In vitro generation of highly infectious recombinant prions adopting structural architectures of bona fide prions

Prions are the protein-only infectious agents responsible for prion diseases. Recent cryo-EM studies have revealed that brain-derived prions adopt Parallel In-Register Intermolecular β-Sheet (PIRIBS) structures and share a remarkably similar global folding architecture, characterized by an overall V shape composed of distinct N-terminal and C-terminal lobes. Despite this advance, the molecular basis of prion conversion and propagation remains largely unclear, in part due to the lack of reproducible in vitro platforms capable of generating infectious prions for detailed mechanistic studies. Our previous studies demonstrated the generation of highly infectious prions in vitro using the Protein Misfolding Cyclic Amplification (PMCA) technology; however, it is unknown whether PMCA faithfully replicates the structure of PrPSc. Here, we report an RML prion-seeded, PMCA-based in vitro propagation assay that enables the robust and reproducible production of recombinant infectious PrPSc fibrils, which are biologically as pathogenic as the RML prion. Moreover, these PMCA-amplified recombinant PrPSc fibrils adopt a V-shaped architecture with two distinct lobes, reminiscent of the RML PrPSc structure. Despite highly conserved biological activity and overall structural similarity, recombinant PrPSc differs structurally from brain-derived prions, particularly in the C-terminal lobe, suggesting that complete structural identity is not needed for high infectivity. Overall, this recombinant PMCA system provides a powerful and versatile platform for investigating the structure-activity relationship of prion infectivity.