Converting normal prion protein (PrP C ) to the pathogenic PrP Sc isoform is central to prion disease. We previously showed that, in the presence of lipids, recombinant mouse PrP (rPrP) can be converted into the highly infectious conformation, suggesting a crucial role of lipid-rPrP interaction in PrP conversion. To understand the mechanism of lipid-rPrP interaction, we analyzed the capability of various rPrP mutants to bind anionic lipids and to gain the lipid-induced proteinase K (PK)-resistance. We found that the N-terminal positively charged region contributes to the electrostatic rPrP-lipid binding, but does not affect lipid-induced PK-resistance. In contrast, the highly conserved middle region of PrP, consisting of a positively charged region and a hydrophobic domain, is essential for lipid-induced rPrP conversion. The hydrophobic domain deletion mutant significantly weakened the hydrophobic rPrP-lipid interaction and abolished the lipid-induced C-terminal PK-resistance. The rPrP mutant without positive charges in the middle region reduced the amount of lipid-induced PK-resistant rPrP form. Consistent with a critical role of the middle region in lipid-induced rPrP conversion, both disease-associated P105L and P102L mutations, localized between lysine residues in the positively charged region, significantly affected lipid-induced rPrP conversion. The hydrophobic domain localized 129 polymorphism altered the strength of hydrophobic rPrP-lipid interaction. Collectively, our results suggest that the interaction between the middle region of PrP and lipid is essential for the formation of PKresistant conformation. Moreover, the influence of disease-associated PrP mutations and 129 polymorphism on PrP-lipid interaction supports the relevance of PrP-lipid interaction to the pathogenesis of prion disease.Prion diseases are a group of transmissible neurodegenerative disorders including Creutzfeldt-Jakob Disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS) in humans, scrapie in sheep, and bovine spongiform encephalopathy in cows (1-3). The transmission of prion disease is mediated by an unusual infectious agent, which is composed of a pathogenic conformer of the normal prion protein (PrP) (1,(4)(5)(6). The conformational change of PrP, converting the normal protease sensitive PrP C to the pathogenic protease resistant PrP Sc form, is a critical pathogenic event in prion disease (7)(8)(9)(10)(11). We recently reported that in the presence of anionic phospholipid POPG (1-palmitoyl-2-oleoylphosphatidylglycerol) and total mouse liver RNA, bacterially expressed recombinant PrP (rPrP) can be converted in vitro into the infectious conformer, causing prion disease in wild type animals (12). The high prion infectivity associated with the recombinant prion (16). Notably, the purified PrP C used in that study contains a stoichiometric amount of co-purified lipid molecules, indicating a role of lipid in PrP conversion (16). The role of lipid in PrP conversion has also been implicated by several previous studi...

Whether a genetic informational nucleic acid is required for the infectivity of transmissible spongiform encephalopathies is central to the debate about the infectious agent. Here we report that an infectious prion formed with bacterially expressed recombinant prion protein plus synthetic polyriboadenylic acid and synthetic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol is competent to infect cultured cells and cause prion disease in wild-type mice. Our results show that genetic informational RNA is not required for recombinant prion infectivity.

The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50 / μg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210 – 220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

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