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      Influence of Mabs on PrP Sc Formation Using In Vitro and Cell-Free Systems


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          PrP Sc is believed to serve as a template for the conversion of PrP C to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP cor PrP Sc would hinder or prevent the formation of the PrP C–PrP Sc complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP Sc formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrP Sc as a template or seed, and a series of incubations and sonications, to convert PrP C to PrP Sc. The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrP C–PrP Sc conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP C conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy).

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          In vitro generation of infectious scrapie prions.

          Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the cellular prion protein (PrPC). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.
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            Formation of native prions from minimal components in vitro.

            The conformational change of a host protein, PrP(C), into a disease-associated isoform, PrP(Sc), appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt-Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP(C) and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP(Sc) molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP(Sc) molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP(Sc) molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP(C) molecules, copurified lipid molecules, and a synthetic polyanion.
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              Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein.

              The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                27 July 2012
                : 7
                : 7
                : e41626
                [1 ]Departments of Neurology and Physiology/Pharmacology, State University New York Downstate Medical Center, Brooklyn, New York, United States of America
                [2 ]Departments of Pathology, Neuroscience, and Neurology, Case Western Reserve University, Cleveland, Ohio, United States of America
                [3 ]Departments of Neurology, Psychiatry and Pathology, New York University School of Medicine, New York, New York, United States of America
                National Institute for Agricultural Research, France
                Author notes

                Competing Interests: TW is a PLoS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

                Conceived and designed the experiments: RR TW. Performed the experiments: BC RP TW RR. Analyzed the data: BC RP TW RR. Contributed reagents/materials/analysis tools: RP TW RR. Wrote the paper: RR.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                : 1 May 2012
                : 22 June 2012
                Page count
                Pages: 7
                This work was supported in part by the State University New York Downstate Medical Center, National Institutes of Health grant NS47433 (TW) and grant NP020048 from the United States Department of Defense National Prion Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Transmembrane Proteins
                Immunologic Subspecialties
                Infectious Diseases
                Prion Diseases
                Infectious Diseases of the Nervous System
                Neurodegenerative Diseases



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