Autocatalytic Conversion of Recombinant Prion Proteins Displays a Species Barrier

Abstract

The most unorthodox feature of the prion disease is the existence of an abnormal infectious isoform of the prion protein, PrPSc. According to the “protein-only” hypothesis, PrPSc propagates its abnormal conformation in an autocatalytic manner using the normal isoform, PrPC, as a substrate. Because autocatalytic conversion is considered a key element of prion replication, in this study I tested whether in vitro conversion of recombinant PrP into abnormal isoform displays specific features of an autocatalytic process. I found that recombinant human PrP formed two distinct β-sheet rich isoforms, the β-oligomer and the amyloid fibrils. The kinetics of the fibrils formation measured at different pH values were consistent with a model in which the β-oligomer was not on the kinetic pathway to the fibrillar form. As judged by electron microscopy, an acidic pH favored to the long fibrils, whereas short fibrils morphologically similar to “prion rods” were formed at neutral pH. At neutral pH the conversion to the fibrils can be seeded with small aliquots of preformed fibrils. As small as 0.001% aliquot displayed seeding activity. The conversion of human PrP was seeded with high efficacy only with the preformed fibrils of human but not mouse PrP and vice versa. These studies illustrate that in vitro conversion of recombinant PrP displays specific features of an autocatalytic process and mimics the transmission barrier of prion propagation observed in vivo. I speculate that this model can be used as a rapid assay for assessing the intrinsic propensities of prion transmission between different species. The most unorthodox feature of the prion disease is the existence of an abnormal infectious isoform of the prion protein, PrPSc. According to the “protein-only” hypothesis, PrPSc propagates its abnormal conformation in an autocatalytic manner using the normal isoform, PrPC, as a substrate. Because autocatalytic conversion is considered a key element of prion replication, in this study I tested whether in vitro conversion of recombinant PrP into abnormal isoform displays specific features of an autocatalytic process. I found that recombinant human PrP formed two distinct β-sheet rich isoforms, the β-oligomer and the amyloid fibrils. The kinetics of the fibrils formation measured at different pH values were consistent with a model in which the β-oligomer was not on the kinetic pathway to the fibrillar form. As judged by electron microscopy, an acidic pH favored to the long fibrils, whereas short fibrils morphologically similar to “prion rods” were formed at neutral pH. At neutral pH the conversion to the fibrils can be seeded with small aliquots of preformed fibrils. As small as 0.001% aliquot displayed seeding activity. The conversion of human PrP was seeded with high efficacy only with the preformed fibrils of human but not mouse PrP and vice versa. These studies illustrate that in vitro conversion of recombinant PrP displays specific features of an autocatalytic process and mimics the transmission barrier of prion propagation observed in vivo. I speculate that this model can be used as a rapid assay for assessing the intrinsic propensities of prion transmission between different species. Spontaneous conversion of the normal cellular isoform of the prion protein, PrPC, into the abnormal pathological isoform, PrPSc, underlies sporadic forms of prion disease, including Creutzfeldt-Jakob disease in humans (1Prusiner S.B. N. Engl. J. Med. 2001; 344: 1516-1526Crossref PubMed Scopus (677) Google Scholar). The occurrence of the sporadic forms of prion maladies is very rare, about one person/million/year (2Masters C.L. Harris J.O. Gajdusek D.C. Gibbs Jr., C.J. Bernouilli C. Asher D.M. Ann. Neurol. 1978; 5: 177-188Crossref Scopus (552) Google Scholar). Such a low occurrence is believed to be because of the extremely slow spontaneous conversion of PrPC into PrPSc (3Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (473) Google Scholar). However, the conversion can be substantially accelerated by administration of an exogenous PrPSc. The “protein-only” hypothesis of prion propagation postulates that the abnormal isoform, PrPSc, acts as a transmissible agent of the disease and self-propagates its pathological conformation using PrPC as a substrate (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (855) Google Scholar). Substantial effort has been devoted to the development of a cell-free system for conversion of non-infectious prion protein (PrP) 1The abbreviations used are: PrP, prion protein; Hu, human; Mo, mouse; ThT, thioflavin T; recPrP, recombinant prion protein; SEC, size-exclusion chromatography; GdnHCl, guanidine hydrochloride. 