Q INSTITUT PASTEUR/ELsEVIER Paris 1992

Res. Virol. 1992, 143, 381-386

TIMES AND TRENDS

Relationship of protease-resistant protein, scrapie-associated fibrils and tubulofilamentous particles to the agent of spongiform encephalopathies H.K. Narang Public Health Laboratory, Institute o f Pathology, Newcastle General Hospital, Westgate Road, Newcastle-Upon-Tyne NE4 6BE (UK)

SUMMARY Tubulofilamentous particles and scrapie-associated fibrils (SAF) are ultrastructural markers, while protease-resistant protein (PrP) is a molecular biological marker for all spongiform encephalopathies. Review of all published work has suggested that PrP molecules aggregate to form a three-dimensional SAF. Further reports have suggested that a single-stranded DNA wraps round SAF and acquires an outer protein coat to form tubulofilamentous particles. As incubation period increases in the infected animals, larger amounts of PrP molecules are committed to form SAF, interfering with the normal supply of PrP to cell membranes which become disrupted and eventually fragment, resulting in vacuoles typical of those found in spongiform encephalopathies.

Key-words: Scrapie, Creutzfeldt-Jakob disease, Prion, Nemavirus, Spongiform encephalopathy; Tubulofilamentous particles, SAF, PrP; Hypotheses.

INTRODUCTION

Scrapie is an infectious, natural disease in sheep, while Creutzfeldt-Jakob disease (CJD) a similar but rare condition in humans (Gajdusek, 1990); in cattle, bovine spongiform encephalopathy (SE) is a similar disease attributed to a scrapie-like agent transmitted by contaminated feed (Wilesmith et al., 1988). Cases of feline spongiform encephalopathy in domestic cats (Wyatt et al., 1990) and zoo tigers have been

Submitted June 12, 1992, accepted October, 1992.

reported (Benbow, 1990). The nature of the agent responsible for these diseases remains controversial, though many hypotheses have been proposed.

Prion/scrapie-associated fibril hypothesis The prion hypothesis proposed by Prusiner (1982) suggested that the infectious agent is composed of molecules of protease-resistant protein

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H.K. N A R A N G

of 27-30 kDa (PrP) without a nucleic acid. To distinguish it from both viruses and viroids, he introduced the term "prion" for the scrapie agent. PrP molecules aggregate to form "prion rods" ('Prusiner et al., 1983), and were previously described as scrapie-associated fibrils by electron microscopy (Merz et al., 1981, 1984).

The backbone of Prusiner's hypothesis

The prion hypothesis was based on two points: no demonstrable nucleic acid in the PrP preparation, and the high resistance of the scrapie agent to inactivation by harsh degradation procedures that specifically hydrolyse or modify nucleic acids, while treatment with protease reduces infectivity (Prusiner, 1991). However, such negative results are always vulnerable to criticism. In the preparation of plasmid DNA, we boil, treat with NaOH, SDS, and many times with phenol and chloroform, but still find it biologically active (Sambrook et al., 1989). In the polymerase chain reaction, DNA is heated several times over 90°C; furthermore, DNA has been amplified after tissue was fixed with formalin and blocked for over 40 years (Shibata et al., 1988). Nucleic acid has been able to withstand these harsh treatment procedures, but not protein. Recently, Prusiner's own group (Kellings et al., 1992) have demonstrated the presence of nucleic acid molecules of a heterogeneous population in preparations of PrP which had extensive hydrolysis treatment with Zn 2+ ions and nuclease. However, this nucleic acid appeared to be much smaller in size than the scrapie-specific ssDNA demonstrated by Narang (1990b). Furthermore, evidence is available that, following nuclease digestion, up to 4-kb polyadenylated sequences have been detected in CJD infectious fractions (Akowitz et al., 1990). Thus it would appear that the extensively Zn2+-ion-treated PrP preparations used as

CJD EM GSSS

= = =

C r e u t z f e l d t - J a k o b disease. electron microscopy.

