In situ photodegradation of incorporated polyanion does not alter prion infectivity

Abstract

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrP(Sc) aggregates in vivo. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrP(Sc), we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrP(C) substrate, PC-oligo was incorporated into physical complexes with PrP(Sc) molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrP(Sc) molecules alone.

Figure 1. Schematic showing the composition of the PC-oligo.

Repeating units of five deoxythymine residues were followed by a photocleavable (PC) linker as shown. Enlarged inset shows the chemical structure of the PC linker (3-(4,4′-Dimethoxytrityl)-1-(2-nitrophenyl)-propan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite). The arrow indicates the location of the cleavage site (the phosphodiester bond between the linker and the 5′ phosphate of the oligonucleotide).

Figure 2. Western blot showing the effect of degrading PC-oligo on its ability to stimulate prion conversion in sPMCA.

Lanes 1 and 6 show non-digested PrP^C used as substrate (-PK). The remaining samples were subjected to limited proteolysis with proteinase K.

Figure 3. Acrylamide gel electrophoresis showing the effects of benzonase and light treatment on oligonucleotide integrity.

A. Experiments performed with dT-oligo. B. Experiments performed with PC-oligo. In both panels, treatments were performed on oligonucleotides in the presence or absence of 1.5 mg/ml recPrP, as indicated.

Figure 4. Western blot showing the final samples from 3 round sPMCA reactions.

Samples containing dT-oligo or PC-oligo were seeded with a dilution of PrP^Sc which was either subjected to light treatment or not, as indicated. All samples were subjected to limited proteolysis with proteinase K.

Figure 5. Representative histological fields of brainstem and hippocampus.

Hemotoxylin and Eosin staining from animals injected with in vitro generated PrP^Sc containing dT-oligo or PC-oligo treated with or without light. Top row: animals injected with prions treated in dark. Middle row: animals injected with prions treated with light. Bottom row: control animals. A. Brain stem. (Scale bar, 100 µm). B. Hippocampus. (Scale bar, 200 µm).

Figure 6. Regional neuropathology of hamsters infected with light- or dark-treated inocula.

Vacuolation profile scores of animals inoculated with samples containing dark-treated dT-oligo (-◆-), light-treated dT-oligo (-⌑-), dark-treated PC-oligo (-▲-), light-treated PC-oligo (-◯-), PrP^C (-X-), or dT/PC-oligos alone (--). The mean values (n = 5–8 animals/group) are shown ± S.E.M. FC: Frontal cortex. PC: Parietal cortex. H: Hippocampus. C: Cerebellum. BS: Brain stem.