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Mammalian Prions Generated from Bacterially Expressed Prion Protein in the Absence of Any Mammalian Cofactors*

Open AccessPublished:March 19, 2010DOI:https://doi.org/10.1074/jbc.C110.113464
      Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrPC, to a misfolded aggregated form, PrPSc. The protein-only hypothesis asserts that PrPSc itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.

      Introduction

      Transmissible spongiform encephalopathies (TSEs),
      The abbreviations used are: TSE
      transmissible spongiform encephalopathy
      PrPC
      normal cellular prion protein
      PrPSc
      disease-associated scrapie isoform of prion protein
      PMCA
      protein misfolding cyclic amplification
      rPrP
      recombinant PrP
      rShaPrP-(23–231)
      recombinant Syrian hamster full-length prion protein
      rShaPrP-(90–231)
      N-terminally truncated fragment of the recombinant Syrian hamster prion protein
      rShaPrPPMCA
      recombinant Syrian hamster PrP aggregates generated in PMCA reaction
      PK
      proteinase K
      PrP27–30
      PK-resistant core of PrPSc
      GPI
      glycosylphosphatidylinositol
      PBS
      phosphate-buffered saline.
      or prion diseases, are a group of fatal neurodegenerative disorders of humans and animals (
      • Cobb N.J.
      • Surewicz W.K.
      ,
      • Prusiner S.B.
      ,
      • Collinge J.
      ,
      • Aguzzi A.
      • Polymenidou M.
      ,
      • Caughey B.
      • Baron G.S.
      • Chesebro B.
      • Jeffrey M.
      ). The pathogenic process in these diseases is typically associated with conformational conversion of a normal (cellular) prion protein, PrPC, to a misfolded form, PrPSc. The “protein-only” model asserts that this rogue PrPSc conformer itself represents the infectious prion agent, self-propagating by binding to PrPC and inducing its conversion to the abnormal PrPSc isoform (
      • Prusiner S.B.
      ).
      Although this protein-only model is consistent with substantial experimental data (
      • Cobb N.J.
      • Surewicz W.K.
      ,
      • Prusiner S.B.
      ,
      • Collinge J.
      ,
      • Aguzzi A.
      • Polymenidou M.
      ,
      • Caughey B.
      • Baron G.S.
      • Chesebro B.
      • Jeffrey M.
      ), unequivocal proof for the purely proteinaceous nature of the infectious TSE agent has been elusive. Prion infectivity has been propagated, and even initiated de novo, under cell-free conditions using “protein misfolding cyclic amplification” (PMCA) (
      • Castilla J.
      • Saá P.
      • Hetz C.
      • Soto C.
      ,
      • Deleault N.R.
      • Harris B.T.
      • Rees J.R.
      • Supattapone S.
      ,
      • Barria M.A.
      • Mukherjee A.
      • Gonzalez-Romero D.
      • Morales R.
      • Soto C.
      ). These PMCA reactions involved successive rounds of incubation and sonication of crude brain homogenates (
      • Castilla J.
      • Saá P.
      • Hetz C.
      • Soto C.
      ,
      • Barria M.A.
      • Mukherjee A.
      • Gonzalez-Romero D.
      • Morales R.
      • Soto C.
      ) or largely purified brain- or cell-derived PrPC and poly(A) RNA (
      • Deleault N.R.
      • Harris B.T.
      • Rees J.R.
      • Supattapone S.
      ,
      • Geoghegan J.C.
      • Miller M.B.
      • Kwak A.H.
      • Harris B.T.
      • Supattapone S.
      ) and, most recently, bacterially expressed recombinant PrP (rPrP) mixed with mouse-derived RNA and acidic lipids (
      • Wang F.
      • Wang X.
      • Yuan C.G.
      • Ma J.
      ). However, the presence of other mammalian cell-derived molecules in these preparations leaves open the question regarding the precise chemical identity of mammalian prions. It has also been reported that amyloid fibrils formed spontaneously from rPrP in the absence of any added cofactors can cause a transmissible neurological disorder in transgenic mice (
      • Legname G.
      • Baskakov I.V.
      • Nguyen H.O.
      • Riesner D.
      • Cohen F.E.
      • DeArmond S.J.
      • Prusiner S.B.
      ,
      • Colby D.W.
      • Giles K.
      • Legname G.
