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Lower specific infectivity of protease-resistant prion protein generated in cell-free reactions

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 11/2011; 108(48):E1244-53. DOI: 10.1073/pnas.1111255108
Source: PubMed

ABSTRACT Prions are unconventional infectious agents that cause transmissible spongiform encephalopathy (TSE) diseases, or prion diseases. The biochemical nature of the prion infectious agent remains unclear. Previously, using a protein misfolding cyclic amplification (PMCA) reaction, infectivity and disease-associated protease-resistant prion protein (PrPres) were both generated under cell-free conditions, which supported a nonviral hypothesis for the agent. However, these studies lacked comparative quantitation of both infectivity titers and PrPres, which is important both for biological comparison with in vivo-derived infectivity and for excluding contamination to explain the results. Here during four to eight rounds of PMCA, end-point dilution titrations detected a >320-fold increase in infectivity versus that in controls. These results provide strong support for the hypothesis that the agent of prion infectivity is not a virus. PMCA-generated samples caused the same clinical disease and neuropathology with the same rapid incubation period as the input brain-derived scrapie samples, providing no evidence for generation of a new strain in PMCA. However, the ratio of the infectivity titer to the amount of PrPres (specific infectivity) was much lower in PMCA versus brain-derived samples, suggesting the possibility that a substantial portion of PrPres generated in PMCA might be noninfectious.

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    • "brain cofactors that might promote infectivity or (2) the generation of noninfectious PrP res by PMCA, as previously suggested (Kim et al., 2010; Klingeborn et al., 2011). Interestingly and similarly to previous reports (Castilla et al., 2005a; Deleault et al., 2007), this material characterized by lower infectivity was originally seeded with 263K hamster scrapie, leaving open the question as to whether seeding with a different strain would have resulted in the same or higher infectivity levels comparable to brain-derived material. "
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    ABSTRACT: Transmissible spongiform encephalopathies (TSEs) most commonly known as prion diseases are invariably fatal neurological disorders that affect humans and animals. These disorders differ from other neurodegenerative conformational diseases caused by the accumulation in the brain of misfolded proteins, sometimes with amyloid properties, in their ability to infect susceptible species by various routes. While the infectious properties of amyloidogenic proteins, other than misfolded prion protein (PrPTSE), are currently under scrutiny, their potential to transmit from cell to cell, one of the intrinsic properties of the prion, has been recently shown in vitro and in vivo. Over the decades, various cell culture and laboratory animal models have been developed to study TSEs. These assays have been widely used in a variety of applications but showed to be time consuming and entailed elevated costs. Novel economic and fast alternatives became available with the development of in vitro assays that are based on the property of conformationally abnormal PrPTSE to recruit normal cellular PrPC to misfold. These include the cell-free conversion assay, protein misfolding cyclic amplification (PMCA) and quaking induced conversion assay (QuIC), of which the PMCA has been the only technology shown to generate infectious prions. Moreover, it allows indefinite amplification of PrPTSE with strain-specific biochemical and biological properties of the original molecules and under certain conditions may give rise to new spontaneously generated prions. The method also allows addressing the species barrier phenomena and assessing possible risks of animal-to-animal and animal-to-human transmission. Additionally, its unprecedented sensitivity has made possible the detection of as little as one infectious dose of PrPTSE and the biochemical identification of this protein in different tissues and biological fluids, including blood, cerebral spinal fluid (CSF), semen, milk, urine and saliva during the pre-clinical and clinical phases of the disease. The mechanistic similarities between TSEs and other conformational disorders have resulted in the adaptation of the PMCA to the amplification and detection of various amyloidogenic proteins. Here we provide a compelling discussion of the different applications of this technology to the study of TSEs and other neurodegenerative diseases.
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