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

3D atomic structure reveals evolution of prions in templated amplification in vitro | Research Square Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article 3D atomic structure reveals evolution of prions in templated amplification in vitro Jan Bieschke, Adam Wenborn, Ioanna Chatzitheodosiou, Kezia Jack, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9083455/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biophysics/Biopolymers in vivo Biological sciences/Evolution/Experimental evolution Full Text Additional Declarations Yes there is potential Competing Interest. J.C is director, J.D.F.W. and G.S.J. are shareholders of D-Gen Ltd., an academic spin-out company working in the field of prion disease diagnosis, decontamination and therapeutics. D-Gen supplied the ICSM35 and ICSM18 antibodies used for Western blot and ELISA performed in this study. The other authors declare no potential conflicts of interest. Supplementary Files ValidationReportsPMCARMLPDB29BREMD57062.pdf EMDBB / PDB validation report rs.pdf Reporting Summary Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information

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