Original Paper
Intervirology 1992;34:105-111
Public Health Laboratory Institute of Pathology, Newcastle General Hospital, Newcastle-upon-Tyne, UK
Key Words Scrapie Nemavirus Fibrils scrapie-associated Spongiform encephalopathies Tubulofilamentous particles
Scrapie-Associated Tubulofilamentous Particles in Scrapie Hamsters ~
Summary Examination of thin sections from the cerebral cortex of scrapieinfected hamster brains revealed characteristic circular 26-30 nm diameter tubulofilamentous particles, identical to those previously described in both experimentaly induced scrapie in mice, hamsters and natural scrapie of sheep, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease and mice and chimpanzees infected with Creutzfeldt-Jakob disease. Longitudinal forms of tubulofilamentous particles were also observed in dendrites and myelinated axons. Both transverse and longitudinally cut particles were readily distinguished from microtubules and synaptic vesicles, thus there appears to be no relationship between tubulofilamentous particles, and microtu bules or synaptic vesicles.
Introduction Morphologically two structures have con sistently been seen by electron microscopy in all spongiform encephalopathies both in ex perimental and natural diseases: (1) scrapie-as sociated fibrils (SAF) in scrapie-infected brain and human Creutzfeldt-Jakob disease (CJD) fractions using negative contrast staining tech niques [1-2], and (2) tubulofilamentous virus like particles in synaptic terminals by thin sec tions [3-12]. In some publications tubulo-
Received : May 1,1992 Accepted: August 28,1992
filamentous particles have been referred to as tubulovesicular structures [7, 9, 11-13]. By an ‘impression’ negative staining technique, it was demonstrated that the tubulofilamentous par ticles are more complex than expected and that SAF form the core of these particles. Recently, to differentiate tubulofilamentous particles from prion/SAF, the term nemavirus (nema = filamentous) was introduced [14]. Although these particles are readily distin guishable form normal cellular structures [3-5,8,11-13,15], convincing differences from
Harash K. Narang Public Health Laboratory, Institute of Pathology Newcastle General Hospital Wcstgatc Road Newcastle-upon-Tyne NE4 6BE(UK)
€>1992 S. Karger AG, Basel 0300-5526/92/ 0342 0105S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 2:20:42 AM
Harash K. Narang
Fig. 1. Section from scrapie-in fected hamster brain cortex show ing both circular and longitudinally cut lubulofilamentous particles (P), microtubules (M) and synaptic vesicles (V). Grid b ar= 300 nm.
Materials and Methods Weaning female LGV/LAKgolden Syrian hamsters were inoculated with 0.03-ml aliquots of a 10% suspen sion of scrapie-infected brain as previously described [7-16]. Scrapie-inoculated animals had ataxia about 60 days later and died with severe wasting and immobility by about 80 days, while controls remained well. Ham sters showing signs of illness were anaesthetised, and after rapid removal the brains were fixed in 4% glutaraldehyde containing 0.05% ruthenium red as described previously [4, 5], Small blocks of grey and white matter were dissected, fixed for a further 30 min in the same fixative, and post-fixed in 1% osmic acid containing 0.05% ruthenium red for 2 h. After dehydration the tissue was embedded in Epon 812.
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Results Circular forms of tubulofilamentous par ticles identical to those previously described were readily found in postsynaptic terminals and dendrites in all scrapie-infected hamster brains (fig. 1). The diameter of the circular form was about 26-30 nm. On further close examin ation, long tubulofilamentous forms of these particles were found in longitudinally cut den drites and myelinated axons (fig. 2). These par ticles measured about 29 nm across with stri ated and spiked surfaces (fig. 2). However, microtubules measured from the same micro graph were about 22 nm in diameter with a sharply defined margin, and with a clear, prominent central lumen (fig. 2). Similar dif ferences between tubulofilamentous particles and microtubules had been observed by the negative staining technique [17, 18]. The par ticles were readily distinguishable both in transverse and longitudinal sections form nor mal microtubules and synaptic vesicles (fig. 2).
