title: 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.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, USA.
authors.forename: Justin R
authors.initials: JR
authors.lastname: Piro
authors.forename: Brent T
authors.initials: BT
authors.lastname: Harris
authors.forename: Surachai
authors.initials: S
authors.lastname: Supattapone
mesh_terms: D000818:Animals; D001483:Base Sequence; D001933:Brain Stem / Q000378:metabolism / Q000473:pathology; D006224:Cricetinae; D005260:Female; D006624:Hippocampus / Q000378:metabolism / Q000473:pathology; D008027:Light; D008647:Mesocricetus; D008954:Models, Biological; D010782:Photolysis; D011071:Poly T / Q000737:chemistry / Q000528:radiation effects; D000071228:Polyelectrolytes; D011108:Polymers / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D011328:Prions / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity* / Q000528:radiation effects*; D017510:Protein Folding; D012608:Scrapie / Q000378:metabolism / Q000473:pathology*; D014774:Virulence / Q000528:radiation effects
chemical_list: D011071:Poly T; D000071228:Polyelectrolytes; D011108:Polymers; D011328:Prions