Prion propagation is controlled by discrete structural regions of PrP rather than overall stability.

Mutational analysis of the cellular prion protein (PrPC) has revealed various regions of the protein that modulate prion propagation. However, these approaches involve deletions, insertions, or replacements in the presence of the WT PrPC, which may mask the true phenotype. Here, site-directed alanine mutagenesis of the prion protein (PrP) was conducted to identify sites, particularly potential "surface patches" of PrPC required for prion propagation. Mutations were targeted to the helical, sheet, and loop regions of PrPC, or a combination thereof and the mutated proteins expressed in mouse neuroblastoma cells in which the endogenous PrPC had been silenced. Using the scrapie cell assay, a highly sensitive cell culture-based bioassay for quantifying infectious titers of Rocky Mountain Laboratory (RML) prions, we found that all mutations within the structured 119 to 231 domain, irrespective of secondary structure, severely reduced prion propagation. We further analyzed four strongly inhibiting mutations within conformationally variable loop regions of PrPC - residues 123 to 125 (PRP1); 134 to 135 (PRP4); 139,141 (PRP5), and connecting helix residues 146 and 153, respectively, as well as residues 188,191 to 192 (PRP13). Mutations in PRP1, 4 and 5 dominantly inhibited prion propagation even in the presence of WT PrPC, while mutations in PRP13 had no comparable effect, suggesting that the former disrupt prion fibril structure or its replication mechanism. None of the most inhibitory mutations substantially altered PrP stability, and mutant PrPs were able to both form amyloid in vitro and seed fibril formation of WT PrP.