1The abbreviations used are: PrP, prion protein; Hu, human; Mo, mouse; ThT, thioflavin T; recPrP, recombinant prion protein; SEC, size-exclusion chromatography; GdnHCl, guanidine hydrochloride. into the infectious PrPSc isoform (5Bessen R.A. Kocisko D.A. Raymond G.J. Nandan S. Lansbury P.T. Caughey B. Nature. 1995; 375: 698-700Crossref PubMed Scopus (459) Google Scholar, 6Hill A.F. Antoniou M. Collinge J. J. Gen. Virol. 1999; 80: 11-14Crossref PubMed Scopus (175) Google Scholar, 7Raeber A.J. Borchelt D.R. Scott M. Prusiner S.B. J. Virol. 1992; 66: 6155-6163Crossref PubMed Google Scholar, 8Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1004) Google Scholar). To monitor conversion in vitro, many studies exploited conformational differences between PrPC and PrPSc (5Bessen R.A. Kocisko D.A. Raymond G.J. Nandan S. Lansbury P.T. Caughey B. Nature. 1995; 375: 698-700Crossref PubMed Scopus (459) Google Scholar, 9Baskakov I.V. Legname G. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2001; 276: 19687-19690Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 10Swietnicki W. Morillas M. Chen S.G. Gambetti P. Surewicz W.K. Biochemistry. 2000; 39: 424-431Crossref PubMed Scopus (217) Google Scholar, 11Lu B.Y. Chang J.Y. Biochemistry. 2001; 40: 13390-13396Crossref PubMed Scopus (28) Google Scholar, 12Kazlauskaite J. Sanghera N. Sylvester I. Venien-Bryan C. Pinheiro T.J. Biochemistry. 2003; 42: 3295-3304Crossref PubMed Scopus (153) Google Scholar) because PrPC is a proteinase K-sensitive α-helical monomer, whereas PrPSc is a proteinase K-resistant, aggregated β-sheet-rich multimer (13Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2064) Google Scholar). Even though formation of β-sheet-rich proteinase K-resistant multimeric isoforms of PrP have been reported (6Hill A.F. Antoniou M. Collinge J. J. Gen. Virol. 1999; 80: 11-14Crossref PubMed Scopus (175) Google Scholar, 10Swietnicki W. Morillas M. Chen S.G. Gambetti P. Surewicz W.K. Biochemistry. 2000; 39: 424-431Crossref PubMed Scopus (217) Google Scholar, 11Lu B.Y. Chang J.Y. Biochemistry. 2001; 40: 13390-13396Crossref PubMed Scopus (28) Google Scholar, 14Jackson G.S. Hosszu L.L.P. Power A. Hill A.F. Kenney J. Saibil H. Craven C.J. Waltho J.P. Clarke A.R. Collinge J. Science. 1999; 283: 1935-1937Crossref PubMed Scopus (364) Google Scholar, 15Kirby L. Birkett C.R. Rudyk H. Gilbert I.H. Hope J. J. Gen. Virol. 2003; 84: 1013-1020Crossref PubMed Scopus (63) Google Scholar), the ultimate direct proof of the protein-only hypothesis, i.e. the in vitro reconstitution of PrPSc from non-infectious PrP, has not yet been achieved. Despite substantial differences between PrPC and PrPSc, it is still unclear which physical property can be used as a valid probe to monitor formation of infectious PrPSc in a cell-free system. According to the protein-only hypothesis, an autocatalytic conversion from PrPC into PrPSc is a key feature that underlies the molecular basis of the transmissible form of prion diseases (16Gajdusek D.C. J. Neuroimmunol. 1988; 20: 95-110Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Strong support for the idea that certain proteins are capable of self-propagating their abnormal conformations was provided by the prion biology of yeast and fungi (17Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1079) Google Scholar, 18Wickner R.B. Masison D.C. Edskes H.K. Yeast. 1995; 11: 1671-1685Crossref PubMed Scopus (138) Google Scholar, 19Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Acad. Natl. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (234) Google Scholar). In vitro made fibrils of Sup35 and HET-s, the prion proteins of yeast and fungi, respectively, cause prion propagation in vivo and change the phenotype of cells (19Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Acad. Natl. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (234) Google Scholar, 20Sparrer H.E. Santoso A. Szoka Jr., F.C. Weissman J.S. Science. 2000; 289: 595-599Crossref PubMed Scopus (137) Google Scholar). The yeast model displays one of the key features that illustrate an autocatalytic mechanism of conversion, a seeding phenomenon in which abnormal isoforms produced during conversion are able to initiate and facilitate conversion in a fresh reaction. Furthermore, seeding of fibril formation of Sup35 in vitro mimics two other peculiar features of in vivo prion propagation, strain phenomenon and species barrier (21Chien P. Weissman J.S. Nature. 