Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes

Cell, Vol. 73, 979-988, June 4, 1993, Copyright 0 1993 by Cell Press

Propagation of Prions with Artificial Properties in Transgenic Mice Expressing Chimeric PrP Genes Michael Scott, l Darlene Groth, * Dallas Foster, l Marilyn Torchia,’ Shu-Lian Yang,t Stephen J. DeArmond,* and Stanley 6. Prusiner’S l Department of Neurology tDepartment of Pathology *Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143

Summary Transgenic mice expressing chimeric prion protein (PrP) genes derived from Syrian hamster (SHa) and mouse (MO) PrP genes were constructed. One SHa/ MoPrP gene, designated MHSM PrP, contains five amino acid substitutions encoded by SHaPrP, while another construct, designated MHMP PrP, has two substitutions. Transgenic (Tg) (MHPM PrP) mice were susceptible to both Syrian hamster and mouse prions, whereas three lines expressing MHM2 PrP were resistant to Syrian hamster prions. The brains of Tg(MH2M PrP) mice dying of scrapie contained chimeric PrPSC and prions with an artificial host range favoring propagation in mice that express the corresponding chimeric PrP and were also transmissible, at reduced efficiency, to nontransgenic mice and hamsters. Our findings provide genetic evidence for homophilic interactions between PrPr in the inoculum and PrPC synthesized by the host. Introduction The prion protein designated PrPS” is consistently found in fractions enriched for scrapie infectivity (Gabizon et al., 1988; Prusiner et al., 1982a, 1983). A wealth of experimental data argues that infectious scrapie prions are composed largely, if not entirely, of PrPSc molecules. PrP* is believed to represent a posttranslationally modified form of a normal cellular protein denoted PrPC. In contrast with PrPC, PrPS”is resistant to protease digestion, is insoluble in nondenaturing detergents, and polymerizes into amyloid after limited proteolysis, forming rod-shaped structures that are composed primarily of the protease-resistant portion of PrPSC, designated PrP 27-30. These aggregates can be dissociated, with retention of infectivity, by dispersion into detergent-lipid-protein complexes (Gabizon et al., 1987). No covalent modification that might explain the different properties exhibited by PrPC and PrPS”has been identified (Stahl et al., 1993) but it seems likely that PrPC and PrPS” differ in tertiary or quaternary structure. Studies of the aggregation of PrP 27-30 into amyloid (Bazan et al., 1987; Caughey et al., 1991; Gasset et al., 1993; Prusiner et al., 1983) structural predictions of PrP conformation (J.-M. Gabriel, F. Cohen, R. A. Fletterick, and S. B. P., unpublished data), and infrared spectroscopy and analysis of

synthetic peptides (Gasset et al., 1992,1993) suggest that the amyloidogenic properties of PrPSc may arise from the formation of 8 sheet structures in regions that have a high a helical content in PrPC. Transmission of scrapie prions between distantly related species, such as Syrian hamsters and mice, is an inefficient process (Kimberlin et al., 1989; Scott et al., 1989). The comparative resistance to infection with prions derived from another species has been described as the “species barrier” (Pattison and Jones, 1988). In contrast, transmission between more closely related species, for example, among Syrian, Armenian, and Chinese hamsters, is much more easily accomplished (Lowenstein et al., 1990). Such studies are typified by a relatively long incubation time during the first passage in the new species; the incubation time shortens, usually within a single passage, to astablevalue that is maintained during subsequent passages. The discovery of PrP and the subsequent isolation and sequencing of the gene encoding PrP from several species of mammals, including Syrian, Armenian, and Chinese hamsters (Lowenstein et al., 1990) led to the hypothesis that the species barrier might result from differences in the sequence of PrP of different species. Experiments with transgenic mice expressing the Syrian hamster (SHa) PrP gene have clearly implicated PrP as the major determinant of susceptibility of the host species to foreign prions (Scott et al., 1989), as well as the host range of the infecting prions (Prusiner et al., 1990). In addition, transgenic SHaPrP mice inoculated with Syrian hamster-passaged prions exhibited a neuropathologic lesion profile typical of Syrian hamster scrapie, while those inoculated with mouse prions were indistinguishable from normal mice inoculated with mouse scrapie (Prusiner et al., 1990). These studies argue that the specificity of prionhost interaction is determined by the primary structure of PrP* in the inoculum as well as that of PrPC expressed in the host animal (Prusiner et al., 1990). Models of prion propagation and PrP” synthesis propose a direct, homophilic interaction between the “substrate” molecule PrPC and the “template” PrP* (Prusiner, 1991; Prusiner et al., 1990). To test this unorthodox hypothesis and investigate the mechanism of prion propagation, we created chimeric PrP genes by substituting regions of the MoPrP open reading frame (ORF) with homologous segments of SHaPrP (Scott et al., 1992). Transgenic mice expressing these chimeric PrP molecules were produced and challenged with Syrian hamster or mouse scrapie prions. One SHalMoPrP chimeric transgene, designated MHPM PrP, contains five amino acid substitutions encoded by the SHaPrP gene, while another construct, designated MHMP PrP, has two substitutions. As reported here, although MHPM differs from MHM2 PrP by only three amino acids, these two transgenes confer drastically different susceptibilities to foreign prions. MH2M prions showed a distinct preference for infection of mice expressing MHPM PrPC, but they were also able to infect both Syrian hamster and nontransgenic mice. The creation of a new species

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Membrane insertion signal

3F4 13A5 --

GPI addition signal -

of prion with an artificial host range that depends on the sequence of the PrP transgene provides genetic evidence that homophilic interaction between PrPSc molecules within the inoculated prion and PrPC synthesized by the host is apparently a crucial step in the propagation of scrapie prions. We also conclude that the specificity of this putative interaction and hence the tertiary structure of the interacting sites must depend in part upon the side chains of one or more of the amino acids at positions 138, 154, and 169. Results SHa MHPM MHM2

