Ultra-efficient Replication of Infectious Prions by Automated Protein Misfolding Cyclic Amplification

Paula Saá,Claudio Soto,Joaquín Castilla

doi:10.1074/jbc.m603964200

Abstract

Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.

Highlights

Our results demonstrate that protein misfolding cyclic amplification (PMCA) is capable of detecting as little as ϳ26 monomers of PrP, which, according to recent data on the minimal size of the infectious particle [17], would correspond to a single molecule of oligomeric infectious PrPSc
The PMCA Technology and Its Reproducibility—PMCA consists of cycles of accelerated prion replication
The basis for PMCA is the observation that prion replication follows a seeding-nucleation model in which oligomeric PrPSc in the infectious material converts PrPC by integrating monomeric proteins into the ends of the aggregate, inducing and stabilizing its misfolding [13, 23]

Summary

Introduction

During the course of the disease, prions replicate by the autocatalytic conversion of PrPC into PrPSc, triggered by the misfolded protein present in the infectious inoculum. One of the limitations of the cell-free conversion method is the relatively low efficiency of PrPSc formation that diminishes its application to study the nature of the infectious agent and to attempt sensitive detection of the protein. Our results show that PMCA enables the increase of infectivity by around 20 million-fold, converting a sample that is not infectious into a highly infectious one These data demonstrate that PMCA has a similar power of amplification as PCR techniques used to amplify DNA and have great promise for the development of highly sensitive detection of PrPSc and for understanding the molecular basis of prion replication

Methods

Results

Conclusion