Cofactor and glycosylation preferences for in vitro prion conversion are predominantly determined by strain conformation

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrP(C), into a pathogenic conformer, PrP(Sc). Infectious prions can exist as different strains, composed of unique conformations of PrP(Sc) that generate strain-specific biological traits, including distinctive patterns of PrP(Sc) accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrP(C) glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrP(C) substrate or by the conformation of PrP(Sc) seed. To distinguish between these two possibilities, we used bank vole PrP(C) to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di- or un-glycosylated bank vole PrP(C) substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrP(C) substrates when reactions contained the same PrP(C) glycoform or cofactor molecule preferred by the PrP(Sc) seed in its host species. Thus, we conclude that it is the conformation of the input PrP(Sc) seed, not the amino acid sequence of the PrP(C) substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrP(C) glycoforms during prion replication.