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microtubules have not been demonstrated in thin sections particularly in longitudinal sec tions. Epon blocks from previous studies were used to find tubulofilamentous particles in longitudinal thin sections.
Discussion In the present study it has been demonstrat ed that the tubulofilamentous particles are readily differentiated from the normal microtu bules and synaptic vesicles, often seen in the same section. However, Gibson and Doughty [9] speculate that the tubulofilamentous par ticles merely represent normal scrapie-specific microtubules formed due to constriction in the
neuronal processes due to the amyloid core. Tubulofilamentous particles are present in all spongiform encephalopathies and in all strains of scrapie, with or without amyloid plaques and are observed before amyloid plaques can be demonstrated. However, amyloid plaques develop late in the disease process [19]. In the present study measurements of tubulofilamen tous particles in both transverse and longitudi nal sections demonstrated that the particles are
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Fig. 2. a Section from scrapieinfected hamster brain cortex show ing in longitudinally cut myelinated axons the tubulofilamentous par ticles (P), microtubules (M) and sy naptic vesicles (V). b Note striated and spiked surfaces (arrow) of the nemavirus. Grid b ar= 300 nm.
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tion of protease and nucleases suggested that the outer coat of the tubulofilamentous par ticles consists of an outer protein coat digested by proteolytic enzyme, a middle layer of singlestranded DNA (ssDNA) removed by DNase and mung bean nuclease, and an innermost resistant SAF/proteose-resistant protein (PrP), which was immunolabelled with antisera against SAF and PrP [16-18]. It is a strange coincidence, as seen in preparations from scrapie-infected hamster brains, mice and human CJD, that SAF/PrP, once considered to be the agent or part of the agent, forms the core of these tubulofilamentous particles (fig. 3) [16-18]. It has been demonstrated that, after mild detergent treatment, SAF shows differing sedimentation characteristics and titre changes [23]. Furthermore, a number of studies have shown that infectivity is associated with SAF/ PrP [2], but infectivity can be separated from SAF/PrP [24], A feature which argues that spongiform encephalopathy agent(s) are devoid of polynu cleotides, for the prion hypothesis has come largely from the fact that the scrapie agent has been found to resist inactivation by harsh procedures that specifically hydrolyse or mod ify nucleic acids, while treatment with protease reduces the infectivity [25], This clearly demon strates that apart from any other classes of macromolecules, protein is an essential part of the scrapie agent. This property is not unique to the scrapie agent because all conventional viruses normally depend on a protein coat for their integrity. In a number of publications Prusiner [25] suggested that nucleic acid was absent in the PrP preparation. However, re cently his group in a further analysis of the PrP preparations, after treatment with Zn2+ ions and nuclease, demonstrated the presence of nucleic acid [26]. The nucleic acid contents of enriched prep arations of mitochondria/tubulofilamentous particles from the normal and scrapie-infected
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bigger, different in shape and surface structure and have no similarities with microtubules or synaptic vesicles. The tubulofilamentous particles have not been considered to be the infectious agent be cause they were thought to be absent from the brains of hamsters with experimental scrapie [8, 20] in which the highest reported concentra tions of the agent occur [21]. However, with the recent demonstration of these particles in scrapie-infected hamster brains [7], their inde pendent confirmation both in scrapie-infected hamsters [9, II, 12] and their presence in the natural scrapie of sheep and human CJD [3,15], it now appears that the tubulofilamentous par ticles are specific to the spongiform encephalo pathies. The presence of these tubulofilamen tous particles in natural scrapie, CJD [15] and bovine spongiform encephalopathy [22] is un likely to be due to the blind passage of a carrier virus as may be the case in experimental ani mals and consequently these particles can no longer be dismissed as incidental to the disease process. In hamsters the particles appear as early as 3 weeks after inoculation, while vacuolation is observed 8 weeks after inoculation [12]. In mice the particles were observed about 12 weeks after inoculation, well before vacuolation was apparent [12, 13]. Furthermore, in scrapie-in fected hamsters these structures have been demonstrated by negative staining at about 3 weeks after inoculation which precedes the ap pearance of other neurological changes [18]. The number and the density of the tubulo filamentous particles increase during the sub sequent weeks until the particles are readily seen to correlate with infective titres [12, 13]. Their appearance early in the incubation period, and also their numbers and density appear to be correlated with the duration of the incubation periods. All these points suggest that the particles may represent the agent. Treatment of impression grids with a combina
