A Single Hamster PrP Amino Acid Blocks Conversion to Protease-Resistant PrP in Scrapie-Infected Mouse Neuroblastoma Cells

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1 JOURNAL OF VIROLOGY, Dec. 1995, p Vol. 69, No X/95/$ Copyright 1995, American Society for Microbiology A Single Hamster PrP Amino Acid Blocks Conversion to Protease-Resistant PrP in Scrapie-Infected Mouse Neuroblastoma Cells SUZETTE A. PRIOLA* AND BRUCE CHESEBRO Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana Received 25 May 1995/Accepted 5 September 1995 Neurodegeneration caused by the transmissible spongiform encephalopathies is associated with the conversion of a normal host protein, PrP-sen, into an abnormal aggregated protease-resistant form, PrP-res. In scrapie-infected mouse neuroblastoma cells, mouse PrP-sen is converted into PrP-res but recombinant hamster PrP-sen expressed in these cells is not. In the present studies, recombinant hamster/mouse PrP-sen molecules were expressed in these scrapie-infected cells to define specific PrP amino acid residues critical for the conversion to PrP-res. The results showed that homology to the region of mouse PrP-sen from amino acid residues 112 to 138 was required for conversion of recombinant PrP-sen to PrP-res in scrapie-infected mouse cells. Furthermore, a single hamster-specific PrP amino acid at residue 138 could inhibit the conversion of the recombinant PrP-sen into PrP-res. The data are consistent with studies in humans which show that specific amino acid residue changes within PrP can influence disease pathogenesis and transmission of transmissible spongiform encephalopathies across species barriers. Scrapie in sheep, bovine spongiform encephalopathy in cattle, and kuru, Gerstmann-Sträussler-Scheinker syndrome, and Creutzfeldt-Jakob disease in humans are members of a group of diseases known as the transmissible spongiform encephalopathies (TSE). The exact nature of the etiologic agent in TSE is unknown. The unusual resistance of the agent to inactivation by irradiation, heat, and other harsh treatments led to the proposal that the agent is an infectious, self-replicating protein (15, 32). The brains of scrapie-infected animals contain a protease-resistant protein, PrP-res (27), which has been hypothesized to be the etiologic agent of TSE (32). Although its precise relationship to the agent is still unclear (1, 19, 37), the importance of PrP-res in the brain pathology associated with TSE diseases is well established (3). The scrapie species barrier is the resistance of one animal species to infection with scrapie agent from another animal species. For example, hamsters inoculated with mouse-adapted strains of scrapie develop disease only after very prolonged incubation periods, and passage of hamster-adapted scrapie strains into mice usually fails to produce disease at all (20 22). The current outbreak of bovine spongiform encephalopathy in Great Britain and concerns that the bovine spongiform encephalopathy agent could cross the species barrier into humans underscores the importance of determining how transmission of scrapie from one species to another can occur. Early experiments showed that genes identical to or linked with the PrP gene could influence resistance to various scrapie strains in mice and sheep (4, 17, 36). Subsequent experiments showed that transgenic mice expressing high levels of the hamster protease-sensitive protein (PrP-sen) were susceptible to infection and disease induction by hamster scrapie (33, 38). These results indicated that PrP molecules were involved in the interspecies transmission of scrapie. One possible explanation is * Corresponding author. Mailing address: Laboratory of Persistent Viral Diseases, NIAID, Rocky Mountain Laboratories, Hamilton, MT Phone: (406) Fax: (406) Electronic mail address: sap@rml.niaid.pc.niaid.nih.gov. that differences between the primary sequence of the hostspecific PrP-sen and the foreign PrP-res molecule in scrapie agent preparations could influence transmission of scrapie across species barriers. This possibility has been supported by studies of transgenic mice expressing chimeric hamster/mouse PrP genes (39) and by the recent finding that species-specific interactions between PrP-sen and PrP-res molecules influence generation of PrP-res in a cell-free system (25). In previous studies with mouse scrapie-infected murine neuroblastoma (Sc -MNB) cells, we showed that expression of mutant or recombinant mouse PrP molecules containing a single hamster PrP-specific methionine at residue 111 could interfere with the conversion of the endogenous mouse PrPsen molecule