Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification

Universidad Autónoma de Madrid, Madrid, Madrid, Spain
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2006; 281(46):35245-52. DOI: 10.1074/jbc.M603964200
Source: PubMed

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.

Download full-text


Available from: Paula Saá, Dec 09, 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of human prion disease. Transgenic (Tg) mice that express the mouse homolog of this mutation (designated PG14) spontaneously accumulate in their brains an insoluble and weakly protease-resistant form of the mutant protein. This form (designated PG14(Spon)) is highly neurotoxic, but is not infectious in animal bioassays. In contrast, when Tg(PG14) mice are inoculated with the Rocky Mountain Laboratory (RML) strain of prions, they accumulate a different form of PG14 PrP (designated PG14(RML)) that is highly protease resistant and infectious in animal transmission experiments. We have been interested in characterizing the molecular properties of PG14(Spon) and PG14(RML), with a view to identifying features that determine two, apparently distinct properties of PrP aggregates: their infectivity and their pathogenicity. In this paper, we have subjected PG14(Spon) and PG14(RML) to a panel of assays commonly used to distinguish infectious PrP (PrP(Sc)) from cellular PrP (PrP(C)), including immobilized metal affinity chromatography, precipitation with sodium phosphotungstate, and immunoprecipitation with PrP(C)- and PrP(Sc)-specific antibodies. Surprisingly, we found that aggregates of PG14(Spon) and PG14(RML) behave identically to each other, and to authentic PrP(Sc), in each of these biochemical assays. PG14(Spon) however, in contrast to PG14(RML) and PrP(Sc), was unable to seed the misfolding of PrP(C) in an in vitro protein misfolding cyclic amplification reaction. Collectively, these results suggest that infectious and non-infectious aggregates of PrP share common structural features accounting for their toxicity, and that self-propagation of PrP involves more subtle molecular differences.
    Journal of Neurochemistry 04/2008; 104(5):1293-308. DOI:10.1111/j.1471-4159.2007.05082.x · 4.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Prions, infectious agents causing transmissible spongiform encephalopathy (TSE), are composed primarily of the pathogenic form (PrP(Sc)) of the host-encoded prion protein. Although very low levels of infectivity have been detected in urine from scrapie-infected rodents, no reports of urinary PrP(Sc) have been substantiated. Studies on the dynamics of urinary PrP(Sc) during infection are needed to ensure the safety of urine-derived biopharmaceuticals and to assess the possible horizontal transmission of prion diseases. Using the protein misfolding cyclic amplification technique, a time-course study of urinary excretion and blood levels of PrP(Sc) was performed in Sc237-infected hamsters and a high rate of PrP(Sc) excretion was found during the terminal stage of the disease. Following oral administration, PrP(Sc) was present in all buffy coat samples examined; it was also present in most of the plasma samples obtained from hamsters in the symptomatic stage. PrP(Sc) was excreted in urine for a few days after oral administration; subsequently, urinary PrP(Sc) was not detected until the terminal disease stage. These results represent the first biochemical detection of PrP(Sc) in urine from TSE-infected animals.
    Journal of General Virology 11/2007; 88(Pt 10):2890-8. DOI:10.1099/vir.0.82786-0 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal protein-misfolding neurodegenerative diseases. TSEs have been described in several species, including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronicwasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) in mink, and Kuru and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are associated with the accumulation of a protease-resistant, disease-associated isoform of the prion protein (called PrPSc) in the central nervous system and other tissues, depending on the host pecies. Typically, TSEs are acquired through exposure to infectious material, but inherited and spontaneous TSEs also occur. All TSEs share pathologic features and infectious mechanisms but have distinct differences in transmission and epidemiology due to host factors and strain differences encoded within the structure of the misfolded prion protein. The possibility that BSE can be transmitted to humans as the cause of variant Creutzfeldt-Jakob disease has brought attention to this family of diseases. This review is focused on the TSEs of livestock: bovine spongiform encephalopathy in cattle and scrapie in sheep and goats.
    ILAR journal / National Research Council, Institute of Laboratory Animal Resources 05/2015; 56(1):7-25. DOI:10.1093/ilar/ilv008 · 1.05 Impact Factor