50 nm
hamster brains have been analysed by agarose gel electrophoresis [27], The results revealed a normal band corresponding to circular 16-kb mtDNA in both infected and uninfected brains, but slower migrating bands of mul timeric mtDNA were observed only in the scrapie-infected preparation. Treatment of the nucleic acid with mung bean nuclese digested multimeric mtDNA, while the normal mtDNA remained intact [27], Aiken et al. [28] suggested
that the mtDNA D-loop in the fragment may be specifically responsible for infectivity. Examin ation of the same preparation of scrapie nucleic acid as used in the gel electrophoresis, electron microscopy spread technique revealed both 16kb mtDNA, multimeric mtDNA and ssDNA molecules of about 0.49 x 106 D often observed in association with SAF [29]. However, in the normal nucleic acid preparation only mtDNA molecules of 16-kb were visualised by electron
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Fig. 3. A model of a nemavirus, ssDNA could lie as short coils round the SAF or as a spring.
Tubulofilamentous Particle Assembly and Vacuolation Hypothesis In all animal species, normal PrP contains a hydrophobic sequence at the N-terminus [28] and this protein is normally found on the cell surface [25, 30, 31]. The ssDNA is a non-host which codes for a peptide or a small protein which has an ability to bind newly synthesised PrP or precursor and cause aggregation into a chain. As the PrP molecules are added into the chain the morphological assembly of proteaseresistant SAF takes place whilst ssDNA wraps around SAF to form the tubulofilamentous particles. This might be achieved by interlink ing of the ssDNA with the host PrP gene by retrotranscription and integration of resulting DNA [32], and would explain familial inherit ance of the natural scrapie of the sheep and GSS and familial CJD cases. The linked chain of PrP peptide may not be recognised for a number of reasons; in particular, it is very simi lar to the host’s own proteins or it cannot be degraded to be recognised by lamphocytes. Thus the host has a new protein, but there is no antibody production to the altered protein. With increasing incubation time, more ssDNA is synthesised, which codes for more of the peptide which in turn interacts with normal host PrP to form more SAF, thus disrupting the normal supply to cell membrane. Since this is a slow gradual weakening of cell membranes, the disease process may start soon after inocula tion, but clinical symptoms become evident only at the end of a long incubation period.
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This also explains the gradual increase in the infective titre. At some stage of the incubation period supply and demand come into play, the plasma membrane of neurons would be getting an insufficient amount of PrP (an essential housekeeping component) and become weak and eventually break, resulting in the vacuoles typically found associated with the clinical dis ease. This gradual weakening of the cell mem brane may correlate with the long incubation period of these brain disorders. It has been suggested that a specific 102 Pro-Leu point mutation in the PrP gene seen in Gerstmann-Straussler-Scheinker syndrome may be responsible for the spongiform ence phalopathy. A few transgenic mice expressing the specific 102 Pro-Leu point mutation died and neuropathoiogical examination revealed spongiform lesions in the brain [33]. However, the disease in these mice was found to be differ ent from other spongiform encephalopathies in that the pathological appearance differed, there was no associated PrP and no demonstratable transmission has been achieved [34], In conclusion, the evidence presented so far, in particular that associated infectivity can be separated from SAF/PrP and that ampho tericin B treatment also dissociates in vivo re plication of the scrapie agent from PrP accumu lation [35], favours involvement of a non-conventional scrapie nucleic acid genome. Nemavirus with a three-layer structure (fig. 3), SAF/ PrP, ssDNA, and an outer protein coat, strongly suggests that these particles could be the scrapie agent. This brings back the tubulo filamentous particles to the forefront of spongi form encephalopathy research.
