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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

Vole bioassay.A, B and C show the neuropathological and PrPres phenotypes observed in voles after primary transmission (left panels, and indicated as I, roman number, in the blot on the right) and second passage (central panels and indicated as II, roman numbers, in the blot) of sheep 198/9 (A), PMCA-derived 18K (B) and PMCA-derived 14K/1 (C). Brain-scoring areas in lesion profiles are: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), cingulate and adjacent motor cortex (9). For each blot a vole adapted scrapie was added (BvScr, last lane). PrPres was detected by antibody SAF84. D) Graph depicting the denaturation profiles obtained by CSA from PrPSc in sheep 198/9 (Sh198/9) or from voles infected with 18K (Bv18K) and vole-adapted 198/9 (Bv198/9). E) Graph depicting the comparison of denaturation profiles of 18K before (18K) and after (Bv18K) transmission in voles. [GdnHCl]1/2 values are reported in the graph. F) Graph depicting the fate of 18K and 14K/2 after intracerebral inoculation in voles. Two groups of 8 voles were inoculated with 14K/2 or 18K and 2 voles for each group were sacrificed at different time points (0, 3, 14 and 52 dpi). Their brains were homogenized and used as seed for PMCA reactions in 3 independent experiments. The values on y axis represent the overall percentage of positive samples per time point after 3 PMCA rounds.
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ppat.1006016.g003: Vole bioassay.A, B and C show the neuropathological and PrPres phenotypes observed in voles after primary transmission (left panels, and indicated as I, roman number, in the blot on the right) and second passage (central panels and indicated as II, roman numbers, in the blot) of sheep 198/9 (A), PMCA-derived 18K (B) and PMCA-derived 14K/1 (C). Brain-scoring areas in lesion profiles are: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), cingulate and adjacent motor cortex (9). For each blot a vole adapted scrapie was added (BvScr, last lane). PrPres was detected by antibody SAF84. D) Graph depicting the denaturation profiles obtained by CSA from PrPSc in sheep 198/9 (Sh198/9) or from voles infected with 18K (Bv18K) and vole-adapted 198/9 (Bv198/9). E) Graph depicting the comparison of denaturation profiles of 18K before (18K) and after (Bv18K) transmission in voles. [GdnHCl]1/2 values are reported in the graph. F) Graph depicting the fate of 18K and 14K/2 after intracerebral inoculation in voles. Two groups of 8 voles were inoculated with 14K/2 or 18K and 2 voles for each group were sacrificed at different time points (0, 3, 14 and 52 dpi). Their brains were homogenized and used as seed for PMCA reactions in 3 independent experiments. The values on y axis represent the overall percentage of positive samples per time point after 3 PMCA rounds.

Mentions: The scrapie isolate 198/9 transmitted to 100% of voles with a mean survival time of 167 days post infection (dpi). The survival time shortened to 94 dpi on sub-passage, indicating the existence of a transmission barrier for adaptation of sheep scrapie to voles (Table 1). The neuropathological phenotype (Fig 3A) was indistinguishable from that previously observed in other ARQ/ARQ scrapie isolates from Italy and UK [16], and all voles showed the expected 18K PrPres profile with no evidence of shorter PrPres fragments (Fig 3A). In previous studies we showed that PrPSc from several Italian classical scrapie isolates displayed a uniform conformational stability, with GdnHCl1/2 values of ~2 M [19, 20], which was preserved after transmission in voles [19, 21]. Accordingly, the conformational stabilities of 198/9 and vole-adapted 198/9 were similar, with GdnHCl1/2 of ~2 M (Fig 3D).


Isolation of a Defective Prion Mutant from Natural Scrapie
Vole bioassay.A, B and C show the neuropathological and PrPres phenotypes observed in voles after primary transmission (left panels, and indicated as I, roman number, in the blot on the right) and second passage (central panels and indicated as II, roman numbers, in the blot) of sheep 198/9 (A), PMCA-derived 18K (B) and PMCA-derived 14K/1 (C). Brain-scoring areas in lesion profiles are: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), cingulate and adjacent motor cortex (9). For each blot a vole adapted scrapie was added (BvScr, last lane). PrPres was detected by antibody SAF84. D) Graph depicting the denaturation profiles obtained by CSA from PrPSc in sheep 198/9 (Sh198/9) or from voles infected with 18K (Bv18K) and vole-adapted 198/9 (Bv198/9). E) Graph depicting the comparison of denaturation profiles of 18K before (18K) and after (Bv18K) transmission in voles. [GdnHCl]1/2 values are reported in the graph. F) Graph depicting the fate of 18K and 14K/2 after intracerebral inoculation in voles. Two groups of 8 voles were inoculated with 14K/2 or 18K and 2 voles for each group were sacrificed at different time points (0, 3, 14 and 52 dpi). Their brains were homogenized and used as seed for PMCA reactions in 3 independent experiments. The values on y axis represent the overall percentage of positive samples per time point after 3 PMCA rounds.
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Related In: Results  -  Collection

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ppat.1006016.g003: Vole bioassay.A, B and C show the neuropathological and PrPres phenotypes observed in voles after primary transmission (left panels, and indicated as I, roman number, in the blot on the right) and second passage (central panels and indicated as II, roman numbers, in the blot) of sheep 198/9 (A), PMCA-derived 18K (B) and PMCA-derived 14K/1 (C). Brain-scoring areas in lesion profiles are: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), cingulate and adjacent motor cortex (9). For each blot a vole adapted scrapie was added (BvScr, last lane). PrPres was detected by antibody SAF84. D) Graph depicting the denaturation profiles obtained by CSA from PrPSc in sheep 198/9 (Sh198/9) or from voles infected with 18K (Bv18K) and vole-adapted 198/9 (Bv198/9). E) Graph depicting the comparison of denaturation profiles of 18K before (18K) and after (Bv18K) transmission in voles. [GdnHCl]1/2 values are reported in the graph. F) Graph depicting the fate of 18K and 14K/2 after intracerebral inoculation in voles. Two groups of 8 voles were inoculated with 14K/2 or 18K and 2 voles for each group were sacrificed at different time points (0, 3, 14 and 52 dpi). Their brains were homogenized and used as seed for PMCA reactions in 3 independent experiments. The values on y axis represent the overall percentage of positive samples per time point after 3 PMCA rounds.
Mentions: The scrapie isolate 198/9 transmitted to 100% of voles with a mean survival time of 167 days post infection (dpi). The survival time shortened to 94 dpi on sub-passage, indicating the existence of a transmission barrier for adaptation of sheep scrapie to voles (Table 1). The neuropathological phenotype (Fig 3A) was indistinguishable from that previously observed in other ARQ/ARQ scrapie isolates from Italy and UK [16], and all voles showed the expected 18K PrPres profile with no evidence of shorter PrPres fragments (Fig 3A). In previous studies we showed that PrPSc from several Italian classical scrapie isolates displayed a uniform conformational stability, with GdnHCl1/2 values of ~2 M [19, 20], which was preserved after transmission in voles [19, 21]. Accordingly, the conformational stabilities of 198/9 and vole-adapted 198/9 were similar, with GdnHCl1/2 of ~2 M (Fig 3D).

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