PrP PrP ~

= =

protease-resistant protein. scrapie PrP.

Gerstmann-Strfiussler-Scheinker syndrome.

inoculum contained enough nucleic acid to account for the infectivity (Kellings et aL, 1992). Ultraviolet and ionizing radiation inactivation studies have not concluded the absence of a nucleic acid, but rather, have suggested that it must be small, in the order of 0.75 x 106 for ssDNA and 1.6x 106 for dsDNA virus (Rohwer, 1984). It is unwise to use evidence of the failure of UV light inactivation of the scrapie agent as proof of the absence of nucleic acid, since a similar resistance spectrum to UV action can be demonstrated in a number of other microorganisms (Latarjet, 1979).

Evidence that PrP is not essential for infectivity

A number of other studies have suggested that infectivity can be separated from both SAF and protein itself and that PrP is not an essential component of the infectious agent (Sklaviadis et aL. 1989). SAF/PrP can be demonstrated in brains of infected mice and hamsters, but not in their spleen, which has a similar titre of infectivity (Czub et aL, 1986). Recently, Xi et al. (1992) demonstrated that treatment of hamsters inoculated with the 263K strain of the scrapie agent with amphotericin B can retard both clinical symptoms and the appearance of PrP in the brain without affecting replication of the agent. No effect was observed in mice or hamsters infected with two other scrapie strains. These results favour involvement of a non-conventional nucleic acid genome.

Role of PrP mutation in the disease process

Transgenic mice in the presence of foreign hamster PrP gene did not develop SE or any other clinical neurological disorder (Scott et al., 1989). On the contrary, when these animals were inoculated with a known dose of the scrapie

SAF SDS SE vWF

= = = =

s c r a p i e - a s s o c i a t e d fibril. sodium dodecyl sulphate.

spongiform encephalopatby, v o n W i U e b r a n d factor.

ON SCRAPIE AND VARIOUS SPONGIFORM ENCEPHALOPATHIES

agent, they developed SE. It was originally suggested that another line of transgenic mice expressing a PrP gene with a specific 102 Pro-Leu point mutation seen in the Gerstmann-Str~iusslerScheinker syndrome (GSSS) developed a degenerative brain disease, but later it appeared that the pathology was different (Hsiao et al., 1990). It has also been established that this disease differs from other SE in that it is not associated with PrP and is not transmissible (Chesebro, 1992).

Unified theory of prion propagation Recently, the transmissible and mutable nature of the agent led to the hypothesis that the agent replicates with the help of a conjectured normal host nucleic acid serving as coprion, and would be present in a normal cell (Weissmann, 1991). The notion of the prion hypothesis or a conjectured normal host nucleic acid serving as coprion appears to be incompatible both with the existence of at least 15 distinct genetically stable strains of scrapie, with host variation and with the adaptation of the agent from one host species to another by repeated passage, as shown by a reduction in the length of incubation, and the scrapie strains breed true with a precise copying process to ensure stability of strains of the agent (Bruce and Dickinson, 1987). Thus far, PrP as an infectious protein and its role in pathogenesis remains unclear.

Tubulofilamentous particles Let us turn the clock back some years from the molecular solution to pathological observations which may offer a possible explanation for PrP/SAF being involved in the formation of tubulof'flamentous particles. These panicles were originally described in scrapie mice and subsequently have been observed in tissue from human CJD, natural scrapie in sheep and from experimental CJD, kuru and scrapie of mice and rats (Narang, 1974, 1987; Narang et al., 1972, 1987a; Baringer et al., 1981). These results have been independently confirmed by others (Liber-

383

ski et al., 1990; Gibson and Doughty, 1989). Such particles have been consistently detected in the brains of all known SE so far examined including BSE (Narang, 1974, 1975, 1987; Narang et al., 1972; Liberski, 1990; Gibson et al., 1989).