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microscopy [29], The whole length of the tubulofilamentous particle may represent a num ber of linked infective units with repeat mole cules of ssDNA (fig. 3). It remains to be con firmed whether ssDNA is related to the genome o f the scrapie agent. However, it is important to point out that only a few plant and phage viruses contain ssDNA and thus it is unlikely to be a host DNA.
1 Merz PA, Somerville RA, Wisniewski HM. Iqbal K: Abnormal fibril from scrapie infected brains. Acta Neuropathol 1981:54:63-74. 2 Merz PA, Rohwer RG, Kascsak R. Wisniewski HM, Somerville RA, Gibbs CJ Jr, Gajdusek DC: Infec tious specific particles from the un conventional slow virus diseases. Science 1984:225:437-440. 3 Narang HK: An electron micro scopic study of natural scrapie sheep brain: further observations on virus like particles and paramyxovirus like tubules. Acta Neuropathol 1974;28: 317-329. 4 Narang HK: Ruthenium red and lan thanum nitrate: A possible tracer and negative stain for scrapie particles. Acta Neuropathol 1974:29:37-43. 5 Narang HK: An electron micro scopic study of the scrapie mouse and rat. Further observations on virus-like particles with ruthenium red and lanthanum nitrate as a possible tracer and negative stain. Neurobiology 1974;4:347-363. 6 Narang HK, Shenton 13. Giorgi PP. Field EJ: Scrapie agent and neuroses. Nature 1972:24:106-107. 7 Narang HK, Asher DM, Pomeroy KL, Gajdusek DC: Abnormal tubulovesicular particles in brains of ham sters with scrapie. Proc Soc Exp Biol Med 1987;184:504-509. 8 Baringer JR. Prusiner SB, Wong JS: Scrapie associated particles in postsynaptic processes: Further ultrastructural studies. J Neuropathol Exp Neurol 1981;40:281-288. 9 Gibson PH, Doughty LA: An elec tron microscopic study of inclusion bodies in synaptic terminals of scrapie-infected animals. Acta Neu ropathol 1989;77:420-425. 10 Lampen PW, Gajdusek DC, Gibbs CJ Jr: Experimental spongiform encephalopathy (Creutzfeldt-Jakob disease) in chimpanzees. J Neuropa thol Exp Neurol 1972;30:20-32. 11 Liberski PP, Yanagihara R, Gibbs CJ Jr, Gajdusek DC: Tubulovesicular structures in experimental spongi form encephalopathy CreutzfeldtJakob disease and scrapie. Intervirology 1988:29:115-119.
12 Liberski PP. Yanagihara R. Gibbs CJ Jr, Gajdusek DC: Appearance of tubulovesicular structures in ex perimental Creutzfeldt-Jakob dis ease and scrapie proceeds the onset of clinical disease. Acta Neuropathol 1990:79:349-354. 13 Narang HK: A chronological study of experimental scrapie in mice. Virus Res 1988;9:293-306. 14 Narang HK: Bovine Spongiform Encephalopathy. Memorandum to the Agriculture Committee House of Commons Appendix. London, HMSO, 1990, vol 22, pp 237-240. 15 Narang HK: Scrapie an unconven tional virus: The current views. Proc Soc Exp Biol Med 1087:184:375-388. 16 Narang HK. Asher DM, Gajdusek DC : Evidence that DNAis present in abnormal tubulofilamentous struc tures found in scrapie. Proc Natl Acad Sei UA 1988:85:3575-3579. 17 Narang HK: Evidence of ssDNA in tubulofilamentous particles: Their relationship to scrapie-associated fi brils. Intervirology 1991:32:185-192. 18 Narang HK, Asher DM, Gajdusek DC: Tubulofilaments in negatively stained scrapie-infected brains: rela tionship to scrapie-associated fibrils. Proc Natl Acad Sei USA 1987; 84: 7730-7734. 19 Wisniewski HM, Bruce ME. Fraser H : Infectious etiology of neuritic (se nile) plaques in mice. Science 1975: 190:1108-1110. 20 Carp RI. Merz PA. Kascsak RJ. Merz G, Wisniewski HM: Biological pro perties of scrapie, an unconventional slow virus. Gen Virol 1985:53: 596-606. 21 Marsh R. Kimberlin RH: Compari son of scrapie and transmissible mink encephalopathy in hamsters: Clinical signs, pathology and pa thogenesis. J Infect Dis 1975:131: 104-110. 22 Liberski PP: Ultrastructural neuropathological features of bovine spon giform encephalopathy. J Am Vet Med Assoc 1990:196:1682. 23 Somerville RA, Merz PA, Carp Rl: Partial copurification of scrapie-as sociated fibrils and scrapie infectivity Intervirology 1986;25:48-55.