      • Wille H.
      • Baskakov I.V.
      • DeArmond S.J.
      • Prusiner S.B.
      ). However, because the transgenic mice used in these studies overproduce PrPC (4–32-fold when compared with wild-type mice), it is unclear whether these synthetic fibrils indeed carry bona fide infectivity or, rather, accelerate a condition to which transgenic mice could potentially be predisposed due to the high level of PrPC or other factors (
      • Nazor K.E.
      • Kuhn F.
      • Seward T.
      • Green M.
      • Zwald D.
      • Pürro M.
      • Schmid J.
      • Biffiger K.
      • Power A.M.
      • Oesch B.
      • Raeber A.J.
      • Telling G.C.
      ). This question is especially pressing because numerous attempts to infect wild-type mice or hamsters with similar material have not been successful.
      We have shown recently that structurally distinct fibrillar aggregates can be produced from rPrP by PMCA when seeded with scrapie-derived brain homogenate or partially purified PrPSc (
      • Atarashi R.
      • Moore R.A.
      • Sim V.L.
      • Hughson A.G.
      • Dorward D.W.
      • Onwubiko H.A.
      • Priola S.A.
      • Caughey B.
      ,
      • Smirnovas V.
      • Kim J.I.
      • Lu X.
      • Atarashi R.
      • Caughey B.
      • Surewicz W.K.
      ). Importantly, these PrPSc-seeded aggregates (denoted rPrPPMCA or rPrP-res(Sc)) display a proteinase K (PK) digestion pattern that is more closely related to PrPSc than that of spontaneously formed rPrP fibrils (
      • Atarashi R.
      • Moore R.A.
      • Sim V.L.
      • Hughson A.G.
      • Dorward D.W.
      • Onwubiko H.A.
      • Priola S.A.
      • Caughey B.
      ,
      • Smirnovas V.
      • Kim J.I.
      • Lu X.
      • Atarashi R.
      • Caughey B.
      • Surewicz W.K.
      ). Here, we report that these aggregates, prepared solely from highly purified recombinant hamster prion protein (rShaPrP) in the apparent absence of any additional cofactors, are infectious to wild-type hamsters.

      RESULTS AND DISCUSSION

      To test the infectivity of rPrPPMCA fibrils, we performed nine rounds of PMCA using as a substrate recombinant Syrian hamster full-length PrP (rShaPrP-(23–231)) or the 90–231 fragment (rShaPrP-(90–231)). In the first round, the reaction was seeded with partially purified, PK-digested PrPSc (PrP27–30) derived from 263K scrapie-infected hamster brain. Each subsequent round was seeded with a 1/10 or 1/100 volume of PMCA product from the previous round (see “Experimental Procedures”). The cumulative 10−14 dilution of the original PrPSc seed theoretically made its concentration ∼0.01 ag/ml or 0.2 molecules/ml, an amount ∼107-fold below one LD50 (a dose lethal to 50% of intracerebrally inoculated hamsters).
      Multiple independently prepared rShaPrP-(23–231)PMCA or rShaPrP-(90–231)PMCA preparations were harvested and inoculated intracerebrally into groups of hamsters. Remarkably, a fraction of inoculated animals in all but one group developed clinical signs of scrapie, although there was considerable variability in the incubation times (ranging from 119 to 401 days) and attack rates (Table 1). In the second passage experiments using brain extract from affected hamsters as the inocula, all hamsters became infected with an incubation time of 84 ± 1 and 75 ± 4 days for rShaPrP-(23–231)PMCA- and rShaPrP-(90–231)PMCA-derived material, respectively (Table 1).
      TABLE 1Summary of infectivity bioassays in hamsters
      Inoculum
      Fifty microliters of each inoculum were used. For inocula derived from rShaPrP, the concentration of protein was 300 μg/ml in each case. For second passage of the rPrPPMCA group, 1% brain homogenate was used. The concentration of undiluted PrP27–30 seed was 1 μg/ml.
      No. of TSE-positive animals
      Animals showed clinical signs for TSE and were positive for PrPSc.