Relationship of tubulofilamentous particles to SAF/PrP It is a strange coincidence that SAF/PrP, once considered to be the agent or part of the agent, forms the core of the abnormal tubulofilamentous particles as seen in preparations from scrapie-infected hamster, mouse and human CJD brains (Narang, 1991 ; Narang and Perry, 1990; Narang et al., 1987b, 1988). By a combination of protease and nuclease treatment, it was possible to demonstrate that the tubulofilamentous particles consisted of an outer protein coat and a middle layer of ssDNA, while an innermost resistant SAF/PrP remained intact (Narang, 1991 ; Narang et al., 1987b, 1988). In contrast to the morphology of common viruses which have a two-layer structure - - a nucleic acid protected by an outer protein coat - - the tubulofilamentous particle has a novel threelayer structure with the ssDNA lying sandwiched between two layers of protein, the inner being the PrP. The nucleic acid contents of enriched preparations from scrapie-infected hamster brains analysed by the spread technique revealed specific ssDNA molecules of about 0.49 x 106 daltons (Narang, 1990b). It is interesting to note that the scrapie genome size estimation obtained by comparing the chemical and heat inactivation rate constants of the scrapie agent directly with the inactivation rate constants of bacteriophages of known nucleic acid size was in the order of 0.75 x 106 for single-stranded and 1.6 x 106 for double-stranded viruses (Rohwer, 1984).

The proposed hypothesis of tubulofilamentous particle assembly In all animal species, the normal PrP33-35 precursor protein is usually found on the cell surface (Prusiner, 1991) and is a "housekeeping"

384

H.K. N A R A N G

lar recognition site for P r P as that on the cell membrane whereby PrP33-35 loses the leader segment, and thus will not reach its target site on the cell membrane. It is possible that, after cleavage of the leader segment by the non-host accessory protein, the pro-peptide segment is convened into an active enzyme peptide. The active enzyme peptide segments generated act against each other (fig. 2). This phenomenon has been well illustrated by chymotrypsinogen, which is convened by cleavage of a single peptide bond into an active enzyme fragment called ~chymotrypsin then self-acts against other ~chymotrypsin molecules. This cleavage would explain the reduction in the weight of precursor protein from 30-35 to 27-30 kDa scrapie P r P (prpSc). The PrP sc does not act as a template, as suggested previously (Prusiner, 1991 ; Weissmann, 1991).

protein (Brown, 1988). The PrP amino acid peptide sequence can be divided into A-H domains (fig. 1). Domain A contains 34 amino acids with triple repeats of Pro Gly Gly, and domain B contains 32 amino acid with quadruple repeats of 8 amino acids in the order of Trp Gly Gin Pro His Gly Gly Gly, while the next domain C of 12 shares Gly Gly Gly with repeats of B (fig. 1). In the next D-H domains, a number of repeats are also observed and, there is homology among domains. The ssDNA wrapped around P r P / S A F codes for a non-host peptide, an "accessory" protein which acts as an enzyme and has the ability to cleave and bind precursor PrP and cause aggregation into a chain, possibly by a mechanism similar to Willebrand factor (vWF) peptide, a plasma glycoprotein which exists as a single type homomultimeric unit (Verweij et al., 1988). The "accessory" protein may have a very simi-

A B

C D E

Met Ala Asn Leu Ser Tyr Trp Leu Leu Ala Leu Phe Val Ala Mel Trp Thr Asp Val Gly Leu Cys 1 Lys Lys Arg Pro Lys (Pro GIv GIv) 1 Trp Asn Thr Gly Gly Set Arg Tyr Pro Gly Gin Gly Ser (Pro Gly Gly )I (Asn Arg ~ Pro) 2 tPro ~ Gly) I ~.~ Thr ITrD GIv Gin Pro His GIv Gly GIv)3 (TrD GIv Gill Pro His GIv Gly (~ly)3 £rrD GIv Gin Pro His GIv Gly Gly)3 f-lm GIv Gin Pro His Gly Gly Gly) 3 Fro (~ly (~lq Gly ~ (~ly Thr His Asn Gin Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Met (Ala Glv Ala) 4 Ala Ala (Ata (~ly Ala) 4 (,y_~Val GIv)5 Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Met Met His Phe Gly Asn Asp Tyr Gly Asp Arg (Tvr Tvr Arg) 6 Glu Ash Met (Ash Arq ~ Pro) 2 P,sn Gin Val ffyr Tvr Ar0) 6 Pro Val Asp Gin Tyr