24 Sklaviadis TK, Manuelidis L, Ma nuelidis EE: Physical properties o the Creutzfeldt-Jakob disease agem J Virol 1989;63:1212-1222. 25 Prusiner SB: Molecular biology o prion diseases. Science 1991:252 1515-1522. 26 Kellings K, Meyer N, Mirenda C Prusiner SB, Riesner D: Further an alysis of nucleic acids in purifiet scrapie prion preparations by im proved return refocussing gel electro phoresis. J Gen Virol I992;73 1025-1029. 27 Narang HK. Millar NS, Asher DM Gajdusek DC: Increased multimerii mitochondrial DNA in the brain ol scrapie-infected hamsters. Interviro logy 1991:32:316-324. 28 Aiken JM. Williamson JL. Borchardl LM. Marsh RF: Presence of mito chondrial D-loop DNA in scrapieinfected brain preparations enriched for prion protein. J Virol 1990:64: 3265-3268. 29 Narang HK: Detection of singlestranded DNA in scrapie-infected brain by electron microscopy. J Mol Biol 1990;216:469-473. 30 Blalock JE, Smith EM: Hydropathic anti-complementary of amino acids based on the genetic code. Biochem Biophys Res Commun 1984:121: 203-207. 31 Hay B, Barry RA, Licberburg I, Pru siner SB, Lingappa VR: Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein. Mol Cell Biol I987;7: 914-920. 32 Murdoch GH, Sklaviadis TK. Ma nuelidis EE, Manuelidis L: Potential retroviral RNAs in Creutzfeldt-Jakob disease. J Virol 1990:64:1477-86. 33 Hsiao KK, Scott M, Foster D, Growth DF, DeArmond SJ, Prusiner SB: Spontaneous neurodegeneration in transgenic mice with mutant prion protein. Science 1990;250:1587-1590. 34 Chesebro B: PrP and the scrapie agent. Nature 1992:356:560. 35 Xi YG, Ingrosso L, Ladogana A, Masullo C, Pocchiari M : Amphotericin B treatment dissociates in vivo replica tion of the scrapie agent from PrP accumulation. Nature 1992:356: 598-601.
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References
Intervirology 1992;34:105-111
Public Health Laboratory Institute of Pathology, Newcastle General Hospital, Newcastle-upon-Tyne, UK
Key Words Scrapie Nemavirus Fibrils scrapie-associated Spongiform encephalopathies Tubulofilamentous particles
Scrapie-Associated Tubulofilamentous Particles in Scrapie Hamsters ~
Summary Examination of thin sections from the cerebral cortex of scrapieinfected hamster brains revealed characteristic circular 26-30 nm diameter tubulofilamentous particles, identical to those previously described in both experimentaly induced scrapie in mice, hamsters and natural scrapie of sheep, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease and mice and chimpanzees infected with Creutzfeldt-Jakob disease. Longitudinal forms of tubulofilamentous particles were also observed in dendrites and myelinated axons. Both transverse and longitudinally cut particles were readily distinguished from microtubules and synaptic vesicles, thus there appears to be no relationship between tubulofilamentous particles, and microtu bules or synaptic vesicles.
