Abstract
The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro. Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.
Highlights
Proposed that prions are unorthodox infectious agents composed of only PrPSc molecules (3)
We obtained qualitatively similar results with a different PrPSc-specific antibody, mAb 15B3, the immunoprecipitation experiment using this antibody was complicated by background affinity for purified HaPrPC (Fig. 1C) (40). These results indicate that, during protein misfolding cyclic amplification (PMCA), poly(A) RNA molecules can induce HaPrPC to adopt a conformation that is reactive against antibodies specific for the PrPSc conformation
Physical Association between HaPrP and Nucleic Acid Molecules in PMCA-generated Complexes—Our observation that poly(A) RNA molecules can induce a change in HaPrPC conformation led us to investigate whether polyanionic and HaPrPC
Summary
Reagents—Synthetic poly(A), catalog number P9403 (0.2– 6 kb by agarose gel electrophoresis), was purchased from Sigma. 150 l of secondary antibody, sheep anti-mouse conjugated with Alexa Fluor 568 (Invitrogen), diluted 1:250 in PBS ϩ 4% BSA, was applied to each slide and allowed to incubate for 2 h in the dark at room temperature in a humidified chamber. PMCA reactions were prepared on ice. Samples without PrPC contained PrPC dilution buffer (20 mM MOPS, pH 7.5, 0.15 M NaCl, 0.5% Triton X-100) in the place of PrPC. The sample pellets were washed by addition of 950 l of PBS, 0.5% Triton X-100, briefly mixed by vortexing, followed by 100,000 ϫ g centrifugation for 45 min at 4 °C. For PrP immunohistochemistry, tissue sections were blocked with 10% normal goat serum in PBS for 30 min at room temperature and incubated overnight at 4 °C with a 1:50 dilution of 3F4 anti-PrP primary mouse IgG (Covance Research Products, Inc). Molecules alter HaPrP conformation, and to test whether the resulting conformation might further resemble that of HaPrPSc, we conducted an immunoprecipitation experiment using 89-112 mAb, a motif-grafted antibody that recognizes the PrPSc confor-
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