Abstract
Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrPSc aggregates in vivo [1], [2]. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrPSc, we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrPC substrate, PC-oligo was incorporated into physical complexes with PrPSc 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 PrPSc molecules alone.
Highlights
Infectious prion diseases such as Creutzfeldt Jakob disease (CJD) and other related human disorders, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and scrapie are associated with the conversion of a host-encoded glycoprotein (PrPC) into a misfolded conformer, PrPSc [3]
These observations suggest that negatively charged polymers might act as a structural support necessary for prion infectivity
Prions containing this light-sensitive polymer remained infectious after light exposure, indicating that negatively charged polymers are not necessary to maintain the structural shapes of infectious prions
Summary
Infectious prion diseases such as Creutzfeldt Jakob disease (CJD) and other related human disorders, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and scrapie are associated with the conversion of a host-encoded glycoprotein (PrPC) into a misfolded conformer, PrPSc [3]. Several biochemical and cell culture studies have implicated polyanions such as single stranded nucleic acid and glycosaminoglycan (GAG) molecules as potent cofactors in the process of infectious prion propagation [1,5,6,11,12,13,14,15,16,17]. Neuropathological analysis of scrapie-infected animals has shown that nucleic acids and GAG-containing proteoglycans co-localize with PrPSc aggregates in situ [1,2]. These observations raise the possibility that moderately long polyanions might be needed to maintain hamster prion infectivity or strain properties by acting as a structural support for PrPSc molecules
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