2001; 410: 223-227Crossref PubMed Scopus (133) Google Scholar, 22Santoso A. Chien P. Osherovich L.Z. Weissman J.S. Cell. 2000; 100: 277-288Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), offering a powerful system to study the propagation of yeast prions. Development of similar in vitro models for conversion of mammalian PrPs would be beneficial to our understanding of the mechanisms of prion diseases. Using C-terminal fragments of recombinant human and mouse PrPs that encompassed residues 90–231 and 89–230, respectively (designated Hu recPrP and Mo recPrP), I have developed an in vitro conversion system that displays attributes of an autocatalytic process. Thus, the conversion of monomeric recPrP into the amyloid fibrils can be seeded with small aliquots of preformed fibrils. I found that cross-seeding with preformed fibrils of heterologous recPrP was not as efficient as seeding with fibrils of homologous recPrP. The low efficiency of cross-seeding illustrated a high selectivity of the autocatalytic conversion in vitro in the absence of cellular environment. This phenomenon mimicked species barrier of prion transmission observed in vivo. I speculate that this model system can be used for assessing an intrinsic propensity of prion transmission between different species. Protein Expression and Purification—The genes coding for Hu PrP-(90–231) and Mo PrP-(89–231) were amplified by PCR from pcDNA3 plasmids containing the full-length Hu PrP and the full-length Mo PrP genes, correspondingly. The amplified genes were cloned into the pET101/d-TOPO vector (Invitrogen) and transformed into Top10 cells (Invitrogen). The transformants were tested by PCR amplification, and the plasmids isolated from the proper clones were retransformed into BL21 Star (DE3) (Invitrogen). The insertion of the correct gene was confirmed by DNA sequencing (Biopolymer Laboratory, University of Maryland, Baltimore). For the expression of Hu rec PrP-(90–231) and Mo recPrP-(89–230), 2% of an overnight culture was inoculated in fresh LB medium that contained 50 μg/ml carbenicillin (Sigma) and was grown at 37 °C until the A600 nm reached 0.7. was to a of and the were grown for an The cells were in pH and by in and at The were at for and proteins were as by Mehlhorn I. Groth D. J. B. D. D. Baldwin M. Fletterick Cohen F.E. D. Prusiner S.B. Biochemistry. PubMed Scopus Google Scholar). The proteins were confirmed by and to be species with an of in form of Hu recPrP and Mo recPrP into the fibrillar isoform at in in a at 37 °C I.V. Legname G. Baldwin Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the was the but in a the conversion not However, it was to the conversion by The conversion in is considered to be at The mechanism of be To monitor the kinetics of fibril aliquots were and into to a of recPrP the of thioflavin to a of were in with at nm on a and were to the fibrils with the at of the were at °C with a of using a in a of and The was with a high system and was on that were to The were for with 2% and in a electron and of recPrP were in an with a at with a of nm and of the of the of the the kinetics of spontaneous in vitro fibril formation of Hu recPrP at 37 °C a substantial by a rapid of amyloid fibrils as measured by The of the was a of the pH from at pH and to at pH In a using mouse and that acidic pH favored formation of the which is the kinetic pathway to the amyloid fibrils I.V. Legname G. Baldwin Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). between two one of which to the β-oligomer and to the fibrillar the substantial differences in the of the To whether the conversion of Hu recPrP to the β-oligomer with the formation of the fibrils, I the of the using size-exclusion of at 37 Hu recPrP was found in the form at pH whereas only of the protein into the β-oligomer at pH The of the of the fibril formation was a of the of the monomeric the the of the at the of fibril the was the The of our on the pH of fibril formation was consistent with a model for the conversion of mouse and recPrP I.V. Legname G. Baldwin Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This model that the in a slow with the and the β-oligomer is not on the kinetic pathway of the fibril formation The is the β-oligomer at acidic whereas the of Hu recPrP at neutral pH to the formation of fibrils. Because a for between the two abnormal isoforms is is to the β-oligomer with the amyloid fibrils using only because abnormal isoforms have similar and at nm the other a assay provided of the amyloid fibrils the β-oligomer Even though abnormal isoforms ThT, the of the amyloid form is the of the The of of the at pH and the low of the of fibril formation and the long I observed that the of conversion at pH and was the at pH and as judged from the in For the change in was at pH and only at pH The slow of conversion at acidic pH was consistent with the low of the observed the of fibril formation at acidic pH the of the its into the fibrils. the of forms and the the between the and the β-oligomer to the its not the other the substantially of the at pH and the of at pH As judged from electron the fibrils formed at pH and were substantially the fibrils formed at pH of long fibrils in which the was very whereas the low of the provided the substrate for fibril In to acidic the high of the at neutral pH accelerated the of The formation of of was by rapid of fibrils and of the The of the of the in of which the short of fibrils formed at neutral pH. The