of tissues such as brain and spleen. All attempts to purify scrapie agent have failed and it is considered likely that it does not exist as a discrete entity, that is there is no virion. The operational size of the agent (the smallest membrane fragments with high scrapie activity) is very roughly 20-30 nm with a mol. wt. of well over 107. Despite the lack of any direct evidence, it is considered likely that membranes with scrapie activity ‘contain’ a specific scrapie nucleic acid. The results of irradiation studies with y-rays and U.V. light indicate that the putative scrapie nucleic acid may have a mol. wt. of only 105. Moreover it has been suggested that this may be the only scrapiespecific part of the infectious complex, particularly as the coding potential of so small a nucleic acid is very limited. The problem now facing us is how do we develop this hypothesis experimentally and establish it as fact? Chemical treatment of scrapie agent
INDIVIDUAL
GLYCOSIDASES (NUMBERED 1- 11)
Fig. 2. Projiles of glycosidase activity in brain. (A) Chandler source of scrapie in Compton white mice (--C) and in hamsters (--A--) ; data from [lb’] and [23]. (3) Chandler scrapie I--C) and cuprizone toxicity (--0--) in Compton mice; datafrom [I8]. (C) 22C (-0-) and 79 A (--@--I strains ofscrapie in Balblc mice: unpublished data of Kimberlin and Dickinson. Bars represent standard errors of 3 experiments. (D) Chandler scrapie (-*-_I and A774 strain of Semliki Forest virus (--O--) in A2G mice. Unpublished data of Kimberlin. Glycosidase activities were measured on fresh brain from intracerebrally infected animals in the clinical stage of scrapie, from mice fed cuprizone for 6 weeks andfrom mice killed II days after intraperitoneal Iinfection with SF virus [21]. Key to enzymes: I. a-fucosidase; 2. a-galactosidase; 3. N-acetyl-~-D-glucosaminidase; 4. &galactosidase; 5. a-mannosidase (pH 4.1) ; 6. a-glucosidase; 7. N-acetyl-/I-D-galactosaminidaFe; 8. B-glucosidme; 9. B_glucuronidase; 10. a-mannosidase (pH 6.0) ; II. j%xylosidase.
One way of probing the structure of the infectious complex is to expose it to reagents which have some specificity of action and measure their effects on infectivity titres. For example, the sensitivity of viruses to sodium dodecyl sulphate (SDS) has. been used to assess the relative importance of protein-nucleic acid and proteinprotein links in stabilising virus structure [lo]. Many experiments of this type have been carried out using concentrated salt solutions, detergents and organic solvents to reduce scrapie infectivity [6]. However it must be remembered that these scrapie experiments are carried out with very crude tissue extracts, not with purified agent. One consequence of this is that the effects of a given treatment on the nonscrapie components in the inoculum may affect the way the inoculum interacts with the host on subsequent titration, that is, the efficiency of infection may be altered. It is emphasised that the titration assay measures infectivity and this can only be used to indicate relative amounts of agent if the efficiency of infection remains constant. Fig. 1 illustrates one simple experiment carried out to investigate this problem. Treatment of scrapie brain with 6 M LiCl or 0.5 % SDS reduces infectivity by about 90-99x [6]. Let us assume that this loss of infectivity is caused solely by inactivation of agent. Then the curves that relate incubation period to the number of infectious units (dose-response curves) should be identical for treated and untreated homogenates. This is clearly not the case. Both the treated curves are shifted to the right and the shape is considerably altered.
TIBS - October
222 Even more dramatic changes in doseresponse curves can be produced simply by dry heating the ME7 strain of scrapie to temperatures of around 160”C (DickinThis son, personal communication). means that the treatments have modified scrapie pathogenesis in some way (see also [ 111) and it is therefore quite likely that the change of infectivity may reflect an alteration in the efficiency of infection as well as in the amount of agent. In further experiments, normal brain was treated with SDS and mixed with untreated scrapie brain prior to titration. The results shown in Table I indicate that scrapie infectivity was actually increased by this procedure ! Therefore these studies illustrate the complexity of effects caused by SDS (and presumably other reagents) which make it very difficult to interpret altered infectivity titres in terms of agent structure, particularly when titre differences are relatively small. Purification
of agent
If current thinking on the nature of scrapie agent is correct then purification means the removal from scrapie membranes of as much host material as possible, without losing infectivity. Brain microsomes from scrapie mice are a convenient source of high infectivity. It has been shown that treatment with mild detergents, such as lysolecithin, can remove up to 98 % of membrane protein and 90 % of phospholipids to give a lOO-fold increase in specific scrapie infectivity compared to whole brain [6]. However it is emphasised that the final extract is still very complex biochemically, and in view of the results with SDS (Table I), it is possible that the amounts of agent are actually reduced by lysolecithin treatment but the efficiency of infection may be increased to give no apparent loss of infectivity.
