Recovery of Small Infectious PrPres Aggregates from Prion-infected Cultured Cells

Olivier Andréoletti,Zaira E. Arellano Anaya,Jimmy Savistchenko,Caroline Lacroux,Didier Vilette,Véronique Massonneau

doi:10.1074/jbc.m110.165233

Olivier Andréoletti, Zaira E. Arellano Anaya

Open Access

https://doi.org/10.1074/jbc.m110.165233

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Abstract

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

Highlights

Diseased tissues, as exemplified by the characterization of PKsensitive abnormal forms of PrP [11,12,13,14,15]
We investigated the diversity of infectious abnormal PrP species in a cell line and in neuronal primary cultures infected with a sheep scrapie agent
We obtained no evidence for the presence of PK-sensitive infectivity, we identified a previously unnoticed abnormal PrP species

Summary

Introduction

Diseased tissues, as exemplified by the characterization of PKsensitive abnormal forms of PrP [11,12,13,14,15]. Pellets and methanol-precipitated supernatants were resuspended in small volumes of PBS without sonication and analyzed by immunoblotting and in the cell-based assay. Cell lysates (500 ␮g of proteins) were PK-digested and centrifuged at 10,000 ϫ g for 1 h, and pellets were analyzed for insoluble PrPres by Western blotting.

Results

Conclusion