fibrils formed at different pH values a distinct The fibrils at pH and PrPSc “prion and were the distinct which was a of fibrils formed at pH of an autocatalytic conversion is a key of prion propagation, I tested whether in vitro formation of fibrils features of the autocatalytic process. small aliquots of preformed fibrils of Hu recPrP were from a into fresh I observed a in the The to which the was on the of to the fresh the as as an 0.001% aliquot of preformed fibrils displayed a seeding in fresh I observed a seeding the preformed fibrils of Mo recPrP were used to the conversion in a fresh of Mo recPrP These illustrate that in to the formation of the the conversion of recPrP into fibrils specific features of an autocatalytic process. As judged from the of the the efficacy of cross-seeding of Mo recPrP with the fibrillar form of Hu recPrP was the efficacy of seeding of Mo recPrP with the fibrillar Mo recPrP preformed fibrils of Mo recPrP displayed low seeding Hu recPrP Thus, of preformed fibrils of Mo recPrP seeding which was an of the seeding that would be displayed by of preformed fibrils of Hu recPrP This that the differences in the of recPrP from different species substantially the of the process of amyloid formation not but was only of the mechanism of the conversion at be Furthermore, the process of seeded conversion at displayed selectivity with to the of and a substrate. Thus, the conversion of Hu recPrP was only the of Hu but not Mo the of monomeric Mo in a with Hu substantially the of conversion seeded with fibrils of Hu recPrP I observed this of the conversion high of recPrP in the conversion at an autocatalytic mechanism for in vitro amyloid formation and displayed species selectivity of this process. In a study I.V. Legname G. Baldwin Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) an for the of α-helical monomeric recPrP and into two distinct β-sheet rich isoforms, the β-oligomer and amyloid fibrils. The amyloid fibrils of recPrP physical with Using a of specific that the amyloid of recPrP has an similar to that of their I.V. Legname G. Baldwin Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To the that conformational of PrPSc, in vitro fibril formation can into the prion that the Hu recPrP can be to the β-oligomer form and to amyloid fibrils. These that the of Hu recPrP is similar to that of Mo recPrP and recPrP I.V. Legname G. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2001; 276: 19687-19690Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, I.V. Legname G. Baldwin Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the studies of Hu recPrP reported that the protein can be to a β-sheet-rich isoform, to between the and fibrillar forms W. Morillas M. Chen S.G. Gambetti P. Surewicz W.K. Biochemistry. 2000; 39: 424-431Crossref PubMed Scopus (217) Google Scholar). The to two different to the of the was with of the of Hu recPrP. Thus, and Surewicz Surewicz W.K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that Hu into amyloid fibrils at pH as by According to our the two at pH at which the formation of the amyloid fibrils and displays the whereas the formation of the β-oligomer of Because the and fibrillar forms have similar be used as a valid probe to monitor the kinetics of amyloid The assay a for of amyloid The of the amyloid fibrils is the of the of β-oligomer However, because to the to be for of is to the as a change in values as a Because a was used for the of the and the Surewicz W.K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), as to whether the formation of the β-oligomer the amyloid fibrils was measured by According to our of Hu the by and Surewicz Surewicz W.K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) GdnHCl, pH at 37 °C in the formation of the β-oligomer but not amyloid fibrils. GdnHCl, the observed only with the but not with the Hu recPrP Surewicz W.K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, These are consistent with our studies that the conformational from the to the β-oligomer was by a barrier with and I.V. Legname G. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2001; 276: 19687-19690Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). the and facilitate the conversion to the Because the is the the of the to the form as by and Surewicz Surewicz W.K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the study I have that in vitro conversion of Hu recPrP to the fibrillar form an autocatalytic i.e. the of preformed fibrils of recPrP to initiate the process of The process of seeded conversion is consistent with the protein-only hypothesis, which postulates that PrPSc propagates its pathological conformation in an autocatalytic manner and acts as a transmissible agent of the prion diseases. conversion of the form of recombinant prion protein was by and S. D. Biol. 2003; PubMed Scopus Google Scholar). The conversion was of the in the of and at pH by of and at acidic which in the formation of by into fibrils. The seeding to of preformed fibrils. Because the in the amyloid fibrils are to other by it is that of the can be in the of using this of are the form of recPrP has been to form a fibrillar G.S. Hosszu L.L.P. Power A. Hill A.F. Kenney J. Saibil H. Craven C.J. Waltho J.P. Clarke A.R. Collinge J. Science. 