I977
TABLE I Alteration of the infectivity titre of a preparation of scrapie brain by the addition of SDS-treated normal brain. Normal and scrapie extracts were prepared as described in the legend to Fig. 1. A 10% suspension of normal membranes was incubated at 37°C for 2 h with 0.5% SDS. The mixture was diluted in saline to give l:/, normal membranes and 0.05 y0 SDS. Scrapie membranes were added and dilutions for inoculation into mice were performed immediately so that the exposure of agent to SDS was minimal. The data show individual titrations of 2 control and 4 treated preparations performed as a single experiment. Unpublished results of Kimberlin and Walker. Group
Infectivity
Controls Treated
7.50.7.88 8.00,8.25 8.36,8.36
titres (-log,,,
brains of scrapie mice. These changes are thought to be secondary consequences of brain damage [8] and conceivably, the single-stranded DNA could also be the result of brain damage. To prove otherwise may be difficult. The only direct approach is to show that the injection of the singlestranded DNA into mice can cause scrapie. This is unlikely to succeed unless the nucleic acid can at least be protected from nucleases in vivo. Certainly all previous attempts to produce scrapie by the injection of nucleic acids from scrapie tissues have failed [ 161. An alternative approach would be to see if there are any sequences in host nucleic acids which are complementary to the single-stranded DNA or to an RNA copy of it, made in vitro. If the answer was no, and contaminating microbial agents could be eliminated, then this would provide good circumstantial evidence that the single-stranded DNA was part of the infectious scrapie agent. Biochemical
changes in scrapie brain
Although an extraneural phase of agent replication takes place in such lymphoid
LD,,
i.c. units/O.03 Mean Mean
g brain) = 7.73 = 8.27
organs as spleen [9], scrapie is a disease that affects the CNS and it is here that histopathological lesions of scrapie are found [7]. There has been intensive study of the biochemistry of scrapie brain in an attempt to understand how the disease develops. The problems of interpretation are considerable [8]. One of the most important has been to find ways of distinguishing primary events in scrapie pathogenesis from secondary changes. The most throughly studied biochemical change in scrapie brain is the increased activity of a selected group of glycosidases (Fig. 2A). Early studies showed that the altered profile of glycosidase activity in scrapie had many of the expected features of a primary event; for example progressive development in the latter half of the incubation period in parallel with the multiplication of agent in brain and the progressive development of histological lesions [ 171. However this interpretation was changed dramatically when studies were broadened to include scrapie in hamster, and other pathological conditions.
Isolation of a single stranded DNA
The problems with agent purification can be circumvented by trying to isolate the putative scrapie nucleic acid from starting materials that have been depleted of host nucleic acids. One recent study involved the preparation of synaptic plasma membranes from scrapie brain followed by detergent treatment with lysolecithin and Triton X-100 [12]. Minute amounts of a small single-stranded DNA were isolated from this material which were not found in similar preparations of normal brain [13]. The DNA had an apparent mol. wt. of 60,000; close to the expected size of the putative scrapie nucleic acid. However it is well known that there is an abnormal synthesis of DNA [14] and an increased activity of DNAase [ 151in the
The name scrapie originates from the way many animals affected with the disease compulsively rub or scrape themselves against fixed objects. The photograph illustrates the severe loss of wool that can result from prolonged rubbing in a Herdwick sheep experimentally affected with scrapie. Thephotograph is by courtesy ofI. H. Pattbon.
TIBS - October 1977
Secondary changes in scrapie
Fig. 2A shows that the enzyme profiles for hamsters and mice are quite different, suggesting that the shape of the profile is more a feature of the host than the agent. Cuprizone (biscyclohexanone oxaldihydrazone) toxicity mimics many of the biochemical changes of scrapie brain [18] (including the abnormal DNA synthesis and the increased DNAase activity) and Fig. 2B shows that it produces in mice a glycosidase profile almost identical to that found in mouse scrapie. Interestingly, the profile produced by cuprizone in hamsters closely resembles that of hamster scrapie [9], again suggesting that the shape of the profile is characteristic of the host. One of the main histopathological lesions of cuprizone toxicity in mice is vacuolation of the white matter [20], a feature shared by the Chandler strain of scrapie (which was used in the scrapie experiments). This suggested that the similar glycosidase profiles of scrapie and cuprizone brain might be associated with white matter vacuolation. Fig. 2C indicates that’ this suggestion is incorrect because the profiles found with the 22C strain of scrapie, which produces no white matter vacuolation, were almost identical to those of the 79A strain, which produces a great deal of white matter damage [7]. Further attempts to define the underlying basis of the glycosidase changes in scrapie andcuprizone toxicity were confounded by the results of another experiment which showed that remarkably similar profiles could be produced by infection with an avirulent strain of Semliki Forest virus. This is an intriguing finding because there is evidence that the enzyme changes in the latter infection are immunologically mediated [21] whereas all the evidence for scrapie indicates just the opposite ! Polyadenylated
