Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrP(Sc)) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrP(Sc) does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrP(Sc). Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2–α2 loop of the cellular prion protein (PrP(C)). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrP(C) but expressing MoPrP(169F) generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP(169F) prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP(169F) prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP(169F) prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2–α2 loop of PrP(C) affects the sensitivity of PrP(Sc) to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrP(C) can be linked to a physicochemical property of the corresponding PrP(Sc).