      /no. of inoculated
      Incubation time of TSE-positive animals, days
      Mean ± standard deviation.
      rPrPPMCA (seeded with PrP27–30)
      rShaPrP-(23–231) (Experiment I)5/10328 ± 113 (133, 323, 391, 392, 401)
      rShaPrP-(23–231) (Experiment II)6/9251 ± 112 (119, 166, 166, 345, 345, 365)
      rShaPrP-(90–231) (Experiment I)7/9185 ± 83 (129, 129, 143, 145, 188, 196, 362)
      rShaPrP-(90–231) (Experiment II)
      Bioassays were performed at Rocky Mountain Laboratories; all other bioassays were done at Case Western Reserve University.
      6/6162 ± 16 (141, 154, 154, 166, 166, 188)
      rShaPrP-(90–231) (Experiment III)1/7187
      rShaPrP-(90–231) (Experiment IV)0/6NA
      NA, not applicable. Animals in these groups did not show any neurological abnormalities during their life span and were not positive for PrPSc.
      2nd passage of rShaPrP-(23–231) (Experiment I)
      Hamster sacrificed at 133 days after inoculation was used.
      6/684 ± 1
      2nd passage of rShaPrP-(90–231) (Experiment I)
      Hamster sacrificed at 129 days after inoculation was used.
      6/675 ± 4
      2nd passage of rShaPrP-(90–231) (Experiment I)
      Hamster sacrificed at 143 days after inoculation was used.
      6/675 ± 5
      Negative controls
      Diluted PrP27–30 seed
      Sample was prepared by serial 10−14 dilution of PrP27–30 seed.
      0/9NA
      NA, not applicable. Animals in these groups did not show any neurological abnormalities during their life span and were not positive for PrPSc.
      Mock PMCA of PrP27–30 alone
      Sample was prepared by subjecting PrP27–30 alone (no rShaPrP substrate) to a mock PMCA procedure that results in 10−14 dilution of PrP27–30.
      0/6NA
      NA, not applicable. Animals in these groups did not show any neurological abnormalities during their life span and were not positive for PrPSc.
      rShaPrP-(23–231) monomer0/9NA
      NA, not applicable. Animals in these groups did not show any neurological abnormalities during their life span and were not positive for PrPSc.
      rShaPrP-(90–231) monomer0/6NA
      NA, not applicable. Animals in these groups did not show any neurological abnormalities during their life span and were not positive for PrPSc.
      Positive control
      Undiluted PrP27–30 seed9/985 ± 4
      a Fifty microliters of each inoculum were used. For inocula derived from rShaPrP, the concentration of protein was 300 μg/ml in each case. For second passage of the rPrPPMCA group, 1% brain homogenate was used. The concentration of undiluted PrP27–30 seed was 1 μg/ml.
      b Animals showed clinical signs for TSE and were positive for PrPSc.
      c Mean ± standard deviation.
      d Bioassays were performed at Rocky Mountain Laboratories; all other bioassays were done at Case Western Reserve University.
      e NA, not applicable. Animals in these groups did not show any neurological abnormalities during their life span and were not positive for PrPSc.
      f Hamster sacrificed at 133 days after inoculation was used.
      g Hamster sacrificed at 129 days after inoculation was used.
      h Hamster sacrificed at 143 days after inoculation was used.
      i Sample was prepared by serial 10−14 dilution of PrP27–30 seed.
      j Sample was prepared by subjecting PrP27–30 alone (no rShaPrP substrate) to a mock PMCA procedure that results in 10−14 dilution of PrP27–30.
      In control experiments, hamsters were inoculated with either undiluted PrP27–30 seed or a 1014-fold dilution thereof (equivalent to cumulative serial PMCA dilution). As expected, animals inoculated with the undiluted PrP27–30 seed succumbed to scrapie disease with a typical incubation time of 85 ± 4 days. In contrast, all hamsters inoculated with diluted PrP27–30 remained healthy (Table 1), confirming that the serial PMCA procedure was sufficient to dilute out all of the original PrP27–30-associated infectivity. A caveat of any infectivity studies with “synthetic prions” is potential contamination with exogenously derived infectivity during either sample preparation or inoculation of animals. To address this issue, we have performed control experiments using a mock PMCA procedure in which PrP27–30 seed was subjected to the PMCA protocol used to generate rShaPrPPMCA but without any rPrP substrate. No infectivity was detected in this material (Table 1), strongly arguing against the likelihood of contamination. Furthermore, bioassays with rShaPrP-(90–231)PMCA were performed independently in two different laboratories, consistently indicating infectivity of this material (Table 1). Given the possibility of spontaneous (non-seeded) rPrP prion formation by PMCA (see below), mock experiments described above represent the only reliable negative control in this type of study.
