Abstract
Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.
Highlights
Prions are unique pathogens, devoid of significant coding nucleic acid, which cause lethal neurodegenerative diseases in mammals, including scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt – Jakob disease (CJD) in humans [1 –3]
To establish that the prion protein (PrP) structures we observe by electron microscopy (EM) are directly associated with prion infectivity, we applied aliquots of highly purified mouse prions [10] (RML strain; figure 1a,b) to individual carbon-coated gold EM grids and measured surface bound infectivity in cell culture using a novel method that we term the Scrapie Cell Grid Assay (SCGA; figure 1c)
We found that purified prions bind avidly to carbon-coated EM grids, and remarkably the SCGA reported prion infectivity titre with an efficiency equivalent to, or better than, measuring prions in solution
Summary
Devoid of significant coding nucleic acid, which cause lethal neurodegenerative diseases in mammals, including scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt – Jakob disease (CJD) in humans [1 –3]. They are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), which self-propagate by means of seeded protein polymerization [1,2]. We developed new methods [10] to obtain exceptionally pure preparations of intact prions from mouse brain, and showed that pathogenic PrP in these preparations is assembled into rod-like assemblies (PrP rods) akin to those described by Prusiner and colleagues [1,11]
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