Scrapie Infection of Prion Protein-deficient Cell Line upon Ectopic Expression of Mutant Prion Proteins

Martin H. Groschup,Hermann Schätzl,Ina M. Vorberg,Markus Geissen,Romina Rost,Elke Maas,Takashi Onodera

doi:10.1074/jbc.m701309200

Abstract

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.

Highlights

Prion diseases or transmissible spongiform encephalopathies (TSEs)3 are fatal neurological disorders such as Creutzfeldt-Jakob diseases in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE)
Alternative cell culture models for prion diseases have greatly helped us to understand the molecular mechanism of PrPSc formation [13, 14], the role of the PrP amino acid sequence for the TSE species barrier, and how PrP structural domains affect conversion of PrPC to PrPSc [15,16,17,18,19,20]
It was unclear whether ectopic PrPSc by itself could initiate the conformational change of subsequent PrPC molecules in the absence of endogenous wild-type PrPSc molecules

Summary

Introduction

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurological disorders such as Creutzfeldt-Jakob diseases in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE). Comparative studies on the direct influence of PrP alterations on the prion infection process (as opposed to studies in persistently infected cell cultures) were mainly restricted to transgenic animal models [6, 7, 21,22,23] One reason for this is that all so-far-identified cell lines susceptible to prions code for endogenous wild-type PrP. The aim of this study was to establish a novel cell culture system based on PrP0/0 cells that (a) allows for the generation of transgenic PrPSc in the absence of any endogenous wild-type PrP and (b) enables us to test broad sets of PrP mutants for their influence on the prion infection process and PrPSc formation. We provide novel insights in how PrPC and PrPSc interact and in the surfaces of PrPSc responsible for the formation of the PrPSc aggregate

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Conclusion