MO

amino acid residue species/line

PrP

x mAb3F4

IX 2-4X

Tg(MHZM)-92 Tg(MHM2)-285

4x

Tg(MHM2)-321

2x 16-32X

Tg(MHM2)-294

2-4X

Tg(SHa)-81

ox

non-Tg Figure 1. Construction Transgenes

and

Expression

of Chimeric

MolSHaPrP

(Upper panel) Relationship of chimeric PrP constructs to SHaPrP and MoPrP ORFs. The top graphic is a schematic of the SHaPrP and MoPrP ORFs. The approximate locations of amino acid sequence differences are depicted by vertical bars. The exact locations (relative to the sequence of SHaPrP) are as follows, from left to right: 5, 14, 53,71,79, 108, 11 I, 138, 154, 169,202,204,215,232,233, and 252. Those occurring in the 22 amino acid N-terminal signal peptide and within the glycoinositol phospholipid anchor signal are depicted within these lines, Also shown are the approximate locations of restriction enzyme cleavage sites used to construct chimeric molecules. These are, together with the location of their cleavage sites relative to the start of the SHaPrP ORF: open box, OxaNl(151); closed triangle, Kpnl (283); open circle, Bgll(392) (this site is not unique within the SHaPrP and MoPrP ORFs); closed box, BstEll (583). Parentheses indicate enzyme sites that do not exist in the native DNA sequence but were introduced by site-directed mutagenesis in such a way as to avoid disruption of the amino acid sequence. The remaining graphics depict the relationship of various chimeric ORFs. whose names are shown at the right. Closed regions were derived from SHaPrP, open regions from MoPrP. The lightly stippled region is homologous between SHaPrP and MoPrP. The amino acid positions of boundaries defined in this diagram are shown at the bottom. (Lower panel) Levels of chimeric Mo/SHaPrP transgene expression determined by immunoblotting. Brain homogenates were adjusted to 5 mg/ml in Tris-buffered saline and serial 2 x dilutions were applied from left to right and transferred to a nitrocellulose membrane. The blot was exposed to MAb 3F4 and developed using an enhanced

Transgenic Mice Expressing Chimeric PrP Genes We constructed several chimeric PrP gene cassettes by replacing regions of the murine PrP ORF with corresponding sections of the SHaPrP ORF (Scott et al., 1992). One chimeric construct, MHM2 PrP, has been previously described (Rogerset al., 1991; Scottet al., 1992; Taraboulos et al., 1990) and contains two amino acid substitutions from Syrian hamsters, a Leu to Met at position 108 and aVal to Met at position 111 (Figure 1, upper panel). MHM2 PrP appears to behave similarly to murine PrP; it forms recombinant MHM2 PrPS”when expressed in murine neuroblastoma cells infected with the RML isolate of mouse prions (see Experimental Procedures; Scott et al., 1992; Taraboulos et al., 1990). Another chimeric ORF, MH2M PrP, contains a total of five amino acid substitutions from Syrian hamsters. In addition to the two substitutions in MHM2 PrP, MHPM PrP contains an Ile to Met at position 138, a Tyr to Asn at position 154, and a Ser to Asn at position 169. When expressed in scrapie-infected mouse neuroblastoma cells, MH2M PrP also appears to be eligible for conversion into PrP*, but at much lower efficiency than MHM2 PrP (M. S., unpublished data). In contrast, SHaPrP does not form PrPSCwhen expressed in the same cells (Scott et al., 1992). In view of these findings, we considered that MH2M PrP might represent a new, artificial “species” of PrP that would be intermediate between those of hamsters and mice. To test this hypothesis, transgenic mice expressing MHMP PrP and MH2M PrP were constructed. Of three founders originally constructed harboring the MHPM PrP transgene, only one, designated Tg(MH2M PrP)92, proved suitable for further analysis. One line expressed the recombinant protein at low levels, and the other could not be bred. The level of expression of the transgene product in the brains of Tg(MH2M PrP)92 mice is similar to that of Tg(SHaPrP)81, a transgenic mouse line harboring the SHaPrP gene(Figure 1, lower panel) (Prusiner et al., 1990; Scott et al., 1989). We also obtained three lines expressing

chemiluminescence kit (Amersham Corporation) and exposed to X-ray film for 10 s. The transgenic mouse lines used are listed at the left, with the relative expression inferred at the right, expressed as a fraction of the level of PrP found in Syrian hamsters, upper series.

Table 1. Incubation

Times in Tg(Mo/SHaPrP)

Mice after Inoculation with either Mouse Syrian Hamster Scrapie Prions Scrapie Incubation

Times (n/n,)

Death (days f

>622 136 f 2.0

19119

150 f

34134 15115

134 23.6 144 f 3.3

26126 12112

142 f 4.4 151 f 3.5

0120 209 O/IO 15115 20/20 Q/Q

>393 >461b >484 146 f 2.1 119 f 3.3 131 f 2.5

lO/lO 14114 717

162 f 1.4 139 f 3.3 145 f 2.9

24124 26126 20/20 25125

75 51 200 180

Host

(nln0)

Illness (days f

nontransgenic nontransgenic

0110 19/19

Tg(MH2M PrP) mice SHa(Sc237) Mo(RML)

Tg(MH2M PrP)92 Tg(MH2M PrP)92

Tg(MHM2 PrP) mice SHa(Sc237) SHa(Sc237) SHa(Sc237) Mo(RML) Mo(RML) Mo(RML)

Tg(MHM2 Tg(MHM2 Tg(MHM2 Tg(MHM2 Tg(MHM2 Tg(MHM2

Tg(SHaPrP) mice SHa(Sc237) SHa(Sc237) Mo(RML) Mo(RML)

Tg(SHaPrP)El Tg(SHaPrP)7 Tg(SHaPrP)El Tg(SHaPrP)7

lnoculum Nontransgenic SHa(Sc237) Mo(RML)

SE)

SE)

mice

PrP)285 PrP)294 PrP)321 PrP)285 PrP)294 PrP)321

24124 26126 20/20 25125

75 46 194 173

f 1.15 f 1.0’ f 3.Y f 4.0”

2.5

f 1.1 f 0.8 f 3.2 f 4.8

B The reduced number of animals in the death column reflects sacrifice of some animals for immunoblotting and neuropathology. D Four animals developed scrapie at 306, 390, 418, 448 days and died at 311, 404, 419, and 453 days, respectively. ’ From Prusiner et al. (1990).

MHMP PrP, designated Tg(MHM2 PrP)285, Tg(MHM2 PrP)294, and Tg(MHM2 PrP)321. Tg(MHM2 PrP)285 and Tg(MHM2 PrP)321 mice contain levels of transgene product similar to those of Tg(SHaPrP)81 and Tg(MH2M PrP)92 mice. These transgenic mice express 2- to 4-fold more PrPCper microgram of total brain protein than normal hamsters (Figure 1, lower panel).

