3D atomic structure reveals evolution of prions in templated amplification in vitro

Biological and pathological properties of prion strains are determined by fibril structure. We replicated murine RML prions through serial protein misfolding cyclic amplification (PMCA) using recombinant PrP substrate supplemented by PrP null mouse brain homogenate to probe whether fibril structure, infectivity and prion strain characteristics are conserved. Recombinant prion populations were purified and analysed for the sizes of proteinase-K resistant PrP fragments, their infectivity profile in murine cell lines, single particle spectral fingerprinting and through 3D structural reconstruction by cryo-electron microscopy. While recombinant prions retained high specific infectivity, we found that fibril structures and biological strain properties mutated during amplification in vitro, leading to a novel prion strain, which is related to, but distinct from RML. Spectral and infectivity profiles suggested a convergence of prions derived from RML, ME7 and 22L prion seeds when amplified in vitro. These findings suggest a structural evolution of prion strains through imperfect templating, in which replication environment and substrate are major drivers of replication fitness.