Fig. 3: Workflow schematic and exemplifying results for determining infectivity of rec-PrPres generated by PMSA.
To illustrate the procedure employed to assess the bona fide prion nature of the rec-PrPres obtained in PMSA, four examples are shown, carried out with the rec-PrPres of gerbil, Egyptian fruit bat, rabbit, and mink, all belonging to distinct orders and exhibit varying misfolding scores. In each case, a rec-PrPres conformer (C1) was centrally displayed in the middle of each panel alongside the respective undigested substrate control as observed after PK digestion. The left part of each panel showcases the partial purification of the rec-PrPres through ultracentrifugation and how the purified protein halo was used as seed in PMCA diluted 1:100 in TgVole brain homogenates used as substrate. A Western blot depicting the outcomes of the three serial PMCA rounds (the first at 1:100 dilution and the rest at 1:10) performed with each seed is shown. These include an unseeded tube as control for spontaneous misfolding or cross-contamination. For each rec-PrPres tested, two independent PMCA experiments were performed using two replicates in each experiment. As can be seen in the Western blots, all rec-PrPres tested were able to induce misfolding of bank vole PrPC, strongly indicating their potential infectivity in vivo. Notably, the rec-PrPres seed is visible after the first PMCA round, indicated by a black arrow. The right part of each panel details the assessment of infectivity for these PMSA products through bioassay in TgVole. Kaplan–Meier curves depict the survival times with the mean incubation period expressed as days post-inoculation (dpi ± SEM) and the number of animals in each group succumbing to disease indicated below (# of sick animals/# of animals inoculated, being in all cases n = 6 to 7 animals). Additionally, the biochemical analysis of the brains of the inoculated animals is presented. By Western blot using Sha31 antibody (1:4000 dilution), all analyzed animals showed PrPSc, confirming the infectious nature of the recombinant prions form these four different species. Cn: selected conformer number; PK: Proteinase K, MW: Molecular weight marker. All the Source Data used for this figure is publicly available in Zenodo repository (10.5281/zenodo.10579518).
