Full restoration of specific infectivity and strain properties from pure mammalian prion protein

Abstract

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP Sc , a misfolded conformer of the normal prion protein, PrP C . However, protein-only PrP Sc preparations lack significant levels of prion infectivity, leading to the alternative hypothesis that cofactor molecules are required to form infectious prions. Here, we show that prions with parental strain properties and full specific infectivity can be restored from protein-only PrP Sc in vitro . The restoration reaction is rapid, potent, and requires bank vole PrP C substrate, post-translational modifications, and cofactor molecules. To our knowledge, this represents the first report in which the essential properties of an infectious mammalian prion have been restored from pure PrP without adaptation. These findings provide evidence for a unified hypothesis of prion infectivity in which the global structure of protein-only PrP Sc accurately stores latent infectious and strain information, but cofactor molecules control a reversible switch that unmasks biological infectivity.

Cofactor and glycosylation preferences for in vitro prion conversion are predominantly determined by strain conformation

“…However, it has been shown that PrP Sc glycosylation is not absolutely required for strain-dependent prion neurotropism, as unglycosylated RML and 301C inoculum maintain their strain-specific PrP Sc distribution and vacuolation [48]. Additionally, we recently showed that a recombinant PrP Sc conformer lacking glycosylation displays same pattern of neurotropism as the same strain of native PrP Sc [34]. Finally, the "coprion" hypothesis [49] posits that small, replicating nucleic acids encode prion strain information, but no such molecules have ever been discovered.…”

“…Finally, we also investigated the cofactor preferences for a pair of matched recPrP Sc conformers which are derived from the same original seed, but subsequently propagated either in the presence or absence of phospholipid cofactor, and therefore termed "cofactor" and "protein-only" PrP Sc , respectively [16,33,34]. The results show that both conformers are able to use either purified phospholipid or poly(A) RNA as cofactors in sPMCA reactions using immunopurified native bank vole PrP C substrate (Fig 5, columns three and four).…”

“…We envision that specific cofactor molecules are differentially distributed throughout the brain, causing specific prion strains to preferentially replicate in brain regions containing higher levels of their preferred cofactor molecule. This model builds upon abundant evidence that cofactor molecules play important roles in prion replication and biological infectivity [11,25,34,43]. To test the cofactor selection model, it will be necessary to first identify cofactor molecule(s) that are able to maintain the strain properties of specific prion strains.…”

“…M109 cofactor recPrP Sc and M109 protein-only recPrP Sc were generated by sPMCA based on a previously established protocol [16]. Expression and purification of bank vole PrP M109 recPrP 23-231 was performed as previously described [34]. sPMCA reactions were performed using a previously established protocol with minor modifications [34].…”

“…Expression and purification of bank vole PrP M109 recPrP 23-231 was performed as previously described [34]. sPMCA reactions were performed using a previously established protocol with minor modifications [34]. Two-hundred microliter reactions containing 6 μg/mL mouse recPrP 23-230 in conversion buffer (20 mM Tris pH 7.5, 135 mM NaCl, 5 mM EDTA pH 7.5, 0.15% (v/v) Triton X-100) were supplemented with purified brain-derived phospholipid cofactor [11] for cofactor recPrP Sc propagation or water for protein-only recPrP Sc propagation.…”