Selective incorporation of polyanionic molecules into hamster prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrP^C, into a pathogenic isoform, PrP^Sc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrP^C 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. In a PMCA reaction lacking PrP^Sc template seed, synthetic poly(A) RNA molecules induce hamster (Ha)PrP^C to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrP^Sc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrP^C molecules subjected to PMCA selectively incorporate an ∼1-2.5-kb subset of [³²P]poly(A) RNA molecules from a heterogeneous mixture ranging in size from ∼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.