Scrapie Incubation Times in Mice Expressing Chimeric PrP Transgenes By comparing transgenic lineswith similar levelsof foreign PrP expression, we sought to minimize the influence of gene dosage on incubation time, which has been described for Tg(SHaPrP) mice (Prusiner et al., 1990). When Tg(MH2M PrP)92 mice had been inoculated with Syrian hamster-passaged Sc237 prions or mouse-passaged RML prions, abbreviated SHa(Sc237) and Mo(RML), respectively, all of the animals developed scrapie at - 140 days. In contrast, the Tg(MHM2 PrP)285 and Tg(MHM2 PrP)321 lines behaved like nontransgenic mice and were resistant to infection with SHa(Sc237) prions, showing no evidence of disease at >395 and >484 days, respectively (Table 1). The third Tg(MHM2 PrP) line, Tg(MHM2 PrP)294, yielded four animals that developed scrapie, at 308, 390, 418, and 448 days. However, the vast majority of animals did not show any signs of disease at >481 days. Since Tg(MHM2 PrP)294 mice expressed by far the highest levels of MHM2 PrPC (Figure 1, lower panel), we believe that the slightly increased susceptibility to SHa(Sc237) prions may be related to the level of expression of chimeric PrPC.

Brains of Tg(MH2M PrP)92 animals inoculated with SHa(Sc237) prions contained large quantities of proteaseresistant MHPM PrP* (Figure 2A, lane 4), at levels similar to those in Tg(SHaPrP)81 mice, which develop scrapie at - 75 daysafter inoculation with SHa(Sc237) prions (Figure 2A, lane 8), and Tg(SHaPrP)7 mice, which develop scrapie at - 50 days after inoculation with SHa(Sc237) prions (Figure 2A, lane 10). The presence of MH2M PrPSc suggests that MH2M (Sc237) prions are produced in these animals as well. That Tg(MH2M PrP) and Tg(MHM2 PrP) mice display markedly different susceptibility to infection with SHa(Sc237) prions argues that the larger region of identity with SHaPrP contained in MHPM PrP, as compared with MHM2 PrP, is apparently able to confer sensitivity to Syrian hamster prions. All of the transgenic mouse lines were susceptible to infection with Mo(RML) prions and succumbed to the disease at 11 O-l 50 days (Table 1). In previous studies with Tg(SHaPrP) mice, expression of SHaPrP caused a prolongation of the incubation period after inoculation with Mo(RML) scrapie prions. The most pronounced effects were with lines expressing the highest levels of SHaPrP; those same lines exhibited the shortest incubation periods following inoculation with SHa(Sc237) prions (Prusiner et al., 1990). In contrast with the Tg(SHaPrP) mice, Tg(MH2M PrP) and Tg(MHM2 PrP) mice exhibited incubation times after inoculation with Mo(RML) prions similar to those of nontransgenic littermatecontrols(Table l).Thesefindings are consistent with the hypothesis that the inhibition of Mo(RML) prion replication in Tg(SHaPrP) mice is due to inhibition by SHaPrPC of the conversion of MoPrPC to

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A

MoPrPSc, perhaps via competitive inhibition (Prusiner et al., 1990; Scott et al., 1992). MH2M PrPSc was readily detected in the brains of Mo(RML)-infected Tg(MH2M PrP)92 mice, in quantities similar to those of MHM2 PrPSc observed in the brains of Tg(MHM2 PrP)285 mice inoculated with Mo(RML) prions (Figure 28). Protease-resistant SHaPrPS” was not obsewed in Tg(SHaPrP) mice infected with mouse prions, as judged by measuring protease-resistant SHaPrP on immunoblots (Prusiner et al., 1990). Thus Tg(MH2M PrP) mice clearly differ from Tg(SHaPrP) mice and resemble Tg(MHM2 PrP) mice when inoculated with mouse prions (Table 1; Figure 28). The incubation period of Tg(MH2M PrP)92 mice following inoculation with Mo(RML) prions (Table 1) was similar to that observed for Tg(MHM2 PrP)285 mice, which express similar levels of chimeric PrP (Figure 1, lower panel). The chimeric MH2M PrPSc was indistinguishable in these animals, whether they were inoculated with Mo(RML) or SHa(Sc237) prions (Figures 2A and 28, lanes 4).

1 2 3 4 5 6 7 8 9101112

B 12

3 4 5 6 789101112

Synthesis of Chimeric Prions with Artificial Properties Brain extracts of Tg(MH2M PrP)92 mice containing chimerit MH2M(Sc237) prions were serially passaged in Tg(MH2M PrP)92 mice and Syrian hamsters (Table 2). The incubation period in Tg(MH2M PrP)92 mice shrank from - 135 days after inoculation with SHa(Sc237) prions to - 70 days after a single passage in Tg(MH2M PrP)92 mice. After a second passage in Tg(MH2M PrP)92 mice, the incubation period was reduced to -65 days (Table 2). As expected, MH2M PrPSCwas detected in the brains of these animals after all three passages (Figure 3A, lanes 4 and 8). In contrast, when MH2M(Sc237) prions derived by a single passage in Tg(MH2M PrP)92 mice were transmitted to hamsters, the incubation period was - 115 days, significantly more protracted than the incubation period in a homologous passage of SHa(Sc237) prions in hamsters, - 75 days (Table 2). After a second homologous passage in Tg(MH2M PrP)92 mice, the MH2M(Sc237) inoculum gave an even longer incubation period when reintroduced into hamsters, lengthening to - 160 days. The presence of SHaPrPSc in the recipient hamsters was confirmed by

Figure 2. Chimeric Mo/SHaPrPSc Molecules in the Brains of Tg(Mo/ SHaPrP) Mice Aliquots of brain homogenates, each containing 50 ug of total protein, were either treated with protease (even-numbered lanes) or left untreated (odd-numbered lanes) and analyzed by Western blot. Immunoblots were developed with anti-PrP MAb 3F4. (A) Inoculated with SHa(Sc237) prions. Lanes 1 and 2, Tg(MH2M PrP)92 mouse, uninoculated; lanes 3 and 4. Tg(MH2M PrP)92 mouse infected with SHa(Sc237) prions; lanes 5 and 6, Tg(SHaPrP)81 mouse, uninoculated; lanes 7 and 8, Tg(SHaPrP)81 mouse infected with SHa(Sc237) prions; lanes 9 and IO, Tg(SHaPrP)7 mouse infected with SHa(Sc237) prions; lanes 11 and 12, nontransgenic mouse control. (6) Inoculated with Mo(RML) prions. Lanes 1 and 2, Tg(MH2M PrP)92 mouse, uninoculated; lanes 3 and 4, Tg(MH2M PrP)92 mouse infected with Mo(RML) prions; lanes 5 and 6, Tg(MHM2 PrP)285 mouse, uninoculated; lanes 7and 8, Tg(MHM2 PrP)285 mouse infected with Mo(RML) prions; lanes 9 and 10, Tg(MHM2 PrP)294 mouse, uninoculated; lanes 11 and 12, Tg(MHM2 PrP)294 mouse infected with Mo(RML) prions. In both (A) and (B), the positions of protein molecular weight markers (Bio-Rad Laboratories) are shown at either side, corresponding to molecular masses (from top to bottom) of 45,31, and 24 kd. The blots were exposed to MAb 3F4 and developed using an alkaline phosphatasecoupled second antibody provided by the manufacturer (Promega).

