Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

Michael B Miller,Surachai Supattapone,James C Geoghegan,David Westaway

doi:10.1371/journal.ppat.1002128

Abstract

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrPSc, suggesting that these domains of cellular prion protein (PrPC) serve as docking sites for PrPSc during prion propagation. To examine the role of polybasic domains in the context of full-length PrPC, we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ∼5 rounds of sPMCA, PrPSc molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrPSc prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrPSc molecules. Remarkably, ΔC-PrPSc and other polybasic domain PrPSc molecules displayed diminished or absent biological infectivity relative to wild-type PrPSc, despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrPSc prions interact with PrPC molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrPSc propagation. Furthermore, polybasic domain deficient PrPSc molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrPSc molecules may not depend on a single stereotypic mechanism, but that normal PrPC/PrPSc interaction through polybasic domains may be required to generate prion infectivity.

Highlights

Prions are infectious proteinaceous particles that cause fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD)
Previous studies have suggested that prion infectivity depends upon the ability of a sample to change the shape of a normal brain protein called the prion protein (PrP) into a disease-associated shape
Other studies have identified a pair of positively charged domains within the structure of PrP that appear to be important for the interaction between the normal and disease-associated shapes of the prion protein

Summary

Introduction

Prions are infectious proteinaceous particles that cause fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD). Prions contain PrPSc, a protease-resistant detergent-insoluble b-sheet-rich conformer of the normal cellular protein PrPC [1,2]. Cell-free in vitro propagation systems have emerged as valuable tools to investigate PrPSc and prion propagation [7]. By serial protein misfolding cyclic amplification (sPMCA), prion infectivity can be propagated in vitro [8]. More rapid and less costly than the gold standard inoculation bioassay, sPMCA has been proposed as an in vitro method to detect prion infectivity [9]. Samples which seed robust sPMCA propagation have been previously associated with biological infectivity [8,10]. It is unknown if PrPSc molecules that robustly seed PMCA propagation in wild type brain homogenate are always associated with appropriate levels of specific infectivity

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Conclusion