Introduction Morphologically two structures have con sistently been seen by electron microscopy in all spongiform encephalopathies both in ex perimental and natural diseases: (1) scrapie-as sociated fibrils (SAF) in scrapie-infected brain and human Creutzfeldt-Jakob disease (CJD) fractions using negative contrast staining tech niques [1-2], and (2) tubulofilamentous virus like particles in synaptic terminals by thin sec tions [3-12]. In some publications tubulo-
Received : May 1,1992 Accepted: August 28,1992
filamentous particles have been referred to as tubulovesicular structures [7, 9, 11-13]. By an ‘impression’ negative staining technique, it was demonstrated that the tubulofilamentous par ticles are more complex than expected and that SAF form the core of these particles. Recently, to differentiate tubulofilamentous particles from prion/SAF, the term nemavirus (nema = filamentous) was introduced [14]. Although these particles are readily distin guishable form normal cellular structures [3-5,8,11-13,15], convincing differences from
Harash K. Narang Public Health Laboratory, Institute of Pathology Newcastle General Hospital Wcstgatc Road Newcastle-upon-Tyne NE4 6BE(UK)
€>1992 S. Karger AG, Basel 0300-5526/92/ 0342 0105S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 2:20:42 AM
Harash K. Narang
Fig. 1. Section from scrapie-in fected hamster brain cortex show ing both circular and longitudinally cut lubulofilamentous particles (P), microtubules (M) and synaptic vesicles (V). Grid b ar= 300 nm.
Materials and Methods Weaning female LGV/LAKgolden Syrian hamsters were inoculated with 0.03-ml aliquots of a 10% suspen sion of scrapie-infected brain as previously described [7-16]. Scrapie-inoculated animals had ataxia about 60 days later and died with severe wasting and immobility by about 80 days, while controls remained well. Ham sters showing signs of illness were anaesthetised, and after rapid removal the brains were fixed in 4% glutaraldehyde containing 0.05% ruthenium red as described previously [4, 5], Small blocks of grey and white matter were dissected, fixed for a further 30 min in the same fixative, and post-fixed in 1% osmic acid containing 0.05% ruthenium red for 2 h. After dehydration the tissue was embedded in Epon 812.
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Results Circular forms of tubulofilamentous par ticles identical to those previously described were readily found in postsynaptic terminals and dendrites in all scrapie-infected hamster brains (fig. 1). The diameter of the circular form was about 26-30 nm. On further close examin ation, long tubulofilamentous forms of these particles were found in longitudinally cut den drites and myelinated axons (fig. 2). These par ticles measured about 29 nm across with stri ated and spiked surfaces (fig. 2). However, microtubules measured from the same micro graph were about 22 nm in diameter with a sharply defined margin, and with a clear, prominent central lumen (fig. 2). Similar dif ferences between tubulofilamentous particles and microtubules had been observed by the negative staining technique [17, 18]. The par ticles were readily distinguishable both in transverse and longitudinal sections form nor mal microtubules and synaptic vesicles (fig. 2).
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microtubules have not been demonstrated in thin sections particularly in longitudinal sec tions. Epon blocks from previous studies were used to find tubulofilamentous particles in longitudinal thin sections.