expressed in Sc -MNB cells to PrP-res (30). We have now expressed a series of mouse/hamster recombinant PrP molecules in these cells and assayed the conversion of the recombinant PrP-sen molecules into PrP-res. Surprisingly, despite their ability to interfere with the conversion of mouse PrP-sen to mouse PrP-res, some of these recombinant PrP-sen molecules were themselves converted into PrP-res. The data indicated that homology with mouse PrP amino acid sequence from residues 112 to 138 was necessary for conversion of the recombinant PrP-sen to PrP-res. Furthermore, a single hamster PrP-specific amino acid at residue 138 could block conversion of the recombinant PrP-sen to PrP-res, whereas a mouse PrP-specific amino acid at residue 138 favored the conversion of the recombinant PrP-sen to PrP-res. MATERIALS AND METHODS Cells. Murine neuroblastoma cells persistently infected with mouse scrapie (Sc -MNB cells) have been described previously (35). These cells express mouse PrP-sen, accumulate mouse PrP-res, and replicate mouse scrapie infectivity (34, 35). Antibodies. The anti-hamster PrP mouse monoclonal antibody 3F4 recognizes an epitope within hamster PrP containing methionines at residues 109 and 112 (2, 18). All of the recombinant PrP molecules used in this study expressed this antibody epitope. The 3F4 mouse monoclonal antibody does not react with mouse PrP, which has leucine and valine at residues 108 and 111. (Hamster PrP has a single amino acid insertion at position 54 and a single amino acid deletion at position 233 as compared with mouse PrP [26, 28]. Therefore, homologous 7754

2 VOL. 69, 1995 SINGLE PrP AMINO ACID BLOCKS CONVERSION TO PrP-res 7755 TABLE 1. A single amino acid change influences the conversion of recombinant PrP-sen to PrP-res Amino acid residue at position b : Clone Background a 109 (108) 112 (111) 139 (138) 155 (154) 170 (169) HaPrP Hamster MET MET MET ASN ASN SP66 Hamster MET MET Ile Tyr Ser SP66-M139 Hamster MET MET MET Tyr Ser SP66-N155 Hamster MET MET Ile ASN Ser SP66-N170 Hamster MET MET Ile Tyr ASN 3F4-positive PrP-res c MoPrP Mouse Leu Val Ile Tyr Ser NA MoPrP-M108/111 Mouse MET MET Ile Tyr Ser MoPrP-M108/M111/M138 Mouse MET MET MET Tyr Ser a Background indicates the species derivation of the recombinant PrP amino acids which are not individually indicated. b The five amino acid residues altered for this study are numbered. Residues in capital letters are hamster PrP specific. Because of a single amino acid deletion in mouse PrP at residue 54 (26, 28), homologous residues between mouse and hamster PrP differ by 1 in their numbering at the positions shown; the residues for mouse PrP are given in parentheses. Boxed residues are associated with a lack of conversion of the recombinant PrP molecule into PrP-res as detected by the monoclonal antibody 3F4. c, 3F4-positive PrP-res;, no 3F4-positive PrP-res; NA, not applicable. mouse and hamster PrP amino acids between these two residues differ by 1 in their numbering.) The anti-prp peptide rabbit polyclonal antibody R.27 was raised to a PrP peptide encompassing amino acid residues 89 to 103 of the mouse PrP molecule and has been described previously (8). The R.27 antibody recognizes both mouse and hamster PrP. Clones. Mouse PrP (MoPrP) mutated to contain the 3F4 epitope and a unique NaeI restriction endonuclease site, MoPrP-M108/M111, and normal hamster PrP (HaPrP) have been described previously (9). Two mouse/hamster PrP chimeras, SP33 and SP40 (see Fig. 2), were derived by utilizing the unique NaeI site within HaPrP and MoPrP-M108/M111 as described previously (30). Additional recombinants were generated with a unique BstEII restriction endonuclease site at nucleotide 659 within the wild-type MoPrP sequence (26). HaPrP does not have this BstEII site. To generate a chimeric PrP molecule with HaPrP sequence between the NaeI-BstEII sites, an NaeI-BstEII fragment derived from HaPrP was generated by PCR mutagenesis of wild-type HaPrP with the HaPrP specific primers 5 -CCAAAAACCAACATGAAGCAGATGGCCGGC GCTGC-3 and 5 -GGTGGTGAGCGTGTGCTGCTT-3. This generated a BstEII site in the HaPrP DNA sequence by changing the A at position 663 to a G. The amino acid sequence remained unchanged. The single-base-pair mutation was confirmed by DNA sequencing and subcloned into SP40. The final chimeric Mo/HaPrP gene (SP67; see Fig. 2) was subcloned into the retroviral expression vector psff (9). A chimeric PrP with HaPrP DNA sequence at the 3 end of PrP was generated by PCR mutagenesis of a wild-type HaPrP cloned into the Bluescript-KS( ) vector (Stratagene), using the HaPrP-specific primer 5 -A