Seeding specificity and ultrastructural characteristics of infectious recombinant prions

Abstract

Infectious mouse prions can be produced from a mixture of bacterially expressed recombinant prion protein (recPrP), palmitoyloleoylphosphatidylglycerol (POPG), and RNA [Wang, F.; et al. (2010) Science 327, 1132]. In contrast, amyloid fibers produced from pure recPrP without POPG or RNA (recPrP fibers) fail to infect wild type mice [Colby, D.W.; et al. (2010) PLoS Pathog. 387, e1000736]. We compared the seeding specificity and ultrastructural features of infectious recombinant prions (recPrP(Sc)) with those of recPrP fibers. Our results indicate that PrP fibers are not able to induce the formation of PrP(Sc) molecules from wild type mouse brain homogenate substrate in serial protein misfolding cyclic amplification (sPMCA) reactions. Conversely, recPrP(Sc) molecules did not accelerate the formation of amyloid in vitro, under conditions that produce recPrP fibers spontaneously. Ultrastructurally, recombinant prions appear to be small spherical aggregates rather than elongated fibers, as determined by atomic force and electron microscopy. Taken together, our results show that recPrP(Sc) molecules and PrP fibers have different ultrastructural features and seeding specificities, suggesting that prion infectivity may be propagated by a specific and unique assembly pathway facilitated by cofactors.

Figure 1

Western blots of sPMCA reactions using normal brain homogenate substrate, seeded with various samples, as indicated. In each blot, the first lane shows the initial round mixture sample not subjected to protease digestion (−PK). The remaining samples were subjected to limited proteolysis with proteinase K.

Figure 2

Graph showing the results of the amyloid seeding assay. Samples: (■) mock-seeded; (●) seeded with preformed recPrP fibers; (▲) seeded with recPrP^Sc. Error bars represent SD.

Figure 3

Representative AFM amplitude images. (A) Two-dimensional images of and recPrP fibers (left) and recPrP^Sc (right). Images at the bottom are smaller scan sizes captured by using a sharper AFM tip. (B) Three-dimensional images of recPrP fibers (left) and recPrP^Sc (right). Images at the bottom are smaller scan sizes captured by using a sharper AFM tip. (C) AFM amplitude images of recPrP fibers treated with a 30 s pulse of sonication before imaging taken from different regions.

Figure 4

Graphs showing the frequency distribution of heights from either (A) recPrP fibers or (B) recPrP^Sc. The data were collected by analyzing AFM images collected in tapping mode. More than 1000 individuals particles were analyzed from each group.

Figure 5

Representative transmission electron microscopic images of recPrP fibers (left) and recPrP^Sc (right). Scale bars equal 100 nm.