PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

Unlabelled: Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP) influences PrP misfolding into the disease-associated isoform, PrP, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP away from rafts. Previous studies showed that nonraft transmembrane PrP variants resist conversion to PrP when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP and GPI-anchored PrP in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP might convert to PrP if seeded by an exogenous source of PrP not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP supported persistent PrP propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP is required for persistent PrP propagation, implicating raft microdomains as a location for conversion.

Importance: Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrP or transmembrane-anchored PrP, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrP We show that only GPI-anchored PrP was able to convert to PrP and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrP to rafts are crucial to the ability of PrP to propagate as a prion.