AGCAGCACACGGTCACCACC-3 and the Bluescript-specific primer 5 -TGA CCGGCAGCAAAATG-3. This generated the BstEII site as described above. The mutation was confirmed by DNA sequencing and subcloned into SP40 at the BstEII site and a unique NotI site in Bluescript KS( ). The complete Mo/HaPrP chimera (SP66; see Fig. 2) was then subcloned into the psff vector. To mutate single amino acid residues within the 224-bp NaeI-BstEII fragment of MoPrP, a set of 10 overlapping oligonucleotides was made. These oligonucleotides spanned the region of MoPrP from nucleotides 436 to 660. A pair of oligonucleotides in which the C at nucleotide 573 was replaced with a G, changing an isoleucine to a methionine, was made. These mutant oligonucleotides were then mixed in equimolar ratios with eight nonmutated oligonucleotides, treated with kinase, and ligated as described previously (10) into a Bluescript KS( ) vector containing a mouse PrP gene from which the NaeI-BstEII fragment had been deleted. The resultant 224-bp fragment contained a 5 NaeI blunt end, a3 BstEII end, and the MoPrP amino acid sequence except for the HaPrPspecific methionine at position 138. The mutation was confirmed by DNA sequencing and subcloned into the NaeI-BstEII sites of SP66 (SP66-M139; see Fig. 4). The same protocol was used to mutate the MoPrP amino acid residues at 155 (tyrosine to asparagine) and 170 (serine to asparagine) to their HaPrP equivalents. These clones are identical to hamster PrP except for two mouse PrP amino acids in the NaeI-BstEII fragment and are shown in Table 1. Infection and analysis of Sc -MNB cells expressing chimeric PrPs. Expression of recombinant mouse/hamster PrP molecules in Sc -MNB cells by using the retroviral expression vector psff and the assay of these cells for expression of PrP-res have been described previously (9, 30). PrP-res derived from exogenous chimeric PrP molecules was detected with the mouse monoclonal antibody 3F4 (18), while PrP-res derived from both endogenous and exogenous PrP molecules was detected with the anti-prp peptide rabbit polyclonal antiserum R.27 (8). Each experiment was repeated at least twice. The Western blot (immunoblot) was developed with the enhanced chemiluminescence reagent system (ECL; Amersham) as specified by the manufacturer. Nonspecific background derived from the ECL development procedure was not uncommon and accounted for the regional background variation observed in the Western blots. Radioimmunoprecipitation of PrP-sen. The expression level of the chimeric PrP-sen molecules in Sc -MNB cells was assayed by immunoprecipitation of radiolabeled cell lysates and by live-cell membrane immunofluorescence with the 3F4 antibody as described previously (5, 30). RESULTS Specificity of conversion of PrP-sen to PrP-res in Sc -MNB cells. HaPrP and MoPrP-M108/M111 were expressed in Sc - MNB cells, and the cells were assayed for the presence of 3F4-reactive PrP-res. The endogenous MoPrP in Sc -MNB cells does not contain the 3F4 antibody epitope and does not react with the 3F4 antibody. Therefore, any 3F4-reactive PrP can be derived only from the exogenous recombinant PrP molecule. Similar levels of recombinant PrP-sen were synthesized by the HaPrP and MoPrP-M108/M111 recombinants (Fig. 1A). As we have described previously, expression of either HaPrP or MoPrP-M108/M111 in Sc -MNB cells interfered with the overall level of expression of PrP-res, as detected by the rabbit polyclonal antiserum R.27 (30). However, when PrP-res was assayed with monoclonal antibody 3F4, a substantial amount of 3F4-positive PrP-res was found in cells which expressed MoPrP-M108/M111, whereas none was detected in cells which expressed HaPrP (Fig. 1B). Therefore, both the mutant MoPrP-M108/M111 and HaPrP blocked the generation of PrP-res from the endogenous MoPrP, but only MoPrP-M108/M111 was itself incorporated into PrP-res. Homology in a region of MoPrP from amino acid residues 112 to 187 is required for the conversion of PrP-sen to PrP-res. To determine the region of PrP which was essential for conversion of the exogenous PrP-sen to PrP-res, several recombinants between MoPrP-M108/M111 and HaPrP (Fig. 2) were expressed in Sc -MNB cells. These recombinants expressed PrP-sen at similar levels (Fig. 3A). However, when PrP-res was assayed with monoclonal antibody 3F4, only MoPrP-M108/ M111, SP40, and SP66 were converted into PrP-res (Fig. 3B). As summarized in Fig. 2, the only MoPrP DNA sequence common to all three of the constructs which converted into PrP-res was a 224-bp NaeI-BstEII fragment. This sequence encodes amino acid residues 112 to 187 of MoPrP. Amino acid residue 139 influences the conversion of PrP-sen to PrP-res. Mouse PrP and hamster PrP differ at only 3 of the 75 amino acid residues encoded by the NaeI-BstEII fragment (Fig. 2) (26). Therefore, we changed each of these amino acids separately to test their influence on the conversion of PrP-sen to PrP-res (Table 1). Conversion of the mutant PrP into PrP-

