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: 9.67). 11/2011; 108(48):E1244-53. DOI: 10.1073/pnas.1111255108
Source: PubMed


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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Available from: Mikael Klingeborn, Oct 07, 2015
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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.
    Virus Research 11/2014; 207. DOI:10.1016/j.virusres.2014.11.007 · 2.32 Impact Factor
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    • "Such transformation, however, does not necessarily indicate a decline in infectivity titer as claimed by Klingeborn and coauthors [35]. Animal bioassays of PMCA-derived PrPSc were terminated at approximately 300 days postinoculation [35], a time-frame not sufficient to establish the infectivity titers by the limiting dilution approach. At PrPSc concentrations approaching limiting dilutions, a significant fraction of animals develops disease after 300 days even for strains with short incubation times. "
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    ABSTRACT: Prion replication is believed to consist of two components, a growth or elongation of infectious isoform of the prion protein (PrP(Sc)) particles and their fragmentation, a process that provides new replication centers. The current study introduced an experimental approach that employs Protein Misfolding Cyclic Amplification with beads (PMCAb) and relies on a series of kinetic experiments for assessing elongation rates of PrP(Sc) particles. Four prion strains including two strains with short incubation times to disease (263K and Hyper) and two strains with very long incubation times (SSLOW and LOTSS) were tested. The elongation rate of brain-derived PrP(Sc) was found to be strain-specific. Strains with short incubation times had higher rates than strains with long incubation times. Surprisingly, the strain-specific elongation rates increased substantially for all four strains after they were subjected to six rounds of serial PMCAb. In parallel to an increase in elongation rates, the percentages of diglycosylated PrP glycoforms increased in PMCAb-derived PrP(Sc) comparing to those of brain-derived PrP(Sc). These results suggest that PMCAb selects the same molecular features regardless of strain initial characteristics and that convergent evolution of PrP(Sc) properties occurred during in vitro amplification. These results are consistent with the hypothesis that each prion strain is comprised of a variety of conformers or 'quasi-species' and that change in the prion replication environment gives selective advantage to those conformers that replicate most effectively under specific environment.
    PLoS ONE 07/2012; 7(7):e41210. DOI:10.1371/journal.pone.0041210 · 3.23 Impact Factor
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    • "While PrP-res is a valuable diagnostic marker for TSE disease, analytic experiments have repeatedly shown that PrP and PrP-res quantities are poor predictors of infectious titer. For example, infectious ∼25nm virus-like particles reproducibly separate from the majority of PrP and PrP-res during centrifugation (reviewed in [11]), and test-tube PrP misfolding-conversion “PMCA” assays typically show enormous amounts of de novo PrP-res can be generated with low or absent infectivity (e.g., [12], [13]). PrP-res itself appears to be insufficient for infection and most PMCA reactions need to be repeatedly primed with complex brain homogenates. "
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    ABSTRACT: Rat septal cells, induced to enter a terminal differentiation-like state by temperature shift, produce prion protein (PrP) levels 7x higher than their proliferative counterparts. Host PrP accumulates on the plasma membrane, newly elaborated nanotubes, and cell-to-cell junctions, important conduits for viral spread. To find if elevated PrP increased susceptibility to FU-CJD infection, we determined agent titers under both proliferating and arresting conditions. A short 5 day arrest and a prolonged 140 day arrest increased infectivity by 5x and 122x (>2 logs) respectively as compared to proliferating cells. Total PrP rapidly increased 7x and was even more elevated in proliferating cells that escaped chronic arrest conditions. Amyloid generating PrP (PrP-res), the "infectious prion" form, present at ~100,000 copies per infectious particle, also increased proportionately by 140 days. However, when these highly infectious cells were switched back to proliferative conditions for 60 days, abundant PrP-res continued to be generated even though 4 logs of titer was lost. An identical 4 log loss was found with maximal PrP and PrP-res production in parallel cells under arresting conditions. While host PrP is essential for TSE agent spread and replication, excessive production of all forms of PrP can be inappropriately perpetuated by living cells, even after the initiating infectious agent is eliminated. Host PrP changes can start as a protective innate immune response that ultimately escapes control. A subset of other neurodegenerative and amyloid diseases, including non-transmissible AD, may be initiated by environmental infectious agents that are no longer present.
    PLoS ONE 04/2012; 7(4):e35471. DOI:10.1371/journal.pone.0035471 · 3.23 Impact Factor
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