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Author(s): Byron Caughey (corresponding author) [1]; Gerald S. Baron (corresponding author) [1] The transmissible spongiform encephalopathies (TSEs) first attracted widespread scientific attention almost half a century ago. The infectious agents [...]

Analysis of several insertion mutants of Francisella novicida, synthesized by in vitro cassette mutagenesis reveals the presence of two mutants, KM14 and KM14S, which are sensitive to serum. The amino acid sequences of these mutants are similar to that of msbA and orfE proteins in Escherichia coli, as indicated by analysis of a cloned genetic locus. The msbA protein belongs to the class of ABC transporter proteins.

The molecularly defined mammalian transmissible spongiform encephalopathies (TSE) prions and their three-dimensional structure, biogenesis and neuropathogenic effects are described. The studies have emphasized on the unique aspect of mammalian TSE diseases, the anchoring of the protein of interest, PrP, to membranes by a glycophosphatidylinositol (GPI) moiety.

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrPSc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrPSc propagation involves conversion from its normal isoform, PrPC, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrPSen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrPSc (PrPRes). Scrapie brain dilutions up to 10¯8 and 10¯13 were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrPRes levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrPC. Although the brains of 263K-affected mice had little immunoblot-detectable PrPRes, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrPRes strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrPSc. [ABSTRACT FROM AUTHOR]

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal proteasesensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6-7, and acid pre-treating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP-res into the raft domains of recipient cells. [ABSTRACT FROM AUTHOR]

Western immunoblotting is a workhorse technique used in the prion field to analyze disease-associated forms of the prion protein, termed PrP Sc . The biochemical stability of PrP Sc aggregates combined with the increased resistance of prion infectivity to inactivation by various treatments that inactivate most other pathogens complicates the use of Western immunoblotting as a means to characterize PrP Sc samples. In this chapter, we describe a method for Western immunoblot analysis of PrP Sc with an emphasis on precautions to address the biochemical and biosafety considerations associated with this procedure.