3 7756 PRIOLA AND CHESEBRO J. VIROL. FIG. 1. The conversion of PrP-sen to PrP-res in Sc -MNB cells is species specific. Shown is analysis of PrP-sen (A) or PrP-res (B) in Sc -MNB cells expressing MoPrP-M108/M111 and HaPrP. (A) PrP-sen radioimmunoprecipitated with the 3F4 mouse monoclonal antibody. The sizes (in kilodaltons) of the specific PrP-sen species are indicated on the left. The 60-kDa HaPrP molecule has been described previously (31). (B) Western blot analysis of 3F4-reactive PrP-res. The immunoblot was developed with the mouse monoclonal antibody 3F4. PrP-res-specific bands are indicated by the arrows and brackets on the left. Molecular mass markers (in kilodaltons) are shown on the right. Both panels are from the same experiment. Lanes in each panel are from the same gel exposed for a constant time. res was unaffected when residue 155 was altered from tyrosine to asparagine (SP66-N155) or when residue 170 was altered from serine to asparagine (SP66-N170) (Fig. 4B). However, when residue 139 was altered from isoleucine to methionine (SP66-M139), the level of 3F4-reactive PrP-res decreased significantly (Fig. 4B) despite high levels of SP66-M139 expression (Fig. 4A). The data, summarized in Table 1, indicated that homology at residue 139 was critical in the conversion of PrPsen to PrP-res. In a reciprocal manner, we tested the effect of this change in the context of the MoPrP amino acid sequence. Residue 138 in MoPrP-M108/M111 (homologous to residue 139 in HaPrP and FIG. 3. Conversion of chimeric Mo/HaPrP-sen to PrP-res in Sc -MNB cells is dependent on the presence of specific MoPrP protein sequence. Shown is analysis of PrP-sen by radioimmunoprecipitation with the 3F4 antibody (A) or PrP-res by immunoblot with the 3F4 antibody (B) in Sc -MNB cells expressing MoPrP-M108/M111, a series of Mo/HaPrP chimeras (SP33 through SP66), the envelope gene of Friend murine leukemia virus (FE), or no construct (mock). The FE gene encodes a nonspecific membrane glycoprotein expressed in the psff vector (30) and was used in these experiments as a negative control. Both panels are from the same experiment, and each panel was derived from a single gel. Exposure time for panel A was 7 days. SP66-M139) was changed from isoleucine to methionine. The resultant recombinant, MoPrP-M108/M111/M138, contained HaPrP-derived amino acid residues only at positions 108 and 111 (i.e., the 3F4 epitope) and at position 138 (Table 1). Recombinants MoPrP-M108/M111 and MoPrP-M108/M111/ M138, which differed by only one amino acid, were expressed at equivalent levels in Sc -MNB cells (Fig. 5A). However, only MoPrP-M108/M111 was converted into PrP-res. Little or no 3F4-reactive PrP-res was detected in cells which expressed the MoPrP-M108/M111/M138 construct (Fig. 5B). The data supported the above results with clone SP66-M139 (Table 1) and demonstrated that the HaPrP-specific methionine at position FIG. 2. Recombinant mouse and hamster PrPs. DNA sequence derived from MoPrP is shown by the solid bars, while DNA sequence derived from HaPrP is shown by the open bars. The tick marks represent amino acid differences between mouse PrP and hamster PrP. Clone names are given on the left. The two hamster PrP-specific methionine residues involved in 3F4 epitope reactivity are indicated by an asterisk. The NaeI and BstEII cloning sites used to generate the clones are shown. Conversion of the designated clone into PrP-res in Sc -MNB cells is summarized on the right:, 3F4-positive PrP-res;, no 3F4-positive PrP-res. FIG. 4. A single amino acid residue change in PrP-sen affects the conversion of PrP-sen to PrP-res in Sc -MNB cells. Shown is analysis of PrP-sen by radioimmunoprecipitation with the 3F4 antibody (A) or PrP-res by immunoblot with the 3F4 monoclonal antibody (B) in Sc -MNB cells expressing recombinant Mo/HaPrP chimeras which differ at a single amino acid residue. The parent clone, SP66, contained only three MoPrP-derived amino acid residues, while clones SP66-M139, SP66-N155, and SP66-N170 contained only two MoPrPderived amino acid residues (Table 1). The data are from a single experiment, and each panel represents data from a single gel. Exposure time for panel A was 24 h. Molecular mass markers (in kilodaltons) on the right are for panel B only.