Table 2. Incubation

Times for Chimeric Prions Passaged in Tg(MH2M PrP)92 Mice or Syrian Hamsters Scrapie Incubation Times Illness

Death

(nln$

(days f SE)

134

f 3.6 f 0.7 64 f 1.9

26/26

142

73

12/12

65

* f k 77 f

l.lb 1.9

48148

3.8

414

0.6

414

Host

@Ino)

(days * SE)

SHa(Sc237) SHa(Sc237)+MHZM SHa(Sc237)-MH2M+MH2M

Tg(MH2M PrP)92 mice Tg(MH2M PrP)92 mice Tg(MH2M PrP)92 mice

34134 22l22

SHa(Sc237) SHa(Sc237)+MH2M SHa(Sc237)-MH2M+MH2M SHa(Sc237)-MH2M-SHa

Syrian Syrian Syrian Syrian

48i48

77

23123 6/6

116 161

a See footnote ’ in Table 1. ’ From Scott et al , (1989).

hamsters hamsters hamsters hamsters

10110

818

818

10110

f 4.4 + 1.2 76 f 2.9

89 -c 1.7 136 f 3.5 187 T 2.4 85 f 1.2

Artificial Prions 963

A 12345678910

6 12

3456

78910

Figure 3. PrP= in the Brains of Affected Animals after Inoculation with Brain Extracts from Tg(MH2M PrP)92 Mice Inoculated with either SHa(Sc237). Mo(RML), or MH2M(Sc237) Prions Aliquots of brain homogenates, each containing 50 ug of total protein, were either treated with protease (even-numbered lanes) or left untreated (odd-numbered lanes) and analyzed by Western blot. (A) Probed with monoclonal antibody MAb 3F4 to identify chimeric PrP molecules. Lanes 1 and 2, Tg(MH2M PrP)92 mouse, uninoculated. Lanes 3 and 4, Tg(MH2M PrP)92 mouse infected with MH2M(Sc237) prions obtained from a single passage of SHa(Sc237) in Tg(MH2M PrP)92 mice. Lanes 5 and 6, Syrian hamster infected with MH2M(Sc237) prions obtained from a single passage of SHa(Sc237) in Tg(MH2M PrP)92 mice. Lanes 7 and 6, Tg(MH2M PrP)92 mouse infected with MH2M(Sc237) prions obtained from two consecutive serial transmissions of SHa(Sc237) prions in Tg(MH2M PrP)92 mice. Lanes 9 and 10, CD-l mice, inoculated with MH2M(Sc237) prions obtained from a single passage of SHa(Sc237) in Tg(MH2M PrP)92 mice. (B) Probed with polyclonal antibody R073 to identify all PrP molecules, including endogenous MoPrP. Samples are as in (A). The position of protein molecular weight markers (Bio-Rad Laboratories) are shown at either side of each blot, corresponding to molecular masses (from top to bottom) of 45, 31, and 24 kd.

Western blotting (Figure 3A, lane 6; M. S., unpublished data). Passaging in Tg(MH2M PrP)92 mice did not permanently alter the characteristic incubation time of the Sc237 isolate. Reintroduction into hamsters followed by an additional serial transmission in hamsters, i.e., SHa(Sc237)+ MH2M+SHa-SHa, produced incubation times of -75 days, which is characteristic of SHa(Sc237) prions in Syrian hamsters (Table 2). To confirm that the properties of SHa(Sc237) prions were not permanently altered by passage through Tg(MH2M PrP)92 mice, the patterns of PrPS”accumulation were determined by histoblotting after transmission back into Syrian hamsters (Taraboulos et al., 1992). The distribution of PrPS” in Syrian hamsters inoculated with MH2M(Sc237) prions obtained following a single passage in Tg(MH2M PrP)92 mice was similar to that in Syrian hamsters inoculated with SHa(Sc237) prions (data not shown).

Different Properties of Distinct Prion Isolates Passaged in Tg(MH2M PrP)92 Mice When Tg(MH2M PrP)92 mice were inoculated with a second distinct isolate or “strain” of Syrian hamster-passaged scrapie prions, 139H (Hecker et al., 1992; Kimberlin et al., 1989), these animals developed signs of illness at - 110 days after inoculation (Table 3). Western blotting experiments confirmed the presence of MHPM PrPSC(data not shown). Interestingly, the SHa(139H) isolate showed a somewhat different behavior during serial transmission in Tg(MH2M PrP)92 mice. Passage of SHa(l39H) prions in Tg(MH2M PrP)92 mice gave a slightly shorter incubation time of - 105 days on second passage (Table 3). These incubation times contrast with those produced by the SHa(Sc237) isolate. On first passage in Tg(MH2M PrP)92 mice, SHa(Sc237) prions gave incubation times of - 135 days (see Table l), while second passage was - 70 days (see Table 2). It will be important to learn whether the properties of the SHa(l39H) isolate are permanently altered following repeated passage through Tg(MH2M PrP)92 mice by subsequent passaging of MH2M(139H) prions back into Syrian hamsters. We also serially passaged extracts from Tg(MH2M PrP)92 mice infected with Mo(RML) prions into Tg(MH2M PrP)92 mice (Table 3). Although the significance of these data is clouded by the presence of endogenous MoPrP, some shortening of incubation period, from - 140 to - 120 days, was detected during the homologous passage (Table 3). Construction of transgenic mice devoid of an endogenous PrP gene (Biieler et al., 1992) will obviate this problem. Chimeric Prions Cross the Species Barrier MH2M prions were found to infect both mice and hamsters efficiently (Table 3). Following a single passage in Tg(MH2M PrP)92, Sc237 prions originally derived from Syrian hamsters were able to infect mice, with an average incubation period of - 185 days (Table 3). MoPrPSc was observed in CD-1 mice infected with MH2M(Sc237) prions (Figure 38, lane 10). Similarly, MH2M(RML) prions were able to infect Syrian hamsters efficiently, with an incubation period of -215 days for the first passage. The transmission of MH2M(RML) prions to hamsters was characterized by an extremely long course from first clinical signs to death, being in excess of 70 days (Table 3). PrPScdeposition in the brains of Syrian hamsters inoculated with MHPM(RML) prions was substantially different from that induced by SHa(Sc237) or MH2M(Sc237) prions. Histoblots showed, first, increased intensity and thickness of PrPS” deposition in the region of the hippocampal fissure and the molecular layer of the dentate gyrus of the hippocampus; second, prominent subpial deposition of PrP*, particularly in the molecular layer of the cerebral cortex; and third, minimal PrPScdeposition in the subependyma (data not shown). PrP Amylold Plaques in Tg(MH2M PrP)92 Mice and Syrian Hamsters In Syrian hamsters inoculated with SHa(Sc237) prions, numerous amyloid plaques are found, particularly in the