      Western blot analysis revealed that brains of all hamsters showing clinical TSE signs accumulated PrPSc. Electrophoretic profiles of PrPSc from hamsters inoculated with rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA, as well as those inoculated in second passage experiments (i.e. using brain homogenates of rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA-infected animals), were indistinguishable from those infected with 263K-PrP27–30 (Fig. 1A), indicating similar PK cleavage site(s) as well as a similar ratio of different PrPSc glycoforms.
      Figure thumbnail gr1
      FIGURE 1Characterization of PrPSc and brain histopathology of rShaPrPPMCA-inoculated and control hamsters. A, Western blot analysis of PrPSc in brain homogenates from hamsters inoculated with PrP27–30 (lane 1), PrP27–30 seed after 10−14 dilution (lane 2), PrP27–30 seed subjected to mock PMCA (resulting in 10−14 dilution) in the absence of any rShaPrP substrate (lane 3), rShaPrP-(23–231)PMCA (lane 4), rShaPrP-(90–231)PMCA (lane 5), brain homogenate from rShaPrP-(23–231)PMCA-inoculated hamster (lane 6), and brain homogenate from rShaPrP-(90–231)PMCA-inoculated hamster (lane 7). Samples were analyzed before (−) and after (+) PK digestion (100 μg/ml, 1 h at 37 °C). The blot was probed with anti-PrP 3F4 antibody (1:10,000). B, lesion profiles in brains of hamsters inoculated with 263K scrapie PrP27–30 (red circle, n = 3), rShaPrP-(23–231)PMCA (blue triangle, n = 10), and rShaPrP-(90–231)PMCA (green square, n = 5). The profiles for hamsters inoculated with rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA were very similar to each other, but both were significantly different from that of hamsters inoculated with 263K scrapie PrP27–30. Specific brain regions showing statistically significant differences between hamsters inoculated with 263K scrapie PrP27–30 and rShaPrP-(90–231)PMCA are indicated by an asterisk (p < 0.001–0.04). For rShaPrP-(23–231)PMCA-inoculated hamsters, statistically significant differences (p < 0.001–0.02) from 263K-inoculated hamsters were observed in the same regions except for the thalamus. Symbols used for brain regions are: Cx, cerebral cortex; HI, hippocampus; Sub, subiculum; BG, basal ganglia; TH, thalamus; CE, cerebellum; Sept n, septal nuclei; BS, brainstem. Error bars indicate S.E. Statistical significance was determined using a two-tailed Student's t test. C–F, spongiform degeneration in basal ganglia of hamsters inoculated with 263K scrapie PrP27–30 (C), rShaPrP-(90–231)PMCA (sacrificed at 129 days after inoculation) (D), and brain homogenate of rShaPrP-(90–231)PMCA-infected hamsters (E). The histology of basal ganglia from control hamster inoculated with normal brain homogenate and sacrificed 368 days after inoculation is shown in panel F. Circles in panels D and E exemplify small clusters of typical vacuoles. Although 263K scrapie PrP27–30-inoculated hamsters showed widespread spongiform degeneration, the spongiform degeneration in rShaPrP-(90–231)PMCA-inoculated hamsters was much less severe, and this difference was maintained in the second passage. No spongiform degeneration was observed in control hamsters. Bar = 100 μm.
      rShaPrPPMCA-inoculated hamsters showed TSE brain pathology, although spongiform degeneration was less severe when compared with hamsters inoculated with 263K scrapie (Fig. 1, B–F, and supplemental Fig. 1). Importantly, the lesion profiles for hamsters inoculated with rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA were very similar to each other but were both significantly different from that of 263K scrapie-inoculated hamsters (Fig. 1B). These distinct lesion profiles found in rShaPrPPMCA-inoculated hamsters were preserved upon second passage (Fig. 2, A and B), indicating that rShaPrPPMCA represents a stable prion strain that is different from 263K scrapie. The overall pattern of PrPSc immunostaining in rShaPrPPMCA-inoculated hamsters was similar to hamsters inoculated with 263K scrapie. However, the PrPSc staining intensity was markedly lower in hamsters inoculated with rShaPrPPMCA, and this low staining intensity was maintained in the second passage (Fig. 2, C–E). Again, this points to distinct strain properties of rShaPrPPMCA, strongly arguing against the possibility that contamination could be responsible for the infectivity of the rPrPPMCA preparations. The apparent emergence of a new prion strain (that is, distinct from the 263K scrapie seed) in our PMCA reaction is puzzling, especially given the previous findings that PMCA can preserve strain characteristics of the seed (
      • Castilla J.
