title: PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.
abstract: Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPCmisfolding into the disease-associated isoform, PrPres, 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 PrPCaway from rafts. Previous studies showed that nonraft transmembrane PrPCvariants resist conversion to PrPreswhen transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPCand GPI-anchored PrPresin distinct membrane environments. Thus, it remained unclear whether transmembrane PrPCmight convert to PrPresif seeded by an exogenous source of PrPresnot associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPCTo 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 PrPCsupported persistent PrPrespropagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPCis required for persistent PrPrespropagation, implicating raft microdomains as a location for conversion.
  
  
  OBJECTIVE
  Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) 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 PrPCor transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPresWe show that only GPI-anchored PrPCwas able to convert to PrPresand able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPCto rafts are crucial to the ability of PrPCto propagate as a prion.
authors.affiliation: Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Marshall
authors.affiliation: Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hughson
authors.affiliation: Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Vascellari
authors.affiliation: Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Suzette A
authors.initials: SA
authors.lastname: Priola
authors.affiliation: Research Center for Food Safety, School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
authors.forename: Akikazu
authors.initials: A
authors.lastname: Sakudo
authors.affiliation: Research Center for Food Safety, School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Onodera
authors.affiliation: Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA geraldsbaron@gmail.com.
authors.forename: Gerald S
authors.initials: GS
authors.lastname: Baron
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D055786:Gene Knockout Techniques; D021962:Membrane Microdomains; D008565:Membrane Proteins; D051379:Mice; D018621:PrPC Proteins / Q000235:genetics*; D018620:PrPSc Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017096:Prion Diseases / Q000235:genetics / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D020033:Protein Isoforms; D021381:Protein Transport
chemical_list: D008565:Membrane Proteins; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions; D020033:Protein Isoforms
keywords: GPI; NpL2; PrP; neuron; prion; raft; transmembrane; transmissible spongiform encephalopathy