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Table 3. Incubation Times for One Mouse and Two Syrian Hamster Prion Isolates Passaged in CD-1 Swiss Mice, Syrian Hamsters, and Tg(MH2M PrP)92 Mice Scrapie Incubation

Times Illness (days + SE)

lnoculum

Host

(nh)

Mo(RML) MHPM(RML) MHSM(RML) MHPM(RML)

Tg(MH2M PrP)92 mice Tg(MH2M PrP)92 mice Syrian hamsters CD-1 mice

15115 23123 24124 29129

144 124 216 145

+ + k +

3.3 2.9 2.9 1.0

SHa(Sc237) MH2M(Sc237) MH2M(Sc237) MH2M(Sc237) Mo(Sc237)

Tg(MH2M PrP)92 mice Tg(MH2M PrP)92 mice Syrian hamsters CD-1 mice CD-1 mice

34134 22/22 23123 18118 919

134 73 116 186 152

k + IT k f

3.6 0.7 1.9 4.2b 4.0

SHa(139H) MHPM(139H) MHPM(l39H) MH2M(139H)

Tg(MH2M PrP)92 mice Tg(MH2M PrP)92 mice Syrian hamsters CD-l mice

13113 414 717

a See footnote ’ in Table 1. b Average of two experiments,

A third transmission

110 f 4.7 103 + 2.9 194 + 6.9 >232

Death (days f

SE)

12112 1202 717 19/19

151 151 293 165

f f f f

3.5 3.1 7.3 2.5

26126 12112 10110 919 919

142 65 136 217 160

f f f + +

4.4 1.2 3.5 3.7 4.4

(rho)

515 212 212

135 f 4.9 143 -c 3.0 >262 f 2.0

has not yielded any sick animals at >328 days.

subcallosal region. These plaques stained strongly with anti-PrP antiserum designated R073 (Figure 4A). Similarly, when SHa(Sc237) prions were inoculated into Tg(MH2M PrP)92 mice, kuru-type amyloid plaques developed thatwerestronglyR073 immunopositive(Figure48). Surprisingly, although inoculation of SHa(Sc237) prions into Tg(MH2M PrP)92 mice produced cerebral amyloid plaques (Figure 48) brain extract8 from Tg(MH2M PrP)92 mice inoculated with SHa(Sc237) failed to produce amyloid plaques either upon a second passage in Tg(MH2M PrP)92 mice (Figure 4C) or upon passage back into Syrian hamsters (Figure 4D). However, upon a second passage through Syrian hamsters, the ability to create PrP amyloid plaques was restored, indicating that the properties of the Sc237 isolate were not permanently changed by passage through Tg(MH2M PrP)92 mice (Figure 4E). No PrP amyloid plaques were found in the brains of Tg(MH2M PrP)92 mice inoculated with Mo(RML) prions (Figure 4F). However, a few, weakly antigenic amyloid plaques were found in 1 of 3 Tg(MH2M PrP)92 mice inoculated with MHPM(RML) prions derived by a single passage in Tg(MH2M PrP)92 mice (Figure 4G). In contrast, MH2M(RML) prions produced multiple PrP-immunopositive amyloid plaques when passaged in Syrian hamsters (Figure 4H). Discussion The SHaPrP and MoPrP genes differ at 16 positions within the ORF. Two chimeric SHa/MoPrP transgenes were created by exchanging segments of the two rodent PrP genes (Scott et al., 1992). One SHa/MoPrP transgene, designated MH2M, contains five amino acid substitutions encoded by SHaPrP, while another construct, designated MHM2, has two substitutions (see Figure 1). Although MH2M differs from MHMP PrP by only three amino acids, these two transgenes were found to confer markedly different susceptibilities to Syrian hamster prions (see Table 1).

Transgenic mice expressing chimeric MH2M PrP were susceptible to infection with SHa(Sc237) prions, whereas three other lines expressing a different chimeric gene, MHM2 PrP, were completely resistant to SHa(Sc237) prions (see Table 1). Lines containing both chimeric PrP genes were susceptible to infection with Mo(RML) prions. MHPM prions showed a distinct preference for infection of mice expressing MH2M PrPC; in addition, they were able to infect both Syrian hamsters and mice. The creation of a new species of prion with an artificial host range that is dependent on the sequence of PrP (see Table 2) argues for recognition between PrPSC molecules within the infecting prion particle and PrPC synthesized by the host. Unique Properties of a New Isolate of Syrian Hamster Scrapie Prions The successful transmission of mousederived MH2M(RML) prions to Syrian hamsters provided an opportunity to study a new Syrian hamster prion isolate. Although the incubation period for first passage into hamsters was - 215 days, we do not yet know whether this value will shorten significantly or, if so, how it will compare with known hamster prion isolates, such as Sc237 and 139H. However, we believe that SHa(RML) prions are distinct from SHa(Sc237) prions. First, the length of the course between first clinical signs and death was unusually long, in excess of 70 days (Table 3). Second, histoblotting experiments revealed several marked differences in the pattern of accumulation of PrP* (data not shown). The changes are most evident in the region of the hippocampal fissure, in which we observed a marked accumulation of PrPS” in hamsters infected with SHa(RML) prions. On the basis of these results, we conclude that SHa(RML) represents a distinct isolate or strain of hamster prions. Neuropathologic Characteristics of Prion Isolates Passaged in Tg(MH2M PrP)92 Mice The changes in amyloidogenicity of the Sc237 and RML

;$cial

Prions

Figure 4. PrP Amyloid Plaque Formation as a Function of Passage History for the Sc237 and RML Prion Isolates Each photomicrograph is from aformalin-fixed, paraffin-embedded histological section immunostained with the anti-PrP polyclonal antiserum R073. (A) Syrian hamsters inoculated with SHa(Sc237) prions. (6) Tg(MH2M PrP)92 mice inoculated with SHa(Sc237) prions. (C) Tg(MHPM PrP)92 mice inoculated with MHPM(Sc237) prions obtained from a single passage of SHa(Sc237) in Tg(MH2M PrP)92 mice. (D) Syrian hamsters inoculated with MH2M(Sc237) prions obtained from a single passage of SHa(Sc237) in Tg(MH2M PrP)92 mice. (E) Syrian hamsters inoculated with SHa(Sc237) obtained by a single serial transmission of SHa(Sc237) prions in Tg(MH2M PrP)92 mice followed by a single passage back into Syrian hamsters. (F) Tg(MH2M PrP)92 mice inoculated with Mo(RML) prions. (G) Tg(MH2M PrP)92 mice inoculated with MHPM(RML) prions obtained from a single passage of Mo(RML) prions in Tg(MH2M PrP)92 mice. (H) Syrian hamsters inoculated with MHPM(RML) prions obtained from a single passage of Mo(RML) prions in Tg(MH2M PrP)92 mice.