      • Saá P.
      • Hetz C.
      • Soto C.
      ,
      • Deleault N.R.
      • Harris B.T.
      • Rees J.R.
      • Supattapone S.
      ), even in the absence of glycosylation (
      • Piro J.R.
      • Harris B.T.
      • Nishina K.
      • Soto C.
      • Morales R.
      • Rees J.R.
      • Supattapone S.
      ). However, our bacterially expressed rPrP substrate differs from the brain-derived PrPC used in previous studies in at least two additional aspects that might influence the fidelity of strain characteristics. One is the absence of any additional brain components that copurify with brain-derived PrPC (
      • Deleault N.R.
      • Harris B.T.
      • Rees J.R.
      • Supattapone S.
      ), and the other is the lack of the glycosylphosphatidylinositol (GPI) anchor. The latter modification may modify strain properties of in vitro-generated prions, consistent with the recent findings regarding the role of the lipid moiety of the substrate in PrPSc amplification by PMCA (
      • Kim J.I.
      • Surewicz K.
      • Gambetti P.
      • Surewicz W.K.
      ).
      Figure thumbnail gr2
      FIGURE 2Comparison of lesion profiles and PrPSc staining observed in first and second passage of rShaPrPPMCA prions. A, lesion profile in first (blue triangle, n = 10) and second (black open squares, n = 5) passage of rShaPrP-(23–231)PMCA; B, lesion profile in first (blue triangle, n = 5) and second (black open squares, n = 12) passage of rShaPrP-(90–231)PMCA. Lesion profiles for 263K scrapie-inoculated hamsters (red circle, n = 3) are shown for comparison. No statistically significant differences were found between profiles for first and second passages of both rShaPrP-(23–231)PMCA and rShaPrP-(90–231)PMCA (p > 0.05), but each of these profiles was significantly different from that for 263K scrapie (p < 0.00001–0.02). Symbols used for brain regions are: Cx, cerebral cortex; HI, hippocampus; Sub, subiculum; BG, basal ganglia; TH, thalamus; CE, cerebellum; Sept n, septal nuclei; BS, brainstem. Error bars indicate S.E. Statistical significance was determined using a two-tailed Student's t test. C–E, immunohistochemical staining of PrPSc in the white matter of corpus callosum (CC) and gray matter of cerebral cortex (Cor). C, hamsters inoculated with 263K scrapie PrP27–30; D, first passage of rShaPrP-(90–231)PMCA; E, second passage of rShaPrP-(90–231)PMCA. The square in panel C indicates perivascular deposits of PrPSc, and arrows indicate plaque-like deposits. Bar = 100 μm.
      The apparent lack of strain-specific fidelity in rPrPPMCA propagation as observed in the present study contrasts with the observations in studies with yeast prions. In the latter system, conformation-encoded strain properties of infectious amyloid fibrils prepared in vitro from recombinant yeast prion protein were fully preserved upon transfection into living cells, and this strain-specific fidelity was considered important for formally proving the concept of protein conformation-based inheritance in yeast (
      • King C.Y.
      • Diaz-Avalos R.
      ,
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      ). Such perfect reproduction of strain-specific patterns in experiments with synthetic mammalian prions may require the use of pure PrP substrate that is chemically identical to brain PrPC (i.e. with posttranslational modifications such as glycosylation and the GPI anchor).
      Given the above caveat of studies with aggregates generated in vitro from bacterially expressed recombinant PrP, one cannot formally rule out the possibility that these preparations trigger infectious TSE disease by some unknown mechanism other than acting as a physical template for the conversion of brain PrPC into the PrPSc state. However, the latter possibility is purely theoretical and highly unlikely.
      The recently reported de novo generation of infectivity (i.e. in the absence of any preexisting PrPSc seed) from brain-derived PrPC and poly(A) RNA (
      • Deleault N.R.
