Relationship between Conformational Stability and Amplification Efficiency of Prions

Recent studies demonstrated that the efficiency, rate, and yield of prion amplification in vitro could be substantially improved by supplementing protein misfolding cyclic amplification (PMCA) with Teflon beads [Gonzalez-Montalban et al. (2011) PLoS Pathog. 7, e1001277]. Here we employed the new PMCA format with beads (PMCAb) to gain insight into the mechanism of prion amplification. Using a panel of six hamster prion strains, the effect of beads on amplification was found to be strain-specific, with the largest improvements in efficiency observed for strains with the highest conformational stability. This result suggests a link between PrP^Sc conformational stability and its fragmentation rate and that beads improved amplification by assisting fragmentation. Furthermore, while exploring the PrP^Sc-independent bead effect mechanism, a synergy between the effects of RNA and beads on amplification was observed. Consistent with previous studies, amplification of all six hamster strains tested here was found to be RNA-dependent. Under sonication conditions used for PMCA, large RNA molecules were found to degrade into smaller fragments of a size that was previously shown to be the most effective in facilitating prion conversion. We speculate that sonication-induced changes in RNA size distribution could be one of the rate-limiting steps in prion amplification.