4 VOL. 69, 1995 SINGLE PrP AMINO ACID BLOCKS CONVERSION TO PrP-res 7757 FIG. 5. MoPrP amino acid residue 138 is important in the conversion of PrP-sen to PrP-res in Sc -MNB cells. All data are from the same experiment. Shown is analysis of PrP-sen by radioimmunoprecipitation with the 3F4 antibody (A) or PrP-res by immunoblot analysis with the 3F4 antibody (B) in Sc -MNB cells expressing the MoPrP recombinants MoPrP-M108/M111 or MoPrP-M108/ M111/M138. These constructs differ by a single amino acid residue at position 138 in the MoPrP gene (Table 1). Molecular mass markers (in kilodaltons) on the right are for panel B only. 138 in MoPrP or position 139 in SP66-M139 prevented conversion of the mutant PrP-sen to the PrP-res form. DISCUSSION Expression of recombinant heterologous PrP molecules in mouse scrapie-infected murine neuroblastoma cells has enabled us to define an amino acid in the PrP gene sequence which has a significant effect on the conversion of PrP-sen to PrP-res. A previous study by Scott et al. had mapped the specific amino acids important in the conversion of PrP-sen into PrP-res to two regions of the PrP-sen molecule which encompassed over 100 amino acid residues (40). Recently, we defined a single region of hamster PrP extending from residues 109 to 188 that is important in the conversion of hamster PrP-sen to hamster PrP-res in a cell-free conversion system (25). Transgenic mouse models had also indicated that this region was important in the susceptibility of the transgenic mice to hamster scrapie (39). In this study, we have demonstrated not only that homology in a single region of mouse PrP from amino acid residues 112 to 138 is important in the conversion of mouse PrP-sen to mouse PrP-res but also that residue 138 alone is capable of modulating the conversion process. Consistent with our finding that homology with the primary amino acid sequence of PrP in this region is important in the formation of PrP-res, several mutations in the PrP gene associated with naturally occurring human TSE, any one of which can affect the disease phenotype, are located within the region of PrP from residues 112 to 187. Within the human PrP gene, mutation of an alanine to a valine at residue 117 is associated with a dementing form of Gerstmann-Sträussler-Scheinker syndrome (16), a change from a methionine to a valine at position 129 predisposes an individual to Creûtzfeldt-Jakob disease (29), an amber mutation at residue 145 is associated with an atypical form of Gerstmann-Sträuŝsler-Scheinker syndrome (23), while a change from an asparagine to an aspartic acid at codon 178 has been associated with both Creutzfeldt- Jakob disease and fatal familial insomnia (13, 14). Our data are the first to directly demonstrate that a single amino acid change within this region of PrP can have a significant effect on the process by which PrP-sen is converted into PrP-res, which may in turn have a profound effect on TSE pathogenesis. Previous results showed that in mouse scrapie-infected cells, recombinant PrP molecules with hamster-specific methionine residues at positions 108 and 111 blocked the conversion of normal MoPrP-sen to PrP-res (30). The experiments in the present study show that these recombinant PrP molecules were themselves converted to PrP-res (Fig. 1; MoPrP-M108/M111). Thus, recombinant PrP molecules with methionines at 108 and 111 appeared to competitively inhibit incorporation of normal MoPrP into PrP-res. Our data are similar to results from in vivo studies of human PrP amyloid, which have shown that in heterozygous Gerstmann-Sträussler-Scheinker syndrome patients expressing PrP molecules with a methionine or valine at codon 129, only the PrP molecules with a valine at 129 were incorporated into PrP amyloid fibrils (41). Taken together, the data strongly suggest that depending on the homology at critical residues in PrP, competitive interactions between PrP molecules can lead to