H

isolates upon passage through Tg(MH2M PrP)92 mice (Figure4) were unexpected. A single passage of SHa(Sc237) prions through Tg(MH2M PrP)92 mice yielded MH2M(Sc237) that gave an incubation period of - 120 days when reintroduced into Syrian hamsters (see Table 2). A subsequent passage of MH2M(Sc237) through Tg(MH2M PrP)92 mice produced MH2M(Sc237) prions that have a much longer incubation period in Syrian hamstersof - 160 days (see Table 2). Although this difference in incubation period might have been caused by the presence of residual SHa(Sc237) inoculum in the brains of Tg(MH2M PrP)92 mice infected with SHa(Sc237) it is also possible that multiple serial passages caused the isolate-specific properties to change. We note that passaging of MH2M(Sc237) prions into mice may be accompanied by some variability (Table 3). With two groups, the inocula produced disease at - 185 days (Table 3). In a third experiment, the mice were still devoid of clinical signs at the time of writing (>330 days). These data suggest that the

transmission between different species or transgenic mice with chimeric PrP genes might sometimes select for variant isolates. On the other hand, the amyloidogenic properties of the Sc237 isolate and the incubation period, at - 75 days, reappeared after two passages in Syrian hamsters (Figures 4D and 4E and see Table 2). Homophilic Interactions Feature in Prion Propagation The unique host range of MH2M prions, especially those derived by infection with SHa(Sc237) prions, have several important implications for understanding scrapie prion rep lication. Homologous transmissions, back into Tg(MH2M PrP)92 animals, are clearly favored, although the chimeric prions may also be passaged into Syrian hamsters and mice. We conclude this by observing the lengthening and shortening of incubation period during homologous or heterologous transmissions (see Tables l-3). This preference for infection of animals expressing a homologous

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PrP argues persuasively for an interaction of PrPScmolecules in the inoculum with the PrPC in cells of the host. Further evidence for the unique tropism of MH2M(Sc237) prions was obtained following serial transmission to CD-l Swiss mice. When Tg(SHaPrP) mice infected with SHa(Sc237) prions were serially passaged into mice, no evidence for transmission to the Syrian hamster prions was observed (Prusiner et al., 1990). In contrast, MH2M(Sc237) prions were able to infect mice, with an incubation period of - 185 days for the first passage, which shortened to - 150 days following a second serial transmission in mice (see Table 3). As expected, mice inoculated with MH2M(Sc237) prions contained PrPSc, as seen from analysis by Western blot (see Figure 38, lane 10). Extracts of brains from Tg(MH2M PrP)92 mice inoculated with Mo(RML) prions transmitted to Syrian hamsters in -215 days. Since Tg(MH2M PrP)92 mice contain an endogenous MoPrP gene, we would expect that the inocula will contain MoPrPSc in addition to MH2M PrPSc,which was observed by Western blotting. Unfortunately, there exists no MoPrP-specific antibody that could be used to confirm this proposal. However, since previous studies have clearly shown that Mo(RML) prions cannot infect hamsters(Prusineret al., 1990; Scott et al., 1989) it seems reasonable to conclude that MH2M(RML) prions are responsible for the transmission to hamsters observed in the experiment (see Table 3). It will be important to perform a detailed analysis of SHa(RML) prions in comparison with other known Syrian hamster prion isolates, such as Sc237 and 139H (Hecker et al., 1992). New Approaches to Studies of Prion Replication and Diversity Although conversion of PrPC to PrPScmight involve a conformational change and the transgenetic data reported here imply homotypic interactions between PrPC and PrPS” during prion replication, the diversity of scrapie prions (Bruce and Dickinson, 1987; Dickinson and Fraser, 1979; Dickinson and Outram, 1988; Kimberlin et al., 1987) poses a conundrum. Some investigators invoke the participation of a hitherto unidentified nucleic acid genome to explain distinct isolates or strains of prions. Since no scrapiespecific polynucleotide has been found and a wealth of data refutes the existence of such a molecule, alternative hypotheses merit consideration. As we have shown here, small variations in the primary structure of PrP can create dramatic variations in the susceptibility of the host animal to scrapie prions, presumably through alterations in the tertiary structure of PrPS”. Other factors, such as Asn-linked oligosaccharides that could convey strain-specific properties, might influence the specificity of the homophilic interactions between PrPC and PrPSc. The proposed interaction of PrPC and PrP* would thus be dependent upon both the PrP sequence and the putative structures responsible for strain-specific behavior. If this were true, it might be expected that the susceptibility of the host to infection with prions derived from another species would vary with the particular strain being studied. This phenomenon is mirrored in experiments de-

scribed here that indicate a marked difference in the relative efficiency of transmission of the Sc237 and 139H isolates between hamsters and Tg(MH2M PrP)92 mice. Indeed, it appears that Tg(MH2M PrP)92 mice are more susceptible to SHa(139H) prions than to SHa(Sc237) (see Table 3). Adaptation of Sc237 to the MH2M background required at least two serial transmissions, shortening from - 135 to - 70 days, whereas the 139H isolate did not appreciably change its incubation period (- 110 days)following the first passage (see Table 3). From our experiments, it appears likely that the Mo(RML) and SHa(Sc237) inocula probably represent different isolates or strains. This is suggested by the contrasting incubation periods and neuropathology observed when these isolates were transmitted to homologous hosts. Although Syrian hamsters are generally preferred as hosts for biochemical studies on scrapie prions, most studies on scrapie strains have been performed in mice. Transgenic mice expressing MHPM PrP may contribute to the analysis of scrapie prion strains by virtue of their ability to act as a “bridge” across the species barrier between hamsters and mice, allowing murine scrapie strains to be adapted to Syrian hamsters. Similar experiments using chimeric PrP genes derived from other species might facilitate the development of murine models for human prion diseases, scrapie of sheep, and transmissible bovine spongiform encephalopathy. Our data demonstrate that it is possible to manipulate to properties of scrapie prions, the clinical manifestation of the disease, and the susceptibility of the host by changing the side chains of a few amino acids encoded by the PrP gene. In concert with biochemical studies on purified proteins, it may become feasible to reconcile the effects of PrP gene manipulation directly with the biochemical characteristics of the genetically engineered prion proteins, suggesting a new approach by which “artificial” prions with contrived properties may be used to unravel the complexities of scrapie prion structure and replication. Experimental