      • Harris B.T.
      • Rees J.R.
      • Supattapone S.
      ), total brain homogenate (
      • Barria M.A.
      • Mukherjee A.
      • Gonzalez-Romero D.
      • Morales R.
      • Soto C.
      ), or recombinant PrP in the presence of mouse RNA and lipids (
      • Wang F.
      • Wang X.
      • Yuan C.G.
      • Ma J.
      ) raises the question as to whether this could also be accomplished using highly purified rPrP in the absence of any cofactors. Our preliminary data suggest that infectious material can indeed be generated de novo from pure rShaPrP, although the probability of such an event appears to be very low. These experiments with de novo generated rPrP prions are still ongoing; after completion, they will be reported elsewhere.
      Altogether, the present data demonstrate that infectious prions can be generated by PMCA from highly purified rPrP in the absence of any apparent cofactors. rPrP differs from typical brain-derived PrPC by its lack of N-linked glycans and a GPI moiety, and these data confirm previous indications that these post-translational modifications are not essential for prion propagation in vivo (
      • Tuzi N.L.
      • Cancellotti E.
      • Baybutt H.
      • Blackford L.
      • Bradford B.
      • Plinston C.
      • Coghill A.
      • Hart P.
      • Piccardo P.
      • Barron R.M.
      • Manson J.C.
      ,
      • Chesebro B.
      • Trifilo M.
      • Race R.
      • Meade-White K.
      • Teng C.
      • LaCasse R.
      • Raymond L.
      • Favara C.
      • Baron G.
      • Priola S.
      • Caughey B.
      • Masliah E.
      • Oldstone M.
      ). Variable attack rates and long incubation times indicate that the infectivity of rShaPrPPMCA is very low relative to brain-derived PrPSc. This is consistent with our observation that inoculation of hamsters with rShaPrP-(90–231)PMCA at ∼25-fold lower dose than that described in Table 1 did not cause any signs of TSE (data not shown). Although the reasons for much lower infectivity of our conversion product (per unit of protein) when compared with that of brain-derived PrPSc are at present unclear, this could be due to one of the following factors (or a combination thereof). (i) The infectious material might constitute only a small subfraction of our PMCA product; (ii) other cofactors that are supplied in vivo, and not in our scrapie-seeded rPrP-PMCA reactions, might promote the infectivity of the conversion product; (iii) non-infectious off-pathway products of the rPrP-PMCA reaction might interfere with the infectivity, or promote the clearance, of the infectious scrapie-seeded rPrP PMCA product. The complete attack rate and greatly reduced incubation period on second passage indicate that the artificial rShaPrPPMCA prions adapt to hamsters, as takes place with archetypal prion diseases.
      It was recently reported that material infectious to wild-type mice can be formed de novo from the mixture of mouse rPrP and relatively large quantities of mouse-derived RNA and an acidic lipid, phosphatidylglycerol (
      • Wang F.
      • Wang X.
      • Yuan C.G.
      • Ma J.
      ). Some apparent infectivity was also reported to be associated with spontaneously formed recombinant PrP amyloid fibrils subjected to “annealing” by incubation with normal brain homogenate, although in this case, clinical signs were observed only in the second passage and only after very long incubation times (481–565 days) (
      • Makarava N.
      • Kovacs G.G.
      • Bocharova O.
      • Savtchenko R.
      • Alexeeva I.
      • Budka H.
      • Rohwer R.G.
      • Baskakov I.V.
      ). In contrast to these recent studies on de novo generated infectivity, the infectious material in our work was generated in a PrPSc-seeded PMCA reaction and, most importantly, in the absence of any added cofactors. The finding that truly infectious prions causing clinical TSE disease in a wild-type host can be generated from pure bacterially expressed prion protein provides a strong support for the protein-only model of these neurological disorders. Furthermore, our data demonstrate that additional cofactors that have been used in previous studies (nucleic acids, lipids) are not obligatory for generation of infectious mammalian prions. However, given the apparent low infectivity titer of the material generated in the present study, it appears that these (and/or other) cofactors may enhance or accelerate prion protein misfolding to the infectious form in vitro as well as in vivo.

      Acknowledgments

      We thank Fusong Chen and Michael Payne for excellent handling of animals and Phyllis Scalzo and Diane Kofskey for expert histological preparations.

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