Discussion In the present study it has been demonstrat ed that the tubulofilamentous particles are readily differentiated from the normal microtu bules and synaptic vesicles, often seen in the same section. However, Gibson and Doughty [9] speculate that the tubulofilamentous par ticles merely represent normal scrapie-specific microtubules formed due to constriction in the
neuronal processes due to the amyloid core. Tubulofilamentous particles are present in all spongiform encephalopathies and in all strains of scrapie, with or without amyloid plaques and are observed before amyloid plaques can be demonstrated. However, amyloid plaques develop late in the disease process [19]. In the present study measurements of tubulofilamen tous particles in both transverse and longitudi nal sections demonstrated that the particles are
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Fig. 2. a Section from scrapieinfected hamster brain cortex show ing in longitudinally cut myelinated axons the tubulofilamentous par ticles (P), microtubules (M) and sy naptic vesicles (V). b Note striated and spiked surfaces (arrow) of the nemavirus. Grid b ar= 300 nm.
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tion of protease and nucleases suggested that the outer coat of the tubulofilamentous par ticles consists of an outer protein coat digested by proteolytic enzyme, a middle layer of singlestranded DNA (ssDNA) removed by DNase and mung bean nuclease, and an innermost resistant SAF/proteose-resistant protein (PrP), which was immunolabelled with antisera against SAF and PrP [16-18]. It is a strange coincidence, as seen in preparations from scrapie-infected hamster brains, mice and human CJD, that SAF/PrP, once considered to be the agent or part of the agent, forms the core of these tubulofilamentous particles (fig. 3) [16-18]. It has been demonstrated that, after mild detergent treatment, SAF shows differing sedimentation characteristics and titre changes [23]. Furthermore, a number of studies have shown that infectivity is associated with SAF/ PrP [2], but infectivity can be separated from SAF/PrP [24], A feature which argues that spongiform encephalopathy agent(s) are devoid of polynu cleotides, for the prion hypothesis has come largely from the fact that the scrapie agent has been found to resist inactivation by harsh procedures that specifically hydrolyse or mod ify nucleic acids, while treatment with protease reduces the infectivity [25], This clearly demon strates that apart from any other classes of macromolecules, protein is an essential part of the scrapie agent. This property is not unique to the scrapie agent because all conventional viruses normally depend on a protein coat for their integrity. In a number of publications Prusiner [25] suggested that nucleic acid was absent in the PrP preparation. However, re cently his group in a further analysis of the PrP preparations, after treatment with Zn2+ ions and nuclease, demonstrated the presence of nucleic acid [26]. The nucleic acid contents of enriched prep arations of mitochondria/tubulofilamentous particles from the normal and scrapie-infected
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bigger, different in shape and surface structure and have no similarities with microtubules or synaptic vesicles. The tubulofilamentous particles have not been considered to be the infectious agent be cause they were thought to be absent from the brains of hamsters with experimental scrapie [8, 20] in which the highest reported concentra tions of the agent occur [21]. However, with the recent demonstration of these particles in scrapie-infected hamster brains [7], their inde pendent confirmation both in scrapie-infected hamsters [9, II, 12] and their presence in the natural scrapie of sheep and human CJD [3,15], it now appears that the tubulofilamentous par ticles are specific to the spongiform encephalo pathies. The presence of these tubulofilamen tous particles in natural scrapie, CJD [15] and bovine spongiform encephalopathy [22] is un likely to be due to the blind passage of a carrier virus as may be the case in experimental ani mals and consequently these particles can no longer be dismissed as incidental to the disease process. In hamsters the particles appear as early as 3 weeks after inoculation, while vacuolation is observed 8 weeks after inoculation [12]. In mice the particles were observed about 12 weeks after inoculation, well before vacuolation was apparent [12, 13]. Furthermore, in scrapie-in fected hamsters these structures have been demonstrated by negative staining at about 3 weeks after inoculation which precedes the ap pearance of other neurological changes [18]. The number and the density of the tubulo filamentous particles increase during the sub sequent weeks until the particles are readily seen to correlate with infective titres [12, 13]. Their appearance early in the incubation period, and also their numbers and density appear to be correlated with the duration of the incubation periods. All these points suggest that the particles may represent the agent. Treatment of impression grids with a combina
50 nm
hamster brains have been analysed by agarose gel electrophoresis [27], The results revealed a normal band corresponding to circular 16-kb mtDNA in both infected and uninfected brains, but slower migrating bands of mul timeric mtDNA were observed only in the scrapie-infected preparation. Treatment of the nucleic acid with mung bean nuclese digested multimeric mtDNA, while the normal mtDNA remained intact [27], Aiken et al. [28] suggested
that the mtDNA D-loop in the fragment may be specifically responsible for infectivity. Examin ation of the same preparation of scrapie nucleic acid as used in the gel electrophoresis, electron microscopy spread technique revealed both 16kb mtDNA, multimeric mtDNA and ssDNA molecules of about 0.49 x 106 D often observed in association with SAF [29]. However, in the normal nucleic acid preparation only mtDNA molecules of 16-kb were visualised by electron
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Fig. 3. A model of a nemavirus, ssDNA could lie as short coils round the SAF or as a spring.
