Selective incorporation of polyanionic molecules into hamster prions

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.

FIGURE 1. Effect of poly(A) RNA on HaPrP^C conformation

A–D, Western blots showing samples subjected to one round of PMCA. Except where indicated, all samples contained ~0.25 µg/ml HaPrP^CA, following PMCA, samples containing HaPrP^C plus other components, as indicated, were subjected to 100,000 × g, and the pellet from each sample was analyzed by Western blot. Samples composed of the input HaPrP^C and HaPrP^Sc are also shown for comparison (lanes 7and 8). B, following PMCA, samples containing HaPrP^C plus other components, as indicated, were immunoprecipitated (IP) with 89–112 mAb. C, following PMCA, samples containing HaPrP^C plus other components, as indicated, were immunoprecipitated with 15B3 mAb Dynabeads. D, samples taken from the PMCA reactions containing HaPrP^C plus other components, as indicated, were subjected to limited proteolysis with 50 µg/ml proteinase K. Although the samples in each figure panel were analyzed on the same Western blot, some sample lanes were rearranged in a different order, as indicated by a space between lanes.

FIGURE 2. Facilitation of HaPrP^Sc propagation by homopolymeric oligonucleotides

Western blots showing purified HaPrP^Sc propagation reactions with various homopolymeric 100-base oligonucleotides (A) or oligo(dT) molecules of various lengths (B). The HaPrP^Sc template used to seed the reactions was generated in a serial propagation reaction containing synthetic (dT)₁₀₀. A sample containing HaPrP^C not subjected to proteinase K digestion is shown in the lane 1 as a reference for comparison of electrophoretic mobility (PrP^C-PK). All the other samples shown on the blots were subjected to limited proteolysis with 50 µg/ml proteinase K(+PK). ℘ = negative control sample containing no polyanion molecules.

FIGURE 3. Fluorescein (dT)₁₀₀-PrP co-localization assay

Fluorescence micrographs showing representative images of samples containing HaPrP^C and fluorescein-labeled (dT)₁₀₀, with (Seeded) or without (Unseeded) HaPrP^Sc. All samples were subjected to one round of PMCA and prepared for analysis as described under “Experimental Procedures.” The sample pellet from a seeded reaction was digested with 50 units of Benzonase before analysis (Seeded + Nuclease). All samples were analyzed by fluorescence microscopy as described under “Experimental Procedures” to visualize HaPrP and oli-go(dT). HaPrP aggregates were immunostained with an anti-PrP mAb and Alexa Fluor 568-labeled secondary antibody and are shown in magenta (PrP). The (dT)₁₀₀ molecules were visualized by the fluorescein label and are shown in green (Fluorescein). Co-localized HaPrP and fluorescein-labeled (dT)₁₀₀were identified by overlaying both images (Merge). Scale bar = 200 µm.

FIGURE 4. Nuclease protection assay

Autoradiograph showing samples containing [³²P]poly(A) RNA with or without HaPrP^C. All samples contained 20 µg/ml (~50,000 cpm) [³²P]poly(A) RNA. Samples were subjected to either one round of PMCA (lanes 1–4) or no PMCA (lanes 5 and 6) and prepared for analysis as described under “Experimental Procedures.” Pellets from each sample were treated with 50 units of Benzonase (lanes2,4, and 6) or without Benzonase (lanes 1,3, and 5). 100pg (1/60,000th) of the total input of [³²P]poly(A) RNA(lane7) was included on the same blotas a reference for poly(A) RNA length, although the position of this sample on the blot was moved as indicated by the space between lanes. The same [³²P]poly(A) RNA preparation was also subjected to a separate analysis to obtain a darker exposure (lane 8).

FIGURE 5. Neuropathological analysis of scrapie-infected hamster brains

Fluorescent micrographs showing representative images of tissue sections from scrapie-infected hamster brains immunostained for HaPrP and histochemically stained with acridine orange. Prior to staining, samples were either not treated (Control) or digested with RNase (RNase) or heparinase III (Heparinase). Immunostained HaPrP aggregates are shown in green (PrP), and acridine orange staining is shown in magenta (Acridine Orange). Scale bar = 10 µm.

FIGURE 6. Selective interaction of poly(A) RNA and HaPrP during PMCA

Without incubation, HaPrP molecules associate with small poly(A) RNA molecules (A) that are not protected from nuclease digestion because of weak intermolecular interaction (C). During PMCA, HaPrP molecules selectively associate with larger poly(A) RNA molecules (B), resulting in the formation of a nuclease-resistant nucleoprotein complex (D).