preferential incorporation of one type of PrP molecule into the growing PrP-res fibril (Fig. 6B). This further suggests that the two different PrP molecules can compete for a common site in the conversion mechanism. Recent in vitro data strongly suggest that this conversion site is either within PrP-res itself or within a molecule tightly associated with PrPres, such as a glycosaminoglycan (6, 7, 24). We also demonstrated that PrP molecules with methionines at codons 108, 111, and 138, in addition to blocking the conversion of MoPrP-sen to MoPrP-res (30), are unable to themselves be converted to PrP-res (Fig. 1, HaPrP; Fig. 5, MoPrP- M108/M111/M138). The methionine at 138 is essential for this effect (Fig. 5). By inhibiting the formation of PrP-res, a PrP gene encoding such recombinant molecules might act to increase resistance to scrapie infection in vivo. Since methionines are present at these positions in normal HaPrP-sen, they may be responsible for the scrapie species barrier which leads to the extended disease incubation times observed when hamsters are inoculated with mouse scrapie agent (20 22). As shown in recent cell-free studies, this species barrier probably involves interactions between heterologous PrP molecules (25) and may FIG. 6. Effects of heterologous PrP-sen molecules on PrP-res accumulation. Shown is a model for the effect of expression of exogenous PrP-sen molecules on PrP-res accumulation in cells persistently infected with mouse scrapie. (A) No inhibition. Conversion of PrP-sen (open circles) to PrP-res (open squares) can occur. (B) Homologous and nonhomologous (striped circles) PrP-sen molecules compete for a common binding site on the growing PrP-res polymer. The nonhomologous PrP-sen can be converted into PrP-res (striped squares), resulting in a decrease in PrP-res derived from the homologous PrP-sen. (C) The nonhomologous PrP-sen (solid circles) cannot be converted to PrP-res and inhibits incorporation of the homologous PrP-sen molecule into PrP-res. PrP-res fibril formation stops, and the amount of PrP-res which accumulates decreases.

5 7758 PRIOLA AND CHESEBRO J. VIROL. be broken if enough PrP-sen is converted to PrP-res to overcome the inhibitory effect of interactions between incompatible PrP molecules. This process might take several in vivo passages of the scrapie agent. The species barrier might remain if insufficient PrP-sen were converted to PrP-res to overcome the inhibitory effect of interactions between incompatible PrP molecules or if the PrP-sen molecule could not be utilized in the conversion process at all (Fig. 6C). The fact that a single amino acid change can influence the conversion process in an in vitro tissue culture system supports results of previous cell-free studies with PrP peptides. Even conservative amino acid changes affect the nucleation-dependent polymerization of mixtures of PrP peptides in vitro (11, 12). In Sc -MNB cells, recombinant PrP molecules with a mutation at residue 138 may act in a similar manner by interacting with the endogenous mouse PrP-res molecules made in these cells, reducing nucleation times, and leading to a decrease in the amount of PrP-res which accumulates over time. ACKNOWLEDGMENTS We thank Sylvia Perryman for doing the DNA sequence analysis, Robert Evans for graphics assistance, and Byron Caughey, Kim Hasenkrug, Nancy Ray, and David Kocisko for critical reading of the manuscript. REFERENCES 1. Aiken, J. M., and R. F. Marsh The search for scrapie agent nucleic acid. Microbiol. Rev. 54: Bolton, D. C., S. J. Seligman, G. Bablanian, D. Windsor, L. J. Scala, K. S. Kim, C. M. J. Chen, R. J. Kascsak, and P. E. Bendheim Molecular location of a species-specific epitope on the hamster scrapie agent protein. J. Virol. 65: Bueler, H., A. Aguzzi, A. Sailer, R.-A. Greiner, P. Autenried, M. Aguet, and C. Weissmann Mice devoid of PrP are resistant to scrapie. 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