Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features

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Abstract

It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.

Section snippets

Experimental procedures

Reagents. Protein-A–Sepharose was from Pharmacia Biotech and PNGase F was purchased from New England BioLabs.

Antibodies. The monoclonal antibodies against PrP were 4F2 [13], ICSM 18 [14]. MH48 is a rabbit polyclonal serum raised against recombinant ovine PrP [15]. Monoclonal antibody CTR433 [16] was used to stain the Golgi apparatus.

Scrapie strain. The isolate originally used to infect Rov cells derives from a naturally scrapie-affected sheep. This isolate was serially propagated and

An improved cell model to study the cell biological properties of the VRQPrP and ARRPrP variants

To minimize as much as possible cell clone-specific versus allele-specific differences, we exploited a recently designed regulatory expression system to express both PrP variants in the same Rov clone. This “dual” regulatory system is based on tetracycline-controlled transactivators that reversibly regulate the activity of two transfected genes in a mutually exclusive manner [19]. In such Rov clones, VRQPrP is expressed in the absence of dox. Addition of 1 μg/ml dox in the cell culture medium

Discussion

Much experimental data indicate that PrP conversion and prion multiplication are complex biological processes that require the coordinated involvement of several cellular and molecular factors [9]. Possible distinct cell biological properties of two PrP polymorphic variants may therefore contribute to the differential capacity of the protein to support prion multiplication. In this study, the cell biology of two ovine PrP variants (ARRPrP and VRQPrP) associated with sheep scrapie susceptibility

Acknowledgments

We thank S. Hawke and J. Collinge (Imperial College, London, United Kingdom) and M. Bornens (Institut Curie, Paris, France) for ICSM18 and CTR433 mAbs, respectively. We acknowledge O. Andreoletti and SAGA for providing us with PrP-genotyped sheep, V. Setola and V. Beringue for critical reading of the manuscript, and R. Morris for helpful discussions. E.S. was partly supported by a fellowship from INRA.

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    These authors contributed equally to the work.

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