F G H

Asn Asn Gin Ash A~q Phe Val His Asp Cys Val Asn lie Thr lie Lys Gin His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Gly Thr Asp lie Lsy lie Met Gly Arg (Val Val Gly) 5 Gin Met Cys Thr Thr Gin Tyr Gin Lys Gly Ser Gin Ala Tyr Tyr Asp Gly Arg Arg Ser---Ser Ala Val Leu Phe Ser Ser Pro Pro Val lie Leu Leu lie Ser Phe Leu lie Phe Leu Met Val Gly

Leader

A

B

C

D

E

22

34

39

25

28

33

F 27

G 26

H 20

Fig. 1. Amino acid sequence of PrP precursor protein with schematic representation from hamster. The segmentsof amino acid are aligned without breaking the order to show similarities(as shown by numbers) or underlining in the domains A-H. Numbers shown underneath are amino acids in each domain.

ON SCRAPIE AND

VARIOUS SPONGIFORM ENCEPHALOPA THIES =sDNA

mRNA GB

~ AP

PrP 33-35

PrP

PrP 33-35+AP Loader is cut

AP

385

d o s e o f SE a g e n t , the b r e a k i n g p o i n t o f cell m e m b r a n e s m a y not be a c h i e v e d in the life s p a n o f the host a n d the disease r e m a i n s subclinical. It is c o n c l u d e d that P r P , a n o r m a l protein, plays a role in the p a t h o g e n e s i s o f SE a n d is closely a s s o c i a t e d with s s D N A , w h i c h is securely anc h o r e d to it, b u t is not the a g e n t itself.

PrP

k--.--. T

Main PfP segment Joins Hoed with hoed of Tail to tail

T

References m

PrP 27-30 dimer T

PrP 27-30 Homomultiman¢ unit

1

Coil membrane with PrP

{A}

Normal

(B}

Infected

Fig. 2. A schematic representation of endoplasmic reticulum (ER) Golgi body (GB) showing synthesis of PrP33-35, and an "accessory" protein CAP) coded by ssDNA. When PrP33-35 molecules make contact with the AP molecules, the latter interacts either as an enzyme or else has a very similar receptor site as cell membrane. The leader segment of PrP33-35 is cut. The two large pro-peptide segments are converted into an active enzyme peptide. They act against each other and join either from head to head or tail to tail position to form a PrP, morphologically seen as SAF. In a normal cell, the cell membrane receives a continuous supply of PrP33-35 (A) whereas a slow disruption in the infected cell causes gaps and weakening of cell membrane (B).

As the P r P m o l e c u l e s are a d d e d to the c h a i n , the m o r p h o l o g i c a l assembly o f protease-resistant S A F takes place whilst s s D N A w r a p s a r o u n d S A F a n d a f t e r a c q u i r i n g a p r o t e i n coat f o r m the t u b u l o f i l a m e n t o u s particles t e r m e d n e m a v i r u s ( N a r a n g , 1990a). As P r P m o l e c u l e s are diverted a w a y f r o m cell m e m b r a n e s t o f o r m S A F , t h e r e results a g r a d u a l w e a k e n i n g o f cell m e m b r a n e s . As the p r o c e s s o f r e p l i c a t i o n o f s s D N A a n d the a c c e s s o r y p r o t e i n accelerates, m o r e a n d m o r e o f the P r P m o l e c u l e s are d i v e r t e d t o f o r m S A F , a n d w e a k cell m e m b r a n e s r e a c h a b r e a k age p o i n t . V a c u o l a t i o n o c c u r s a n d clinical symptoms become evident after a long incubat i o n p e r i o d . In s o m e hosts i n f e c t e d with a low

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