Procedures

Reagents Proteinase K was obtained from Beckman Instruments. Monoclonal antibody 13A5 and polyclonal antibody R073 have been described previously (Barry and Prusiner. 1986; Serban et al., 1990). Monoclonal antibody 3F4 was a generous gift of Dr. R. Kascsak (Kascsak et al., 1987). Alkaline phosphatase-conjugated anti-mouse immunoglobulin and anti-rabbit immunoglobulin antibodies were purchased from Promega. Polyacrylamide gel electrophoresis reagents were obtained from Bio-Rad Laboratories. For the experiment shown in Figure 1 (lower panel), an enhanced chemiluminescence blotting technique was used as described by the manufacturer (Amersham Corporation). Restriction endonucleases and most other enzymes used for construction of recombinant plasmids were obtained from New England Biolabs, Pharmacia LKS, or Bethesda Research Laboratories. T4 DNA ligase was obtained from Collaborative Research. In vitro packaging was performed using a Gigapack kit (Stratagene) and was carried out as recommended by the supplier. Recombinant PrP Plasmld Constructions Routine procedures for recombinant DNA manipulation were performed as described (Sambrook et al., 1989). Construction of the MHMP chimeric PrP cassette has been previously described (Rogers et al., 1991). The MHPM chimeric gene employed in this study was

$t..ficial

Prions

constructed by exchanging the Kpnl-BstEll fragment of a MoPrP ORF cassette (Scott et al., 1992) with the corresponding region from a silent site-mutated SHaPrP ORF cassette (Scott et al., 1992). Preparatlon of Brain Homogenates Brain homogenates (10% [w/v] in phosphate-buffered saline) were prepared by repeated extrusion through syringe needles of successively smaller size, from 16 gauge to 22 gauge. The homogenates were cleared by centrifugation at 5000 x g for 5 min. Samples to be analyzed bywestern blotwereadjusted to0.5%sodiumdeoxycholate, 0.5% Nonidet P-40 and recentrifuged. lmmunoblot Analysis Protein concentration was determined by bicinchoninic acid assay as recommended by the manufacturer (Pierce Chemical). Aliquots (lo50 ug) were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting as previously described (Scott et al., 1969; Towbin et al., 1979). Dot blots were prepared by serially diluting 10 ug aliquots of brain homogenates in 0.01 M Tris-HCI (pH 7.5) 0.1 M NaCI, 0.5% Nonidet P-40, 0.5% sodium deoxycholate in a 96-well microtiter Petri dish. Two-fold dilutions were performed. The samples were then ap plied to a nitrocellulose membrane using a minifold transfer apparatus (Schleicher & Schuell). The dot blot was processed exactly as a Western blot, except that an enhanced chemiluminescence detection method was employed (Amersham Corporation). The blot was exposed to X-ray film (Kodak) for 5-60 s. Construction of Transgenic Mice Recombinant expression cassettes were prepared by cleavage of PrP ORF constructions (Rogers et al., 1991) with Bglll (which cleaves immediately adjacent to the PrP initiation codon). The 5’ protruding terminiwerefilledwithreverse transcriptase,andSalllinkerswereadded. The PrP ORF cassettes were then excised using Sall and Xhol (which cleaves immediately past the end of the ORF). These -0.6 kb fragments (-2 ug) were purified by agarose gel electrophoresis and Iigated in a volume of 10 pl to Sall-digested cosSHa.Tet vector DNA (10 ug) that was treated with bacterial alkaline phosphatase as recommended by the manufacturer (Boehringer Mannheim). Following in vitro packaging, the cosmid phage stocks were used to infect Escherichia coli DHl cells, and plasmid “minipreps” (Sambrook et al., 1989) were screened by restriction digestion analysis for the presence of a -2.2 kb Sall-Xhol fragment corresponding to the recombinant PrP exon II fragment. The Not1 inserts recovered from large-scale DNA preparations were used to construct transgenic mice, and the breeding and screening of transgenic mouse lines was carried out exactly as has been previously described (Scott et al., 1989,1992). For identification of transgenic animals, a probe corresponding to the 3’untranslated region of SHaPrP was used (Scott et al., 1992). Determination of Scrapie Incubation Periods Mice and hamsters were inoculated intracerebrally with 30 ul of brain extract using a 27 gauge disposable hypodermic needle inserted into the right parietal lobe. The preparation of inocula and the criteria for diagnosis of scrapie in mice and hamsters have been described previously(Carlson et al., 1986; Prusineret al., 1982b). Beginning50days after inoculation, the mice and hamsters were examined for neurologic dysfunction every 3 days. Once clinical signs of scrapie were detected, the animals were examined daily. When some animals whose death was clearly imminent were identified, their brains were taken for histologic examination and confirmation of the diagnosis of scrapie. Prion Isolates The Chandler isolate from Swiss mice (Chandler, 1961) was provided by Dr. W. Hadlow and was passaged in Swiss mice from a closed colony at the Rocky Mountain Laboratory or in Swiss CD-1 mice obtained from Charles River Laboratories. This murine isolate was designated RML. After multiple passages in random-bred Syrian golden hamsters (Marsh and Kimberlin, 1975) the scrapie agent was passaged in an Lak/LHC inbred Syrian hamster and the brain was given to us by Dr. R. Marsh (Prusiner et al., 1980). Upon three subsequent passages in Lak/LVG hamsters, the pool of prions was enlarged, and the fourth passage pool was designated Sc237 (Scott et al., 1989). Repeated passage of the scrapie agent in Lak/LVG hamsters at limiting

