Lower specific infectivity of protease-resistant prion protein generated in cell-free reactions

Abstract

Prions are unconventional infectious agents that cause transmissible spongiform encephalopathy (TSE) diseases, or prion diseases. The biochemical nature of the prion infectious agent remains unclear. Previously, using a protein misfolding cyclic amplification (PMCA) reaction, infectivity and disease-associated protease-resistant prion protein (PrPres) were both generated under cell-free conditions, which supported a nonviral hypothesis for the agent. However, these studies lacked comparative quantitation of both infectivity titers and PrPres, which is important both for biological comparison with in vivo-derived infectivity and for excluding contamination to explain the results. Here during four to eight rounds of PMCA, end-point dilution titrations detected a >320-fold increase in infectivity versus that in controls. These results provide strong support for the hypothesis that the agent of prion infectivity is not a virus. PMCA-generated samples caused the same clinical disease and neuropathology with the same rapid incubation period as the input brain-derived scrapie samples, providing no evidence for generation of a new strain in PMCA. However, the ratio of the infectivity titer to the amount of PrPres (specific infectivity) was much lower in PMCA versus brain-derived samples, suggesting the possibility that a substantial portion of PrPres generated in PMCA might be noninfectious.Creutzfeldt-Jakob disease | amyloid | neurodegeneration | protein polymerization | protein aggregation

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

“…A recent study showed evidence of high infectivity titers in PMCA reactions with the hyper strain of transmissible mink encephalopathy, although incubation times per infectious unit were longer than those observed for brain-derived infectivity (51). A related report of a study performing careful quantitative comparisons of the PMCAgenerated infectivity titer versus the amount of PrP res has drawn attention to the fact that a significant fraction of PrP res generated in PMCA reactions may not be infectious (32). Consistent with this observation, albeit in the context of mutant PrP C molecules, PMCA seeding activity can be present in samples with limited infectivity (39).…”

Selective Amplification of Classical and Atypical Prions Using Modified Protein Misfolding Cyclic Amplification

“…On the one hand, PMCA-derived PrP Sc products were shown to produce the same strain-specific disease phenotype in animals as brain-derived PrP Sc (16,18,24). On the other hand, differences in incubation times to disease by brain-and PMCAderived PrP Sc suggest the possibility of change in structure and/or composition of PrP Sc populations in response to replication in vitro (23). To what extent can strains be changed in PMCA?…”

Dissociation of Prion Protein Amyloid Seeding from Transmission of a Spongiform Encephalopathy

“…Recent studies concluded that the ratio of the infectivity titer to the amount of PrP TSE (specific infectivity) is much lower when the PrP TSE is generated by amplification in vitro than in infected brain-derived samples (15). Thus, while misfolded protein is readily generated in these assays, it is clear that not all the misfolded protein is infectious.…”