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Isolation of a Defective Prion Mutant from Natural Scrapie

View Article: PubMed Central - PubMed

ABSTRACT

It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

No MeSH data available.


Related in: MedlinePlus

Identification of 14K from a natural scrapie sample.A) Serial 10-fold dilutions of 2 Italian scrapie samples (ES47/10/3 and 198/9) were used as seeds in serial PMCA reactions using vole brain homogenate substrate. Products from rounds 4°, 6° and 8° (indicated in roman numbers) were digested with PK and analyzed by Western blot with antibody SAF84. After 8 PMCA rounds both samples were positive up to dilution 10−7. An atypical PrPSc with smaller PrPres (indicated by the asterisk) emerged after the sixth round only from the last detectable dilution of sample 198/9, and was propagated until the end of the experiment. B) Three in vitro selected prion populations (18K, 14K/1 and 14K/2, as indicated on the top of each blot) were serially propagated for 4 successive PMCA rounds (represented in roman numbers). After each round, aliquots of the PMCA products were digested with PK and analyzed by Western blot with antibody SAF84. For 14K/1, PK-digested PrPres is also shown after enzymatic removal of N-linked oligosaccarides, which allows to better appreciate the co-presence and evolution of 14K and 18K PrPres (indicated by a single and a double asterisk respectively) during the experiment.
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ppat.1006016.g001: Identification of 14K from a natural scrapie sample.A) Serial 10-fold dilutions of 2 Italian scrapie samples (ES47/10/3 and 198/9) were used as seeds in serial PMCA reactions using vole brain homogenate substrate. Products from rounds 4°, 6° and 8° (indicated in roman numbers) were digested with PK and analyzed by Western blot with antibody SAF84. After 8 PMCA rounds both samples were positive up to dilution 10−7. An atypical PrPSc with smaller PrPres (indicated by the asterisk) emerged after the sixth round only from the last detectable dilution of sample 198/9, and was propagated until the end of the experiment. B) Three in vitro selected prion populations (18K, 14K/1 and 14K/2, as indicated on the top of each blot) were serially propagated for 4 successive PMCA rounds (represented in roman numbers). After each round, aliquots of the PMCA products were digested with PK and analyzed by Western blot with antibody SAF84. For 14K/1, PK-digested PrPres is also shown after enzymatic removal of N-linked oligosaccarides, which allows to better appreciate the co-presence and evolution of 14K and 18K PrPres (indicated by a single and a double asterisk respectively) during the experiment.

Mentions: To test the sensitivity of detection of serial bank vole PMCA (bvPMCA) on natural scrapie isolates, serial ten-fold dilutions of 3 sheep scrapie isolates (198/9, ES47/10/2 and ES47/10/3) were subjected to 10 rounds of PMCA (Fig 1A). The sensitivity observed with the whole set of inocula resembled that reported by Chianini et al. [17], showing a limit of detection of 10−6/10−7 after 8 bvPMCA rounds (Fig 1A). Sixteen unseeded samples were used as negative controls and remained negative up to the 10th round, showing that neither cross-contamination nor spontaneous appearance of PrPSc occurred under these experimental conditions, as previously reported [18]. Moreover, with the only exception of dilution 10−7 of 198/9 (see below), the electrophoretic pattern of the PK-resistant core of the amplified PrPSc was similar to that in the original inocula, as previously observed with other scrapie isolates [17]. We will refer to this pattern as 18K, according to the apparent MW of the unglycosylated PrPres.


Isolation of a Defective Prion Mutant from Natural Scrapie
Identification of 14K from a natural scrapie sample.A) Serial 10-fold dilutions of 2 Italian scrapie samples (ES47/10/3 and 198/9) were used as seeds in serial PMCA reactions using vole brain homogenate substrate. Products from rounds 4°, 6° and 8° (indicated in roman numbers) were digested with PK and analyzed by Western blot with antibody SAF84. After 8 PMCA rounds both samples were positive up to dilution 10−7. An atypical PrPSc with smaller PrPres (indicated by the asterisk) emerged after the sixth round only from the last detectable dilution of sample 198/9, and was propagated until the end of the experiment. B) Three in vitro selected prion populations (18K, 14K/1 and 14K/2, as indicated on the top of each blot) were serially propagated for 4 successive PMCA rounds (represented in roman numbers). After each round, aliquots of the PMCA products were digested with PK and analyzed by Western blot with antibody SAF84. For 14K/1, PK-digested PrPres is also shown after enzymatic removal of N-linked oligosaccarides, which allows to better appreciate the co-presence and evolution of 14K and 18K PrPres (indicated by a single and a double asterisk respectively) during the experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120856&req=5

ppat.1006016.g001: Identification of 14K from a natural scrapie sample.A) Serial 10-fold dilutions of 2 Italian scrapie samples (ES47/10/3 and 198/9) were used as seeds in serial PMCA reactions using vole brain homogenate substrate. Products from rounds 4°, 6° and 8° (indicated in roman numbers) were digested with PK and analyzed by Western blot with antibody SAF84. After 8 PMCA rounds both samples were positive up to dilution 10−7. An atypical PrPSc with smaller PrPres (indicated by the asterisk) emerged after the sixth round only from the last detectable dilution of sample 198/9, and was propagated until the end of the experiment. B) Three in vitro selected prion populations (18K, 14K/1 and 14K/2, as indicated on the top of each blot) were serially propagated for 4 successive PMCA rounds (represented in roman numbers). After each round, aliquots of the PMCA products were digested with PK and analyzed by Western blot with antibody SAF84. For 14K/1, PK-digested PrPres is also shown after enzymatic removal of N-linked oligosaccarides, which allows to better appreciate the co-presence and evolution of 14K and 18K PrPres (indicated by a single and a double asterisk respectively) during the experiment.
Mentions: To test the sensitivity of detection of serial bank vole PMCA (bvPMCA) on natural scrapie isolates, serial ten-fold dilutions of 3 sheep scrapie isolates (198/9, ES47/10/2 and ES47/10/3) were subjected to 10 rounds of PMCA (Fig 1A). The sensitivity observed with the whole set of inocula resembled that reported by Chianini et al. [17], showing a limit of detection of 10−6/10−7 after 8 bvPMCA rounds (Fig 1A). Sixteen unseeded samples were used as negative controls and remained negative up to the 10th round, showing that neither cross-contamination nor spontaneous appearance of PrPSc occurred under these experimental conditions, as previously reported [18]. Moreover, with the only exception of dilution 10−7 of 198/9 (see below), the electrophoretic pattern of the PK-resistant core of the amplified PrPSc was similar to that in the original inocula, as previously observed with other scrapie isolates [17]. We will refer to this pattern as 18K, according to the apparent MW of the unglycosylated PrPres.

View Article: PubMed Central - PubMed

ABSTRACT

It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

No MeSH data available.


Related in: MedlinePlus