Tubulofilamentous Particle Assembly and Vacuolation Hypothesis In all animal species, normal PrP contains a hydrophobic sequence at the N-terminus [28] and this protein is normally found on the cell surface [25, 30, 31]. The ssDNA is a non-host which codes for a peptide or a small protein which has an ability to bind newly synthesised PrP or precursor and cause aggregation into a chain. As the PrP molecules are added into the chain the morphological assembly of proteaseresistant SAF takes place whilst ssDNA wraps around SAF to form the tubulofilamentous particles. This might be achieved by interlink ing of the ssDNA with the host PrP gene by retrotranscription and integration of resulting DNA [32], and would explain familial inherit ance of the natural scrapie of the sheep and GSS and familial CJD cases. The linked chain of PrP peptide may not be recognised for a number of reasons; in particular, it is very simi lar to the host’s own proteins or it cannot be degraded to be recognised by lamphocytes. Thus the host has a new protein, but there is no antibody production to the altered protein. With increasing incubation time, more ssDNA is synthesised, which codes for more of the peptide which in turn interacts with normal host PrP to form more SAF, thus disrupting the normal supply to cell membrane. Since this is a slow gradual weakening of cell membranes, the disease process may start soon after inocula tion, but clinical symptoms become evident only at the end of a long incubation period.
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Narang
This also explains the gradual increase in the infective titre. At some stage of the incubation period supply and demand come into play, the plasma membrane of neurons would be getting an insufficient amount of PrP (an essential housekeeping component) and become weak and eventually break, resulting in the vacuoles typically found associated with the clinical dis ease. This gradual weakening of the cell mem brane may correlate with the long incubation period of these brain disorders. It has been suggested that a specific 102 Pro-Leu point mutation in the PrP gene seen in Gerstmann-Straussler-Scheinker syndrome may be responsible for the spongiform ence phalopathy. A few transgenic mice expressing the specific 102 Pro-Leu point mutation died and neuropathoiogical examination revealed spongiform lesions in the brain [33]. However, the disease in these mice was found to be differ ent from other spongiform encephalopathies in that the pathological appearance differed, there was no associated PrP and no demonstratable transmission has been achieved [34], In conclusion, the evidence presented so far, in particular that associated infectivity can be separated from SAF/PrP and that ampho tericin B treatment also dissociates in vivo re plication of the scrapie agent from PrP accumu lation [35], favours involvement of a non-conventional scrapie nucleic acid genome. Nemavirus with a three-layer structure (fig. 3), SAF/ PrP, ssDNA, and an outer protein coat, strongly suggests that these particles could be the scrapie agent. This brings back the tubulo filamentous particles to the forefront of spongi form encephalopathy research.
Tubulofilamentous Particle in Scrapie
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microscopy [29], The whole length of the tubulofilamentous particle may represent a num ber of linked infective units with repeat mole cules of ssDNA (fig. 3). It remains to be con firmed whether ssDNA is related to the genome o f the scrapie agent. However, it is important to point out that only a few plant and phage viruses contain ssDNA and thus it is unlikely to be a host DNA.
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Ill
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References