RNA
A useful by-product of these experiments has been the development of controls, such as cuprizone toxicity, which can be used to evaluate other biochemical findings in scrapie brain, for example the recently reported [22] decrease in amounts of nuclear polyadenylated RNA (poly(A)RNA). This is a particularly interesting finding because the reduction in poly(A)RNA can be detected within a few weeks of infection. A similar change was found in spleen, possibly indicating an association with the multiplication of agent in this tissue. But reduced amounts of poly(A)RNA were also found in liver. This organ contains very little scrapie infectivity [9], so clearly, alternative explanations must be sought. However it is important to have found a biochemical change which occurs
223 earlier in the incubation period of scrapie than any other abnormality; it is the early changes which are most likely to be of primary importance in understanding the disease ! References 1 Kimberlin, R.H. (1976) in SIow Virus Diseases R. H., ed) pp. of Animals and Man (Kimberlin, 3-13, North-Holland, Amsterdam 2 Kimberlin, R.H. (1976) New Sci. 72, 381-382 3 Dickinson, A.G. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R. H., ed.), pp. 209-241, North-Holland, Amsterdam 4 Kimberlin, R. H. (1976) Sci. Prog. Oxf 63, 461b 481 5 Kimberlin, R. H. (1976) Scrapie in the Mouse, pp. 7-10, Meadowfield Press, Co. Durham, U.K. 6 Millson, G.C., Hunter, G.D. and Kimberlin, R.H. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R.H., ed.), pp. 243-266, North-Holland, Amsterdam 7 Fraser, H. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R. H., ed.), pp. 267305, North-Holland, Amsterdam 8 Kimberlin, R.H. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R. H., ed.), pp. 307-323, North-Holland, Amsterdam 9 Outram, G. W. (1976) in Slow Virus Diseases of Animals and Man (Kimberlin, R.H., ed.), pp. 3255357, North-Holland, Amsterdam
10 Boatman, S. and Kaper, J. M. (1976) Virology 70, l-16 I I Dickinson, A.G. and Fraser, H. (1969) Nature 222, 892-893 12 Somerville, R.A., Millson, G.C. and Hunter, G.D. (1976) Biochem. Sot. Trans. 4, 1112~1113 13 Corp, C. R. and Somerville, R. A. (I 976) Biochem. Sot. Trans. 4, 111~1112 14 Kimberlin, R.H. (1972) J. Neurochem. 19, 27672778 15 Millson, G.C. (1965) J. Neurochem. 12,461468 16 Hunter, G.D., Collis, S.C., Millson, G.C. and Kimberlin, R. H. (1976)J. Gen. Viral. 32,157-162 I7 Kimberlin, R. H., Millson, G. C. and Mackenzie, A. (1971) J. Comp. Pathol. 81,469477 18 Kimberlin, R. H., Millson, G. C., Bountiff, L. and Collis, S.C. (1974) J. Comp. Pathol. 84, 2633270 19 Kimberlin, R. H., Colhs, SC. and Walker, C. A. (1976) J. Comp. Pathol. 86, 135-142 20 Pattison, I. H. and Jebbett, J. N. (1971) Res. Vet. Sci. 12, 378-380 21 Suckling, A.J., Bateman, S. and Webb, H.E. (1976) Biochem. Sot. Trans. 4, 326-328 22 Corp, C. R. and Kimberlin, R. H. (1976) Biochem. Sot. Trans. 4, 113221133 23 Kimberlin, R.H. and Marsh, R.F. (1975) J. Infecf. Dis. 131,977103
On the existence of trimeric enzymes W.A. Wood 2-Keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida has been shown to be a trimeric oligomer, and recently trimeric structures have been reported for a few other enzymes. This development is discussed in the context of the belief that odd-numbered arrangements are rare andprobably not suited to efficientfunction or evolutionary survival.
In the past 15 years there has been a remarkable evolution of thinking about the types of oligomeric structures of proteins which possess suitable properties to function efficiently and to survive in Nature. Initially, as methods for probing subunit composition became available, small oligomers of both odd- and evennumbered protomer composition were considered possible and examples with n= 1 to 6 were reported [l]. However, by the late 1960s a widely held belief had developed that protomer structures which allowed odd-numbered arrangements would not survive evolutionary pressures. This feeling seems to have been generated not from any known inherent chemical instability characteristics of such oligomers, but mainly from implications of the Monod-Wyman-Changeux model of allosteric enzymes. W. A. Wood is Professor of Biochemistry at Michigan State University, East Lansing, MI 48824, U.S.A.
The Monod model [2] requires that symmetry of an oligomer be conserved when one protomer changes conformational state. Monod et al. [2] described two symmetrical arrangements : (a) isologous associations among protomers involving a binding domain with two identical binding sets, and a two-fold rotational axis of symmetry; and (b) heterologuos associations in which the binding domain is made up of two different binding sets. It was further developed that isologous, or isologous plus heterologous associations ‘give rise to evennumbered oligomers’, whereas exclusive use of heterologous domains of binding could lead to oligomers containing any number of protomers greater than two. ‘On this basis, the apparently rather wide prevalence of dimers and tetramers among oligomeric enzymes suggest rather strongly that the quaternary structures of these proteins are mostly built up by isologous polymerization’ [2]. In such consi-