Research Article: Isolation of a Defective Prion Mutant from Natural Scrapie

Date Published: November 23, 2016

Publisher: Public Library of Science

Author(s): Ilaria Vanni, Sergio Migliore, Gian Mario Cosseddu, Michele Angelo Di Bari, Laura Pirisinu, Claudia D’Agostino, Geraldina Riccardi, Umberto Agrimi, Romolo Nonno, Jason C Bartz.


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.

Partial Text

Transmissible spongiform encephalopathies (TSEs) are progressive and fatal neurodegenerative disorders affecting animals and humans, with the most common forms being scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) of cattle, chronic wasting disease (CWD) of cervids, and Creutzfeldt-Jakob disease (CJD) in humans. TSEs are caused by the misfolding of the host-encoded prion protein monomers (PrPC) into autocatalytic self-replicating aggregates (PrPSc). The protein-only hypothesis postulates that the agent responsible for these pathologies, the prion, is exclusively composed of PrPSc [1]. Prion replication can be modelled as a template-based mechanism, where PrPC misfolds into PrPSc and acquires the ability to recruit other PrPC molecules and to trigger their misfolding, through an autocatalytic template-based mechanism [1]. This model is supported by protein misfolding cyclic amplification (PMCA) [2], a technique that mimics in vitro the PrPC-to-PrPSc autocatalytic conversion, leading to the generation of a huge amount of infectious prions in healthy brain homogenates seeded with minute amounts of PrPSc and subjected to multiple cycles of sonication and incubation [3].

In agreement with previous observations [17], our findings show that sheep PrPSc is able to efficiently propagate on vole PrPC by PMCA, even when seeded at extremely high dilutions (up to 10−7), allowing to derive in vitro vole-adapted prion populations starting from a seemingly low number of replicative units. As expected, after several rounds of PMCA we usually recovered a faithful vole-adapted scrapie PrPSc, characterized by a PrPres of 18 kDa; however, from high dilutions of a single sheep isolate, 198/9, we repeatedly derived a PrPSc with a shorter PK-resistant core of 14 kDa. We selected and studied two prion populations, named 18K and 14K/2, both derived in vitro by serial bvPMCA, starting from highly diluted seeds of the same scrapie isolate. PrPres from 18K and 14K/2 populations had different PK-cleavage site and different conformational stability, so that they could be seen as different bona fide PrPSc conformers, potentially encoding for different biological properties. Indeed, 18K was competent for in vivo replication and resulted in a pathological phenotype indistinguishable from vole-adapted 198/9 while, unexpectedly, 14K/2 was unable to infect voles. These results suggest that the in vitro derived prion population containing 18 kDa PrPres aggregates encoded for the faithful scrapie strain, while that exclusively composed of 14K kDa PrPres aggregates behaved like a defective mutant, being unable to replicate in live animals. It is of note, however, that even 18K did not preserve 100% fidelity compared to its in vivo counterpart, sheep and vole-adapted scrapie; indeed, the conformational stability of sheep and vole-adapted PrPSc was higher than that of 18K, which however reverted to the original phenotype upon in vivo propagation.




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