dilution produced the cloned isolate 263K (Kimberlin and Walker, 1978). Many studies have been performed with SC237 and 263K prions; these prions seem to exhibit similar properties in Syrian hamsters (Marsh and Hanson, 1977; Prusiner et al., 1982b). After more than 20 passages of the Chandler isolate in mice, an isolate designated 139A wasobtained (Dickinson, 1976). Passageof mouse 139Aprionsin Lak/ LVG Syrian golden hamsters produced the 139H isolate (Kimberlin et al., 1987). After six passages in Syrian hamsters, 139H prions were provided to us by Dr. R. Kimberlin and R. Carp. Hlstopathology Whole brains from littermates inoculated with the various prion isolates dedicated to neurohistological examination as well as half-brains from other related experiments were used. The brains were removed rapidly at the time of sacrifice, immersion fixed in 10% buffered formalin, and embedded in paraffin. Sections 8 pm thickwerestained with hematoxy lin and eosin for evaluation of spongiform degeneration. Peroxidase immunohistochemistry with antibodies to glial fibrillary acidic protein was used to evaluate the degree of reactive astrocytic gliosis. Acknowledgments We thank D. R. Borchelt, R. Hecker, and C. Weissmann for many helpful discussions; M. Elepano, R. Cotter, S. Huber, R. Kohler, Y. Zerbajadian, D. Rapp, and C. Cruz for expert technical assistance; J. Cayetano-Canlas for preparation of histological sections; J. McCulloch for photomicrography; and L. Gallagher for manuscript preparation. This work was supported by research grants from the National Institutes of Health (AG02132, NS14069, AG08967, and NS22786) and the American Health Assistance Foundation, as well as by gifts from Sherman Fairchild Foundation and National Medical Enterprises, Received February 9, 1993; revised March 31, 1993. References Barry, R. A., and Prusiner, S. 8. (1966). Monoclonal antibodies to the cellular and scrapie prion proteins. J. Infect. Dis. 154, 518-521. Bazan, J. F., Fletterick, R. J., McKinley, M. P., and Prusiner, S. B. (1967). Predicted secondary structure and membrane topology of the scrapie prion protein. Prot. Eng. 1, 125-135. Bruce, M. E., and Dickinson, A. G. (1987). Biological evidence that the scrapie agent has an independent genome. J. Gen. Virol. 68,7989. Bijeler, H., Fischer, M., Lang, Y.. Blt’ithmann, H., Lipp, H.-L., DeArmond, S. J., Prusiner, S. B., Aguet, M., and Weissmann, C. (1992). The neuronal cell surface protein PrP is not essential for normal development and behavior of the mouse. Nature 356, 577-582. Carlson. G. A., Kingsbury, D. T., Goodman, P. A., Coleman, S., Marshall, S. T., DeArmond, S., Westaway, D., and Prusiner, S. B. (1986). Linkage of prion protein and scrapie incubation time genes. Cell 48, 503-U 1. Caughey, Caughey, associated chemistry

B. W., Dong, A., Bhat, K. S., Ernst, D., Hayes, S. F., and W. S. (1991). Secondary structure analysis of the scrapieprotein PrP 27-30 in water by infrared spectroscopy, Bio30,7672-7680.

Chandler, R. L. (1961). Encephalopathy in mice produced by inoculation with scrapie brain material. Lancet 1, 1378-1379. Dickinson, A. G. (1976). Scrapie in sheep and goats. In Slow Virus Diseasesof Animals and Man, R. H. Kimberlin, ed. (Amsterdam: NorthHolland Publishing), pp. 209-241. Dickinson, A. G., and Fraser, H. (1979). An assessment of the genetics of scrapie in sheep and mice. In Slow Transmissible Diseases of the Nervous System, Volume 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 367-386. Dickinson, A. G., and Outram, G. W. (1988). Genetic aspectsof unconventional virus infections: the basis of the virion hypothesis. In Novel Infectious Agents and the Central Nervous System. Ciba Foundation Symposium 735, G. Bock and J. Marsh, eds. (Chichester, England: John Wiley & Sons), pp. 63-83.

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Gabizon, R.. McKinley, M. P., and Prusiner. S. B. (1987). Purified prion proteins and scrapie infectivity copartition into liposomes. Proc. Natl. Acad. Sci. USA 84, 4017-4021. Gabizon, R.. McKinley, M. P., Groth. D. F., and Prusiner, S. B. (1988). lmmunoaffinity purification and neutralization of scrapie prion infectivity. Proc. Natl. Acad. Sci. USA 85, 8617-6621. Gasset, M., Baldwin, M. A., Lloyd, D., Gabriel, J.-M., Holtzman, D. M., Cohen, F., Fletterick, Ft., and Prusiner, S. B. (1992). Predicteda-helical regions of the prion protein when synthesized as peptides form amyloid. Proc. Nab. Acad. Sci. USA 89, 10940-10944. Gasset, M., Baldwin, M. A., Fletterick, R. J., and Prusiner, S. B. (1993). Perturbation of the secondary structure of the scrapie prion protein under conditions associated with changes in infectivity. Proc. Natl. Acad. Sci. USA 90, l-5. Hecker, R., Taraboulos. A., Scott, M., Pan, K.-M., Torchia, M., Jendroska, K.. DeArmond, S. J., and Prusiner, S. B. (1992). Replication of distinct prion isolates is region specific in brains of transgenic mice and hamsters. Genes Dev. 6. 1213-1228. Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M., and Diringer, H. (1987). Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins J. Virol. 61, 3688-3693. Kimberlin, R. H., and Walker, C. A. (1978). Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture. J. Gen. Virol. 39, 487-496. Kimberlin. R. H., Cole, S., and Walker, C. A. (1987). Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters. J. Gen. Virol. 68, 1875-1881. Kimberlin, R. H., Walker, C. A., and Fraser, H. (1989). The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice. J. Gen. Virol. 70, 2017-2025. Lowenstein, D. H., Butler, D.A., Westaway, D.. McKinley, mond, S. J., and Prusiner, S. B. (1990). Three hamster different scrapie incubation times and neuropathological code distinct prion proteins. Mol. Cell. Biol. 10, 1153-l

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Sambrook, J., Fritsch, E. F., and Maniatis. T. (1969). Plasmid vectors. In Molecular Cloning: A Laboratory Manual, Second Edition, C. Nolan, ed. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 1.7-l ,101. Scott, M., Foster, D., Mirenda, C., Serban, D., Coufal, F., Wllchli, M., Torchia, M., Groth, D., Carlson. G., DeArmond, S. J., Westaway, D., and Prusiner, S. B. (1989). Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59. 847-857. Scott, M. R., Kohler, R., Foster, D., and Prusiner, S. B. (1992). Chimeric prion protein expression in cultured cells and transgenic mice. Prot. Sci. 1, 986-997. Serban, D., Taraboulos, A., DeArmond, S. J., and Prusiner, S. B. (1990). Rapid detection of Creutzfeldtdakob disease and scrapie prion proteins. Neurology 40, 110-t 17. Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame. A. L., and Prusiner, S. B. (1993). Structural analysis of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32, 1991-2002. Taraboulos, A., Rogers, M., Borchelt, D. R., McKinley, M. P., Scott, M., Serban, D., and Prusiner, S. 8. (1990). Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation. Proc. Natl. Acad. Sci. USA87,82626266. Taraboulos, A., Jendroska, K., Serban. D., Yang, S.-L., DeArmond, S. J., and Prusiner, S. B. (1992). Regional mapping of prion proteins in brains. Proc. Natl. Acad. Sci. USA 89, 7620-7624. Towbin, H., StaehelinT., andGordon, J. (1979). Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 43504354.