1999; 283: 1935-1937Crossref PubMed Scopus (364) Google Scholar, S. D. Biol. 2003; PubMed Scopus Google Scholar), it is the form that the prion disease Prusiner S.B. J. Biochem. 1988; PubMed Scopus Google Scholar, Raymond Caughey B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The seeding phenomenon has been observed using the form of into an abnormal The seeding of in vitro conversion with preformed fibrils in was I observed the seeding the of Hu recPrP to fibrils to the monomeric Hu recPrP was studies of the that the multimeric isoforms of recPrP with a molecular seeding activity. one that on the of of propagation to the substrate be as high as These that the autocatalytic conversion is efficient the is seeded with fibrils of homologous recPrP. Furthermore, I found that the autocatalytic conversion displayed species fibrils of Hu recPrP were capable of seeding the conversion of Hu recPrP, whereas the conversion of Mo recPrP with the of fibrillar Hu recPrP was and vice versa. This the high selectivity of the autocatalytic conversion with to the of and Using B. Surewicz Surewicz W.K. Proc. Acad. Natl. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar) found that the residues the of PrP are for the formation of amyloid one of the between Hu and Mo PrP is at in Hu PrP and in Mo this other the 90–231 the species selectivity of autocatalytic conversion to be The high selectivity of autocatalytic conversion was confirmed using Strong species selectivity of seeded conversion in vitro the species barrier of prion propagation observed in vivo. our were in with an made on which Hu and Mo PrPC Scott M. J. M. Cohen F.E. S.J. Prusiner S.B. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). with high of Hu PrPC expression were to Hu PrPSc but to human of the Mo PrP gene Scott M. J. M. Cohen F.E. S.J. Prusiner S.B. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). the of Mo and Hu in vitro conversion by Hu Such can be by between Hu and Mo for to the of Hu has yet to be whether the of Mo to Hu the of Hu to the of the and the conversion of Hu recPrP. The species barrier transmission of species I.H. Sci. PubMed Google Scholar) and is considered as one of the most peculiar feature of prion studies in two have been that to the species the in PrP between the species and the species and the conformational of of PrPSc S.B. Scott M. D. K.-M. Groth D. C. M. Serban D. D. S.J. Cell. Full Text PDF PubMed Scopus Google Scholar, Groth D. J. M. P. S.J. Prusiner S.B. J. Virol. 1997; PubMed Google Scholar, M. S. I. J. Hill A.F. Collinge J. J. 2002; PubMed Scopus Google Scholar). the of the PrPC of a is to the of PrPSc of a the disease has the In PrPSc is from one species to the is in the and the and I.H. Sci. PubMed Google Scholar, Groth D. J. M. P. S.J. Prusiner S.B. J. Virol. 1997; PubMed Google Scholar). the species barrier and the strain were using an in vitro conversion system of the prion protein of yeast (21Chien P. Weissman J.S. Nature. 2001; 410: 223-227Crossref PubMed Scopus (133) Google Scholar, Santoso A. P. Weissman J.S. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). amyloid fibrils of Sup35 were capable of seeding the formation of fibrils in fresh and provided that the conformation of the fibrils. As a the strain and conformational of the were to the formed fibrils (21Chien P. Weissman J.S. Nature. 2001; 410: 223-227Crossref PubMed Scopus (133) Google Scholar). For a of a cell-free system has been developed using mammalian recombinant in vitro system displayed high efficiency and species selectivity of the conversion in the absence of cellular environment. In to the differences between the of PrPC and PrPSc, the conformational of PrPSc to the to the species barrier in vivo Groth D. J. M. P. S.J. Prusiner S.B. J. Virol. 1997; PubMed Google Scholar, A.F. M. S. I. Collinge J. P. Nature. 1997; PubMed Scopus Google Scholar). of PrPSc the species barrier to different species. the conformational of and the of the of with conformational in the D. R.A. Legname G. J. D. S. Prusiner S. Scott 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), it would be to the of transmission of different PrPSc from and to Because cellular and are for the in vitro conversion system in our the selectivity of seeding the differences in the of a and a substrate. This system be used as a rapid assay for assessing the intrinsic propensities of prion transmission between different species. I for plasmids containing the genes mouse and human PrPs and for the and expression of recombinant